Title of Invention	AN IN VITRO METHOD FOR REDUCING EFFECTS OF HETEROPHILIC ANTIBODIES DURING SPECIFIC BINDING REACTION OF A BINDING PAIR
Abstract	The invention discloses an in vitro method for reducing effects of heterophilic antibodies, particularly effects due to human anti- mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and triglycerides; during a specific binding reaction of a binding pair, wherein a first binding member recognises its complementary second binding member, wherein the method comprises the use of an aqueous solution as medium for the specific binding reaction which aqueous solution is comprising: a) a buffer to control pH ; b) a compound A selected from the group consisting of: (i) a compound defined by the general formula I R1-[[CR2R3]p-O]q-R4, wherein R1, R2, R3, R4, p and and q are as defined in the specification , (ii) polyol, and (iii) saccharide c) a non-ionic detergent.

Title of Invention

AN IN VITRO METHOD FOR REDUCING EFFECTS OF HETEROPHILIC ANTIBODIES DURING SPECIFIC BINDING REACTION OF A BINDING PAIR

Abstract

The invention discloses an in vitro method for reducing effects of heterophilic antibodies, particularly effects due to human anti- mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and triglycerides; during a specific binding reaction of a binding pair, wherein a first binding member recognises its complementary second binding member, wherein the method comprises the use of an aqueous solution as medium for the specific binding reaction which aqueous solution is comprising: a) a buffer to control pH ; b) a compound A selected from the group consisting of: (i) a compound defined by the general formula I R1-[[CR2R3]p-O]q-R4, wherein R1, R2, R3, R4, p and and q are as defined in the specification , (ii) polyol, and (iii) saccharide c) a non-ionic detergent.

Full Text	[0001] The present invention relates to an aqueo is solution for the use as medium for the specific binding reaction of a binding pair, [OOOP] Background of the invention [0003] Immunoassays where one or more antibodies are used to detect the test substance (analyte) in a test sample are widely known. The evolution of immunoassay methods increased the sensitivity of this test. Despite of the developments within the recent decades, there remans a desire to eliminate unspecific binding reactions, cross-reactivities and the influence of the compounds present in the matrix. [0004] Immunoassays depend upon the ability of i first binding member of a binding member pair, e.g. an antigen or a ligand, to specifically bind to a second binding member of a binding member pair, e.g. an antibody or a receptor. In order to determine the extend of such binding, a conjugate, comprising one of such binding members are labeled with a detectable moiety Such binding member pairs can be an antigen and an antibody directed to such an antigen. [0005] Immunoassays can be performed in a competitive immunoassay format or in a sandwich immunoassay format. In the competitive immunoassay format an antigen can be immobilized to a solid phase material whereby the amount of detectable moiety that is bound to a solic phase material can be detected, measured and correlated to the amount of antibody present in the test sample. Examples of solid phase materials include beads, particles, micro- particles and the like. In the sandwich immunoassa format a test sample, containing for example an antibody, is contacted with a protein such as an antigen. The antigen is immobilized on a solid phase material. Examples of solid phase materials include bear is, particles, micro-particles and the like. The solid phase material is typically cleated with a second antigen or antibody that has been labeled with a detectable moiety. The second antigen or antibody, respectively, then becomes bound to the corresponding antibody or antigen, respectively, on the solid phase mater al and, after one or more washing steps, to remove any unbound material an indicator material such as a chromogenic substance, is introduced to react wi h the detectable moiety to produce the detectable signal, e.g. a color change. The color change is then detected, measured and correlated to the amount of antibody present in the test sample. It should also be noted that various dilutents and suffers are also required to optimize the operation of the micro-particles, antige is, conjugates and other components of the assay that participate in chemical 'eactions. [0006] In order o achieve optimal results in immunoassays the solution which is used for the binding reactions between the binding partners (for example the antibody and antige n reaction or the complex formation of ligand and receptor) must provide a mediim that optimizes the ability of antibodies to bind to the antigen, or must provide a medium that optimizes the ability of ligands to bind to the receptor, while non-specific interactions, low-affinity binding and matrix effects are strongly reduced o even prevented in order to avoid the generation of a false signal. [0007] In order to eliminate non-specific interactions and cross-reactivities detergents have been edded to buffers which are used for washing steps after the binding reaction in order to remove unspecific bindings. [0008] For immunoassays, like western-blot analyses, enzyme-linked immuno-sorbant assa (ELISA) and others, solutions containing phosphate buffered saline (PBS)! upplemented with bovine serum albumin and 0.01 to 0.05 (v/v) Tween 20 is use i as medium for the binding reactions between the binding partners (for example antibody and antigen). It is, however, very often experienced that unspecific or low-affinity binding, cross-reactivities and matrix effects can not be avoided with such buffers of the state of art. For example, when developing a CRP-assay involving the detection of a plurality of analytes it appeared that cross- reactivities due to the use of the plurality of antibodies as well as matrix effects became a problem, which could not be solved by the use of conventional immunoassay buffers. [0009] The object of the present invention therefore was to provide a solution for the use as a medium for the specific bindi ig reaction of a binding member pair wherein the unspecific binding, low affinity binding, cress-reactivities and matrix effects are strongly reduced or even preventec. Furthermore, it was the object of the present invention to provide a method of an immunoassay, wherein unspecific and low affinity binding, cross-reactivities and matrix effects are reduced or prevented. [0010] Summary of the invention [0011] The object of the present invention is solver by an aqueous solution for use as medium for the specific binding reaction of a binding member pair, wherein a first binding member recognizes its complementary second binding member, the solution comprising a) a buffer to control pH; b) a compound A selected from the group consisting of: a compound defined by the general formula I R1-[[CRzR3]p~0]q-R'1 wherein R1 is hydrogen or hydroxy group, R2 for each unit independently is hydrogen or hydroxy group, R3 is hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p is an integer of from 2 to 10 and q is an intet er of from 1 to 100, with the proviso that the compound at least carries two hydroxy groups; polyol; saccharide; c) a non-ionic detergent. [0012] In case F4 in the general formula I of compound A is hydrogen the neighbouring residue Rz is also hydrogen. In case R1 is hydroxy group the neighbouring residue R2 is hydrogen. In a preferred embodiment q in the formula of compound A is an in :eger of from 1 to 50, more preferred from 1 to 30. [0013] The inventors of the present invention surprisingly have found that the aqueous solution according to the present invention reduces the cross- reactivities, matrix e fects, unspecific bindings and low affinity-binding in immunoassays. Furthermore, it was found that even effects due to heterophilic antibodies (human anti mouse-antibody) are prevented when the aqueous solution according to the present invention is used. Additionally, negative effects due to rheuma factors, hemoglobin, bilirubin and triglycerides can be avoided with this buffer even in case of plasma applications. [0014] As used lerein a "binding member pair" comprises a "first binding member" and a "seccnd binding member". Both binding members undergo a specific binding to eacli other. The first binding member of a binding member pair may be an antigen or aligand, respectively. The second binding member (e.g. an antibody or a receptor, respectively) specifically recognizes and binds to the first binding member (e.g. antigen or ligand, respectively). The second binding member is the corresponding binding member and therefore also named "corresponding binding member". The irtisan will understand that the terms "first" binding member and "second" binding nember, respectively, may be for example the antigen and the corresponding antit ody, respectively, or vice versa. [0015] The aque )us solution according to the present invention represents a universal buffer as a rr edium in immunoassays and binding reactions in a variety of matrices, for example blood plasma, blood serum and others. In case of mulli- analyie applications, for example, if protein chips are used, the simultaneous incubation of several (era plurality) of analytes and several antibodies, unspecillc bindings and cross-rea ;tivities very often occur. Such undesired binding reactions have been observed in many cases. The use of standard ELISA buffers known in the state of art could not prevent such cross-reactivity offects. In addition in the state of art, the use of native samples resulted in matrix effects which gave erroneous measurements compared to other methods for reference. As used herein the term "matrix" refers to all compounds presen: in a native sample, like blood scrum; in particular the term "matrix" refers to the Drganic and especially to biological compounds such as proteins. [0016] The aqueous solution according to the piesent invention may be used as medium for the binding reaction of a binding pair, as sample dilution buffers for immunoassays and binding reactions as well as dilution buffer for antibodies and antigens, respectively. Further applications are multi-analyte immunoassays and proteomics, wherein undesired cross-reactivities of antibodies labeled with the fluoraphor can be prevented. Fluorophor tabled antibodies tend to bind other proteins in an unspecific manner. By usirg the aqueous solution according to the present invention such effects can be avoided. [0017] By using the buffer according to the present invention in immunoassays like ELISA and protein chips a further positive effect was shown. When incubating the surface carrying the immobilized sntibodies with the buffer according to the present invention the activity of the immobilized antibodies was increased. This is resulting in an enhancement of the bin ting of the analyte to the immobilized antibody. In conclusion, the buffer according to the present invention in addition to the reduction of unspecific signals and unspecific effects also increases the specific signals due to a positive influence on the specific binding reaction between the analyte and the antibody. The resulting increase of the activity of the immobilized antibodies provides a higher se nsitivity of the respective assay. [0018] The buffer according to the present invention may be used for immunoassays ELISA, EIA, FIA, lateral-flow-test, protein chips, multi-analyte assays, western blots, dot blots, immunohistochemistn,, receptor-ligand-assays and immuno-PCR. Detailed description cf the invention [0019] In a preferred embodiment of the present invention the aqueous solution is further comprising a protein in an amount effective to immunologically block non-specific anti )Ody binding. This protein preferably is selected from the group bovine serum ulbumin, ovalbumin, casein, fetal bovine serum. Further preferred, the protein is present in the aqueous solution in a concentration in the range of 0.1 to 2 % (w,v) and further preferred in the range of 0.5 to 1.5 % (w/v). These proteins are rot recognized by any of the antibodies used in the immunoassays. This uirecognized protein allows the immunological blocking of non-specific antibody-b nding by molecules or compounds which might be present in the sample. [0020] In a furthur embodiment the aqueous solution is comprising a salt selected from the group NaCI, KCI, NH4CI. Further preferred the aqueous solution is having an ionic strength of 100 mM to 1.5 mM, more preferred of 200 mM to 1 M, even further preferred of 200 mM to 800 mM, particularly more preferred of 200 mM to 600 mM and most preferred of 250 mM to 500 mM. The inventors surprisingly have found that a high ionic strength of the buffer used as a medium for binding reactions, fur example, in the range of 200 mM to 600 mM is further reducing unspecific binding and cross-reactivies, while the specific binding reaction is not negativel / influenced. [0021] In a particular preferred embodiment the buffer of the aqueous solution is selected fron the group Tris (Tris(hydroxymethyl)-aminomethane, Pipes (Piperazine-1,4-bis-2-etiane sulfonic acid), Mes (4-Morpholino ethane sulfonic acid), Hepes (4-(2-hycroxyethyl)-1-piperazine-ethane sulfonic acid), phosphate buffer. [0022] In a furthe • preferred embodiment the compound A is selected from the group polyalkylene (lycol, polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol, monosaccharides, disaccharides, trisaccharides, saccharose, mannose, trehalose, polyol, glycerol anc. mixtures thereof. In a preferred embodiment the concentration of the compourd A is in the range of 0.5 to 25 % (v/v), preferably in the range of 2.0 to 20 % (v,v), more preferred in the range of 2.0 to 15 % (v/v), further more preferred in the range of 2.0 to 10 % (v/v), even more preferred in the range of 2.0 to 7 % (v/v), and most preferred around 5 % (v/v). The concentration is given in % (v/v) in case cnumpound A is a liquid. In case the compound A is solid (for example a saccharide ¦ the concentration has to be understood as % (w/v). [0023] In a further preferred embodiment the aqueous solution comprises as i non-ionic detergent a compound of the general formula s elected from the group a) a substituted phenyl residue having substituent:; R1 and R2 (R 1-Ph-R2), wherein R1 is C1-C9 alkyl group, and R2is - wherein "a" is an integer of 5 to 40, wherein R2 ir respect to R1 is in para, meta orortho position. , b) , wherein n, x, y and z together is an integer of 5 to 40, R is a fatty acid residue. [0024] Further preferred the non-ionic detergent is selected from the group Dodecylpoly(ethyleneglycolether)m, wherein m is an infager of 5 to 40; 1-O-n- Octyl-f£-D-gluccpyrano:;ide (n-Octylglucoside); Alkylphenolpoly(ethyleneglycol- ether)m, wherein rn is c n integer of 5 to 40, preferably m=11 (Nonidet P40e); 1 -O- n-Dodecyl-fS-D-glucopyranosyl (1-4)alpha-D-giucopyranoside; Dodecylpoiy- (ethyieneglycolether)m, wherein m is an integer of 5 to 40, preferably m = 23 (Brij35®); Poiy(oxyeth7lene)(20)-sorbitane mono fatty acid ester, preferably selected from Pol/(oxyethylene)(20)-sorbitane monooleate (Tween®80), Poly(oxyethylene)(20)-.' ;orbitane monolaurate (Tween®20), Poly(oxyethylene)(20)- sorbitane monopal nilat (Tween®40), Poly(oxyethylene)(20)-sorbitane monostearate); Octylpl enolpoly(ethyleneglycolether)m, wherein m is an integer of 5 to 40, preferably m=10 (Triton^X-100). [0025] In prefer-ed embodiments the concentration of the non-ionic detergent is in the ranee of 0.1 to 1.0 % (v/v). Preferably, the concentration of the non-ionic detergent in tie range of 0.15 to 1.0 % (v/v), more preferred in the range of 0.2 to 1.0 % (v/v), further more preferrred in the range between 0.2 and 0.8 % (v/v), even more preferred in the range of 0.25 % to 0.6 % (v/v), and most preferred about 0.25 % (v/v). [0026] An imporant feature of the present invention is the presence of compound A as given i n claim 1 in combination with the non-ionic detergent. Each of said both ingredients (compound A and non-ionic detergent) is present in the aqueous solution of the present invention in higher concentrations as it is known in respect to incubation solutions for immunoassays of the state of art. In a further preferred embodiment an aqueous solution is provided, wherein the ratio of the non-ionic detergent to the compound A is from 1:15 to 1:25, preferably around 1:20. [0027] In a pa ticularly preferred embodiment ihe aqueous solution comprises compound / in a concentration in the range of 2 to 7 % (v/v) and a non- ionic detergent in the range of between 0.2 to 0.8 % (v/v). Preferably the aqueous solution is having an icnic strength of 200 mM to 1 M, more preferred of 200 mM to 800 mM, particularly more preferred of 200 mM to 600 mM and most preferred of 250 mM to 500 mM. It is preferred that the aqueous solution comprises as compound A a compound selected from the grcup polyalkylene glycol, polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol, glycerol and mixtures thereof, more preferred a compound selected from polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol and most preferred ethylene glycol. [0028] The aqueous solution according to the present invention preferably does not contain dithiothreitol. Further preferred the aqueous solution according to the present invention does not contain U-mercapto-ethanol. [0029] In a further preferred embodiment the pH cf the aqueous solution is adjusted in the range of 5.6 to 9.6, preferably in the ra nge of 6.0 to 9.0, further preferred in the range of 6.5 to 8.0 and most preferred in he range of 6.8 to 7.4. [0030] A particularly preferred embodiment of ihe aqueous solution is having the capability of reducing unspecific binding, cross-reactivity and disturbing effects of the matrix. The aqueous solution according to the present invention particularly has the capability of preventing the low-affiniiy binding with KD values of up to 10"7 M, compared to standard conditions. Furtfnr preferred the aqueous solution has the capability of preventing the low-affinity binding with Kd values of up to 10'7 M and reducing the mid-range affinity binding with Kd values in the range of between 10"7 M and 10"° M by at least 90 lo compared to standard conditions. Even further preferred, the aqueous solution has the capability of preventing the low-affinity binding with KD values of up to 10"T and reducing the mid-range affinity binding with Kp values in the range of between 10"7 and 10'9 by at least 90 % compared to standard conditions. As used herein "standard conditions" are represented by an aqueous solution cc nsisting of 50 mM PBS (phosphate buffered saline, pH 7.4), 150 mM NaCI, 1 % iw/v) BSA (see also table 1: reference example). The same results were obtained when comparing the measurements with another standard solution, namey an aqueous solution consisting of 50 mM PBS (pH 7.4), 100 mM NaCI, 0.05 % (v/v) Tween®20. [0031] Apart from the effect of reducing the unspecific, low affinity binding as well as matrix effects, the aqueous solution particularly preferred is having the capability to increase the binding activity of antibodies, preferably the binding activity of immobilized antibodies as well as the binding activity between ligands and receptors. The increase of the binding activity of immobilized antibodies by using the solution according to the present invention was about 10 % or more. [0032] The object of the present invention is also solved by a concentrate of the aqueous solution of the present invention desribed before, preferably a 2 to 10 fold concentrate, more preferred a 3 to 5 fold concentrate. [0033] Further, t le object of the present invention is solved by the use of the aqueous solution acccrding to the present invention as a medium for the binding reaction of a binding pair, wherein a first binding member specifically recognizes and binds its compler lenlary second binding member. Preferably the aqueous solution is used as a nedium for the antibody-antigen binding reaction and in an alternative embodiment the aqueous solution is used as a medium for the receptor-ligand binding reaction. In other preferred embodiments the aqueous solution is used as dilution buffer for samples, reagents, ligands, receptors, antigens, antibodies, "'he aqueous solution may also preferably be used as a washing buffer in imrnmoassays after the binding reaction was carried out. [0034] The inve ition further provides a method for reducing unspecific binding and/or cross- eactivity and/or disturbing effects of matrices during a specific binding react on of a binding pair, wherein a first binding member recognises its complementary second binding member, the method comprising the use of the aqueous sc lution of the present invention as medium for the specific binding reaction. [0035] In anothe ¦ aspect of the invention the aqueous solution according to the present invention c in be provided as a component of a kit. As used herein, the term "kit" means a co; lection of reagents and associated materials e.g. buffers, carrier comprising an immobilised binding member and reagents which are required to perform an assay. Therefore, the present ir ivention provides a kit for detection by immunoassay of at least one analyte to be tested, wherein the analyte to be tested is a first binding member of a bincing member pair, wherein i the first binding member binds specifically to its complementary binding member, the kit comprising: a) a vessel containing an aqueous solution of ihe present invention; b) a carrier comprising the complementary binding member immobilised thereon to capture the analyte; and > c) optionally, a reagent which immunologically recognises the analyte bound to the complementary binding member, wherein the reagent (antibody) is conjugated to a means of detection; and d) optionally: reagents which are reactive with said means of detection to produce a detectable reaction product. . [0036] A typical kit for example is used as ELISA lit for the detection of, for example, antibodies in blood serum. In this case the carrier according to b) comprises as complementary binding member immobilissid thereon, for example, a virus antigen to capture the analyte. The analyte which ii the antibody in the blood serum. The reagent according to c) which immunologically recognises the analyte i then is an anti-antibody which is recognising the capturec antibody. Best mode for carrying out the invention [0037] The present invention is characterized in tiat the aqueous solution for use as medium for the specific binding reaction of a binding member pair, wherein a first binding member recognizes its complementary second binding member, comprises: a) a buffer to control pH; b) a compound A selected from the group consisting of: a compound defined by the general formula I R1-f[CR2l l3]p-0]q-R4, wherein R1 is hydrogen or hydroxy group, R2 for each un t independently is hydrogen or hydroxy group, R3 is hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p is an inte jer of from 2 to 10 and q is an integer of from 1 to 100, with the proviso that the compound at least carries two hydroxy groups; po yol; saccharide; [0038] c) a r on-ionic detergent. [0039] The method of the present invention for reducing unspecific binding and/or cross-reactivity and/or disturbing effects of matrices during a specific binding reaction of a t inding pair, wherein a first binding member recognises its complementary seconc binding member, i.e. for a immunoassy, is characterized to comprise the use of the above aqueous solution. [0040] Preferably an aqueous solution was found to be useful which comprises compound A in a concentration in the range of 2 to 7 % (v/v) and a non- ionic detergent in the range of between 0.2 to 0.5 % (v/v). Preferably the aqueous solution is having an ic nic strength of 200 mM to 1 M, more preferred of 200 mM to 800 mM, particularly more preferred of 200 mM to 600 mM and most preferred of 250 mM to 500 mM. It is preferred that the aqueous solution comprises as compound A a con pound selected from the group polyalkylene glycol, polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol, glycerol and mixtures thereof, more preferred a compound selected from polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol and most preferred ethylem glycol. [0041] The present inventions will be explained in more detail in the following examples. However, the examples are only used for illustration and do not limit the scope of the present invention. Accompanying Description of the/Figures [0042] Figure 1 shows the amount of detector ant body C6 bound to CRP in a sandwich assay as absorbance at 450 nm plotted acainst the concentration of CRP [ng/ml]. The binding reaction of detector antibody C6 to the protein CRP was performed as outlined under Example 1 under standari conditions and by using sample buffers according to the present invention, r Sample buffers I to III according to the solution of the present invention reduce the influence of the matrix effects very well. Sample buffer I shows the best sensitivity. The low sensitivity of the reference buffer is caused by a strong matrix effect. [0043] Figure 2 shows the influence of two ilifferent buffers on high background signals caused by unspecific binding o the polyclonal detector antibody P2 which binds unspecific to the capture aitibody P3 (according to Example 2). In this experiment no analyte was present. .'Sample buffer I decreases significantly the unspecific binding compared to the higli background signals with reference example buffer!!. Examples [0044] Example 1: Reduction of matrix effects [0045] 100 ul diluted capture antibody C2 (final concentration 1 ug/ml in PBS-Buffer) was added to each well of a microtiter plnte (C8 StarWell Module, NUNC) and the plate was covered with a plate sealer The capture antibody is directed against CRP (c-reactive protein). Then the p ate was incubated for 5 hours at room temperature. The plate sealer was rerr oved and the plate was washed 4 times with 300 ul washing buffer (10 mM Piosphate, 350 mM NaCI, 0.05 % Tween, pH 7.') per well. Than 200 ul of blocking solution (PBS-Buffer pH 7.4, 1 % BSA) was added to each well. After covering with a plate sealer the plate was incubated avernic ht at 4 °C. The analyte CRP (c-reactive protein) was diluted in rabbit serum (0-5 ng/ml) and incubated for 30 min at room temperature. The biotin-labelled detecto • antibody C6 (directed against CRP) was diluted in different sample buffers and reference example buffers (see table 1). The final concentration was 4 jjg/ml in each preparation. The CRP-containing rabbit serum standards were diluted 1:2 with detector antibody containing sample buffers. Tho preparations were incL bated for 30 min at room temperature. The plate sealer was removed and the plate was washed 4 times with 300 ul washing buffer. Oddments of washing buffer wen: completely removed by taping the plate dry. 100 pi of the CRP-preparations weia added to the wells. The plate was covered with a plate sealer and incubated far 4 h at room temperature under gentle shaking. After that the plate was wash 3d again. 100 ul of diluted NeutrAvidin™'-Horseradish peroxidase ccnjugatec (final concentration 0,05 ug/ml in PBS-Buffer) was added to each well. The plat j was incubated for 1 h at room temperature under gentle shaking. Then the plat a was washed again. Equal volumes of the two solutions of lmmunoPure©TMB Sibstrat were mixed and 100 pi were added immediately to each well. The plate vas incubated at room temperature until the desired colour developed. The colou' changed from clear to brilliant blue. In a final step the reaction was stoppec by adding 150 pi 2 M H2S04 to each well and the absorbance was read out at 450 nm with an ELISA plate reader (Molecular devices). The influence of different buffers on matrix effects are plotted in fig. 1. Table 1 shows the results of the test. In table 1 the reduction of non-specific binding, low-affinity binding and matrix effects is indicated in the column "result" with "+". The number of"+" is indicating the amount of reduction of non-specific binding, low-affinity binding and matrix effects compared to the reference example buffer ("-"). Figure 1 stows the amount of detector antibody C6 bound to CRP as absorbance at 450 nrr plotted against the concentration of CRP [ng/ml]. Sample buffer I shows the be; t sensitivity. The low sensitivity of the reference buffer is caused by a strong ma rix effect. [0046] Example 2: Reduction of an unspecifk binding of a polyclonal detector antibody [0047] For the next assay 250 pi diluted captu 'e antibody P3 (polyclonal rabbit-anti-protease, own preparation, final concentra'ion 1 g/ml in PBS-Buffer) i was added to each well of a microtiter plate (C8 StarW 2ll Module, NUNC) and the plate was covered with a plate sealer. The plate was inc ubated for 4 hours at room temperature- After that the plate sealer was removed a id the plate was washed 4 times with 300 pi washing buffer (10 mM Phosphato, 350 mM NaCI, 0.05 % Tween®, pH 7.4) per well. Then 200 pi of blocking solitlon (PBS-Buffer pH 7.4, 1 i % BSA) was added to each well. The plate was covered again with a plate sealer and incubated overnight at 4 °C. The biotin-labelled p Dlyclonal detector antibody P2 (polyclonal rabbit-anti-protease, own preparation) was diluted in reference example buffer II or in sample buffer I (see taMe 1) respectively (final concentration 10 ug/ml in each preparation) and added to the wells (250 pi per well; fivefold replicates). The plate was incubated for 2 I at room temperature. The plate sealer was removed and the plate was washed 4 times with 300 pi washing buffer. Oddments of washing buffer were completely removed by taping the plate dry. 250 pi of diluted NeutrAvidin71'-Horseradish peroxidase conjugated (final concentration 0,5 pg/ml in PBS-Buffer) was added to each well. The plate was incubated for 1 h at room temperature under gentle shaking. Then the plate was washed again. Equal volumes of the two solutions of lrnmunoPum®TMB Substrat were mixed and 100 pi were added immediately to ?ach well. The plate was Incubated at room temperature until the desired cobur develops. The colour changes from clear to brilliant blue. In a final step the reaction was stopped by . adding 150 pi 2 M H2SO4 to each well and the absorban ;e was read out at 450 nm with an ELISA plate reader (Molecular devices). The inlluence of the two different buffers on high background signals caused by unspecif c binding of the polyclonal detector antibody P2 which binds unspecifically to th= capture antibody P3 is plotted in Fig. 2. Figure 2 shows the reduction of background signals due to the use of sample buffer I according to the present invent on. In this experiment no analyte was used. Further, as antibody a polyclonal serum was used. A polyclonal serum comprises many different antibodies directed against a target protein. Many antibodies will bind with low or lower affinity, while some antibodies will bind with mid-range affinity anc one or only a few antibodies will bind with high affinity. The use of the buffer accc rding to the invention prevents the low-affinity binding and at least reduces the mid-range affinity binding of the respective antibodies as shown in Figure 2. As a resu t, once an analyte will be added in such an assay the signal- to noise-ratio will be improved due to the properties of the aqueous solution according to the prese nt invention. We claim: 1. An in vitro method for reducing any one or more of the following effects: effects of heterophilic antibodies, particularly effects due to human anti- mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and triglycerides; during a specific binding reaction of a binding pair, wherein a first binding member recognises its complementary second binding member, wherein the method comprises the use of an aqueous solution as medium for the specific binding reaction which aqueous solution is comprising: a) a buffer to control pH such as herein described; b) a compound A selected from the group consisting of: a compound defined by the general formula I R1-[[CR2R3]P-O]q,-R4, wherein R1 is hydrogen or hydroxy group, R2 for each unit independently is hydrogen or hydroxy group, R3 is hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p is an integer of from 2 to 10 and q is an integer of from 1 to 100, with the proviso that the compound at least carries two hydroxy groups; polyol; saccharide such as herein described; c) a non-ionic detergent. 2. The method as claimed in claim 1, wherein the aqueous solution is optionally comprising a protein in an amount effective to immunologically block non-specific antibody binding. 3. The method as claimed in claim 2, wherein the protein is selected from the group bovine serum albumin, ovalbumin, casein, fetal bovine serum. 4. The method as claimed in claim 2 or 3, wherein the concentration of the protein is in the range of 0.1 to 2 % (w/v) (preferably in the range of 0.5 to 1.5%(w/v). 5. The method as claimed in any of claims 1 to 4, wherein the aqueous solution is optionally comprising a salt selected from the group NaCI, KCI, NH4CI. 6. The method as claimed in any of claims 1 to 5, wherein the aqueous solution is having an ionic strength of 100 mM to 1.5 M, preferably of 200 mM to 1 M, more preferred of 200 mM to 800 mM, further preferred of 200 mM to 600 mM, most preferred of 250 mM to 500 mM. 7. The method as claimed in any of claims 1 to 6, wherein the buffer is selected from the group Tris (Tris(hydroxymethyl)-aminomethane, Pipes (Piperazine-1,4-bis-2-ethane sulfonic acid), Mes (4-Morpholino ethane sulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid), phosphate buffer. 8. The method as claimed in any of claims 1 to 7, wherein the compound A is selected from the group polyalkylene glycol, polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol, monosaccharides, disaccharides, trisaccharides, saccharose, mannose, trehalose, polyol, glycerol and mixtures thereof. 9. The method as claimed in any of claims 1 to 8, wherein the concentration of the compound A is in the range of 0.5 to 25 % (v/v), preferably in the range of 2.0 to 20 % (v/v), more preferred in the range of 2 to 15 % (v/v), further more preferred in the range of 2.0 to 10 % (v/v), even more preferred in the range of 2.0 to 7.0 % (v/v), and most preferred around 5 % (v/v). 10. The method as claimed in any of claims 1 to 9 wherein the non-ionic detergent is a compound of the general formula selected from the group. a) a substituted phenyl residue having substituents R1 and R2 (R1-Ph- R2), wherein R1 is C1-C9 a alkyl group, and R2 is a -O-[CH2-CH2-O]a-H group, wherein "a" is an integer of 5 to 40, wherein R2 in respect to R1 is in para, meta or ortho position. b) wherein n, x, y and z together is an integer of 5 to 40, R is a fatty acid residue. 11. The method as claimed in any of claims 1 to 9, wherein the non-ionic detergent is selected from the group Dodecylpoly(ethyleneglycolether)m, wherein m is an integer of 5 to 40; 1-O-n-Octyl-ß-D-glucopyranoside (n- Octylglucoside); Alkylphenolpoly(ethylene-glycolether)m, wherein m is an integer of 5 to 40, preferably m=11 (Nonidet P40®); 1-O-n-Dodecyl-B-D- glucopyranosyl (1-4)alpha-D-glucopyranoside; Dodecylpoly- (ethyleneglycolether)m, wherein m is an integer of 5 to 40, preferably m = 23 (Brij35®); Poly(oxyethylene)(20)-sorbitane mono fatty acid ester, preferably selected from Poly(oxyethylene)(20)-sorbitane monooleate (Tween®80), Poly(oxyethylene)(20)-sorbitane monolaurate (Tween®20), Poly(oxyethylene)(20)-sorbitane monopalmitat (Tween®40), Poly(oxyethylene)(20)-sorbitane monostearate); Octylphenolpoly(ethylene- glycolether)m, wherein m is an integer of 5 to 40, preferably m=10 (Triton®X- 100). 12. The method as claimed in any of claims 1 to 11, wherein the concentration of the non-ionic detergent is in the range of 0.1 to 1.0 % (v/v), preferably in the range of 0.15 to 1.0 % (v/v), more preferred in the range of 0.2 to 1.0 % (v/v), further more preferred in the range between 0.2 and 0.8 % (v/v), even more preferred in the range of 0.25 % to 0.6 % (v/v), and most preferred about 0.25 % (v/v). 13. The method as claimed in any of claims 1 to 12, wherein the ratio of the non-ionic detergent to the compound A is from 1:15 to 1:25, preferably around 1:20. 14. The method as claimed in any of claims 1 to 13, wherein the aqueous solution does not contain dithiothreitol. 15. The method as claimed in any of claims 1 to 14, wherein the pH is adjusted in the range of 6.0 to 9.0, more preferred in the range of 6.5 to 8.0, and most preferred in the range of 6.8 to 7.4. 16. The method as claimed in any of claims 1 to 15, wherein the aqueous solution is having the capability of reducing unspecific binding, cross- reactivity, and disturbing effects of the matrices. 17. The method as claimed in any of claims 1 to 16, wherein the aqueous solution is having the capability of preventing the low-affinity binding with Kd values of up to 10-7M. 18. The method as claimed in any of claims 1 to 17, wherein the aqueous solution is having the capability of preventing the low-affinity binding with Kd values of up to 10-7M and reducing the mid-range affinity binding with KD values in the range of between 107 M and 10-8 M by at least 90 %. 19. The method as claimed in any of claims 1 to 18, wherein the aqueous solution is having the capability of preventing the low-affinity binding with KD values of up to 10-7M and reducing the mid-range affinity binding with KD values in the range of between 107 and 109 by at least 90 %. 20. The method as claimed in any of claims 1 to 19, wherein the aqueous solution is having the capability to increase the binding activity (affinity) of antibodies, preferably the binding activity (affinity) of immobilized antibodies. 21. The method as claimed in any one of claims 1 to 20, wherein the binding reaction is an antibody-antigen binding reaction. 22. The method as claimed in any one of claims 1 to 21, wherein the binding reaction is a receptor-ligand binding reaction. 23. The method as claimed in any one of claims 1 to 22, wherein the aqueous solution is used as dilution buffer for samples and reagents, preferably ligands, receptors, antigens, antibodies or as washing buffer. 24. The method as claimed in any one of claims 1 to 23, wherein optionally any one or more of the following effects are reduced: unspecific binding, cross-reactivity, disturbing effects of matrices. The invention discloses an in vitro method for reducing effects of heterophilic antibodies, particularly effects due to human anti- mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and triglycerides; during a specific binding reaction of a binding pair, wherein a first binding member recognises its complementary second binding member, wherein the method comprises the use of an aqueous solution as medium for the specific binding reaction which aqueous solution is comprising: a) a buffer to control pH ; b) a compound A selected from the group consisting of: (i) a compound defined by the general formula I R1-[[CR2R3]p-O]q-R4, wherein R1, R2, R3, R4, p and and q are as defined in the specification , (ii) polyol, and (iii) saccharide c) a non-ionic detergent.

Full Text

[0001] The present invention relates to an aqueo is solution for the use as
medium for the specific binding reaction of a binding pair,
[OOOP] Background of the invention
[0003] Immunoassays where one or more antibodies are used to detect the
test substance (analyte) in a test sample are widely known. The evolution of
immunoassay methods increased the sensitivity of this test. Despite of the
developments within the recent decades, there remans a desire to eliminate
unspecific binding reactions, cross-reactivities and the influence of the compounds
present in the matrix.
[0004] Immunoassays depend upon the ability of i first binding member of a
binding member pair, e.g. an antigen or a ligand, to specifically bind to a second
binding member of a binding member pair, e.g. an antibody or a receptor. In order
to determine the extend of such binding, a conjugate, comprising one of such
binding members are labeled with a detectable moiety Such binding member
pairs can be an antigen and an antibody directed to such an antigen.
[0005] Immunoassays can be performed in a competitive immunoassay
format or in a sandwich immunoassay format. In the competitive immunoassay
format an antigen can be immobilized to a solid phase material whereby the
amount of detectable moiety that is bound to a solic phase material can be
detected, measured and correlated to the amount of antibody present in the test
sample. Examples of solid phase materials include beads, particles, micro-
particles and the like. In the sandwich immunoassa format a test sample,
containing for example an antibody, is contacted with a protein such as an antigen.
The antigen is immobilized on a solid phase material. Examples of solid phase
materials include bear is, particles, micro-particles and the like. The solid phase
material is typically cleated with a second antigen or antibody that has been
labeled with a detectable moiety. The second antigen or antibody, respectively,
then becomes bound to the corresponding antibody or antigen, respectively, on
the solid phase mater al and, after one or more washing steps, to remove any
unbound material an indicator material such as a chromogenic substance, is
introduced to react wi h the detectable moiety to produce the detectable signal,
e.g. a color change. The color change is then detected, measured and correlated
to the amount of antibody present in the test sample. It should also be noted that
various dilutents and suffers are also required to optimize the operation of the
micro-particles, antige is, conjugates and other components of the assay that
participate in chemical 'eactions.
[0006] In order o achieve optimal results in immunoassays the solution
which is used for the binding reactions between the binding partners (for example
the antibody and antige n reaction or the complex formation of ligand and receptor)
must provide a mediim that optimizes the ability of antibodies to bind to the
antigen, or must provide a medium that optimizes the ability of ligands to bind to
the receptor, while non-specific interactions, low-affinity binding and matrix effects
are strongly reduced o even prevented in order to avoid the generation of a false
signal.
[0007] In order to eliminate non-specific interactions and cross-reactivities
detergents have been edded to buffers which are used for washing steps after the
binding reaction in order to remove unspecific bindings.
[0008] For immunoassays, like western-blot analyses, enzyme-linked
immuno-sorbant assa (ELISA) and others, solutions containing phosphate
buffered saline (PBS)! upplemented with bovine serum albumin and 0.01 to 0.05
(v/v) Tween 20 is use i as medium for the binding reactions between the binding
partners (for example antibody and antigen). It is, however, very often experienced
that unspecific or low-affinity binding, cross-reactivities and matrix effects can not
be avoided with such buffers of the state of art. For example, when developing a
CRP-assay involving the detection of a plurality of analytes it appeared that cross-
reactivities due to the use of the plurality of antibodies as well as matrix effects
became a problem, which could not be solved by the use of conventional
immunoassay buffers.
[0009] The object of the present invention therefore was to provide a
solution for the use as a medium for the specific bindi ig reaction of a binding
member pair wherein the unspecific binding, low affinity binding, cress-reactivities
and matrix effects are strongly reduced or even preventec. Furthermore, it was the
object of the present invention to provide a method of an immunoassay, wherein
unspecific and low affinity binding, cross-reactivities and matrix effects are
reduced or prevented.
[0010] Summary of the invention
[0011] The object of the present invention is solver by an aqueous solution
for use as medium for the specific binding reaction of a binding member pair,
wherein a first binding member recognizes its complementary second binding
member, the solution comprising
a) a buffer to control pH;
b) a compound A selected from the group consisting of:
a compound defined by the general formula I
R1-[[CRzR3]p~0]q-R'1 wherein R1 is hydrogen or hydroxy group, R2 for
each unit independently is hydrogen or hydroxy group, R3 is
hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p
is an integer of from 2 to 10 and q is an intet er of from 1 to 100, with
the proviso that the compound at least carries two hydroxy groups;
polyol;
saccharide;
c) a non-ionic detergent.
[0012] In case F4 in the general formula I of compound A is hydrogen the
neighbouring residue Rz is also hydrogen. In case R1 is hydroxy group the
neighbouring residue R2 is hydrogen. In a preferred embodiment q in the formula
of compound A is an in :eger of from 1 to 50, more preferred from 1 to 30.
[0013] The inventors of the present invention surprisingly have found that
the aqueous solution according to the present invention reduces the cross-
reactivities, matrix e fects, unspecific bindings and low affinity-binding in
immunoassays. Furthermore, it was found that even effects due to heterophilic
antibodies (human anti mouse-antibody) are prevented when the aqueous solution
according to the present invention is used. Additionally, negative effects due to
rheuma factors, hemoglobin, bilirubin and triglycerides can be avoided with this
buffer even in case of plasma applications.
[0014] As used lerein a "binding member pair" comprises a "first binding
member" and a "seccnd binding member". Both binding members undergo a
specific binding to eacli other. The first binding member of a binding member pair
may be an antigen or aligand, respectively. The second binding member (e.g. an
antibody or a receptor, respectively) specifically recognizes and binds to the first
binding member (e.g. antigen or ligand, respectively). The second binding member
is the corresponding binding member and therefore also named "corresponding
binding member". The irtisan will understand that the terms "first" binding member
and "second" binding nember, respectively, may be for example the antigen and
the corresponding antit ody, respectively, or vice versa.
[0015] The aque )us solution according to the present invention represents a
universal buffer as a rr edium in immunoassays and binding reactions in a variety
of matrices, for example blood plasma, blood serum and others. In case of mulli-
analyie applications, for example, if protein chips are used, the simultaneous
incubation of several (era plurality) of analytes and several antibodies, unspecillc
bindings and cross-rea ;tivities very often occur. Such undesired binding reactions
have been observed in many cases. The use of standard ELISA buffers known in
the state of art could not prevent such cross-reactivity offects. In addition in the
state of art, the use of native samples resulted in matrix effects which gave
erroneous measurements compared to other methods for reference. As used
herein the term "matrix" refers to all compounds presen: in a native sample, like
blood scrum; in particular the term "matrix" refers to the Drganic and especially to
biological compounds such as proteins.
[0016] The aqueous solution according to the piesent invention may be
used as medium for the binding reaction of a binding pair, as sample dilution
buffers for immunoassays and binding reactions as well as dilution buffer for
antibodies and antigens, respectively. Further applications are multi-analyte
immunoassays and proteomics, wherein undesired cross-reactivities of antibodies
labeled with the fluoraphor can be prevented. Fluorophor tabled antibodies tend to
bind other proteins in an unspecific manner. By usirg the aqueous solution
according to the present invention such effects can be avoided.
[0017] By using the buffer according to the present invention in
immunoassays like ELISA and protein chips a further positive effect was shown.
When incubating the surface carrying the immobilized sntibodies with the buffer
according to the present invention the activity of the immobilized antibodies was
increased. This is resulting in an enhancement of the bin ting of the analyte to the
immobilized antibody. In conclusion, the buffer according to the present invention
in addition to the reduction of unspecific signals and unspecific effects also
increases the specific signals due to a positive influence on the specific binding
reaction between the analyte and the antibody. The resulting increase of the
activity of the immobilized antibodies provides a higher se nsitivity of the respective
assay.
[0018] The buffer according to the present invention may be used for
immunoassays ELISA, EIA, FIA, lateral-flow-test, protein chips, multi-analyte
assays, western blots, dot blots, immunohistochemistn,, receptor-ligand-assays
and immuno-PCR.
Detailed description cf the invention
[0019] In a preferred embodiment of the present invention the aqueous
solution is further comprising a protein in an amount effective to immunologically
block non-specific anti )Ody binding. This protein preferably is selected from the
group bovine serum ulbumin, ovalbumin, casein, fetal bovine serum. Further
preferred, the protein is present in the aqueous solution in a concentration in the
range of 0.1 to 2 % (w,v) and further preferred in the range of 0.5 to 1.5 % (w/v).
These proteins are rot recognized by any of the antibodies used in the
immunoassays. This uirecognized protein allows the immunological blocking of
non-specific antibody-b nding by molecules or compounds which might be present
in the sample.
[0020] In a furthur embodiment the aqueous solution is comprising a salt
selected from the group NaCI, KCI, NH4CI. Further preferred the aqueous solution
is having an ionic strength of 100 mM to 1.5 mM, more preferred of 200 mM to 1
M, even further preferred of 200 mM to 800 mM, particularly more preferred of 200
mM to 600 mM and most preferred of 250 mM to 500 mM. The inventors
surprisingly have found that a high ionic strength of the buffer used as a medium
for binding reactions, fur example, in the range of 200 mM to 600 mM is further
reducing unspecific binding and cross-reactivies, while the specific binding
reaction is not negativel / influenced.
[0021] In a particular preferred embodiment the buffer of the aqueous
solution is selected fron the group Tris (Tris(hydroxymethyl)-aminomethane, Pipes
(Piperazine-1,4-bis-2-etiane sulfonic acid), Mes (4-Morpholino ethane sulfonic
acid), Hepes (4-(2-hycroxyethyl)-1-piperazine-ethane sulfonic acid), phosphate
buffer.
[0022] In a furthe • preferred embodiment the compound A is selected from
the group polyalkylene (lycol, polypropylene glycol, propylene glycol, polyethylene
glycol, ethylene glycol, monosaccharides, disaccharides, trisaccharides,
saccharose, mannose, trehalose, polyol, glycerol anc. mixtures thereof. In a
preferred embodiment the concentration of the compourd A is in the range of 0.5
to 25 % (v/v), preferably in the range of 2.0 to 20 % (v,v), more preferred in the
range of 2.0 to 15 % (v/v), further more preferred in the range of 2.0 to 10 % (v/v),
even more preferred in the range of 2.0 to 7 % (v/v), and most preferred around
5 % (v/v). The concentration is given in % (v/v) in case cnumpound A is a liquid. In
case the compound A is solid (for example a saccharide ¦ the concentration has to
be understood as % (w/v).
[0023] In a further preferred embodiment the aqueous solution comprises as
i non-ionic detergent a compound of the general formula s elected from the group
a) a substituted phenyl residue having substituent:; R1 and R2 (R 1-Ph-R2),
wherein R1 is C1-C9 alkyl group, and R2is - wherein "a" is an integer of 5 to 40, wherein R2 ir respect to R1 is in para,
meta orortho position.
, b)
, wherein n, x, y and z together is an integer of 5 to 40, R is a fatty acid
residue.
[0024] Further preferred the non-ionic detergent is selected from the group
Dodecylpoly(ethyleneglycolether)m, wherein m is an infager of 5 to 40; 1-O-n-
Octyl-f£-D-gluccpyrano:;ide (n-Octylglucoside); Alkylphenolpoly(ethyleneglycol-
ether)m, wherein rn is c n integer of 5 to 40, preferably m=11 (Nonidet P40e); 1 -O-
n-Dodecyl-fS-D-glucopyranosyl (1-4)alpha-D-giucopyranoside; Dodecylpoiy-
(ethyieneglycolether)m, wherein m is an integer of 5 to 40, preferably m = 23
(Brij35®); Poiy(oxyeth7lene)(20)-sorbitane mono fatty acid ester, preferably
selected from Pol/(oxyethylene)(20)-sorbitane monooleate (Tween®80),
Poly(oxyethylene)(20)-.' ;orbitane monolaurate (Tween®20), Poly(oxyethylene)(20)-
sorbitane monopal nilat (Tween®40), Poly(oxyethylene)(20)-sorbitane
monostearate); Octylpl enolpoly(ethyleneglycolether)m, wherein m is an integer of
5 to 40, preferably m=10 (Triton^X-100).
[0025] In prefer-ed embodiments the concentration of the non-ionic
detergent is in the ranee of 0.1 to 1.0 % (v/v). Preferably, the concentration of the
non-ionic detergent in tie range of 0.15 to 1.0 % (v/v), more preferred in the range
of 0.2 to 1.0 % (v/v), further more preferrred in the range between 0.2 and 0.8 %
(v/v), even more preferred in the range of 0.25 % to 0.6 % (v/v), and most
preferred about 0.25 % (v/v).
[0026] An imporant feature of the present invention is the presence of
compound A as given i n claim 1 in combination with the non-ionic detergent. Each
of said both ingredients (compound A and non-ionic detergent) is present in the
aqueous solution of the present invention in higher concentrations as it is known in
respect to incubation solutions for immunoassays of the state of art. In a further
preferred embodiment an aqueous solution is provided, wherein the ratio of the
non-ionic detergent to the compound A is from 1:15 to 1:25, preferably around
1:20.
[0027] In a pa ticularly preferred embodiment ihe aqueous solution
comprises compound / in a concentration in the range of 2 to 7 % (v/v) and a non-
ionic detergent in the range of between 0.2 to 0.8 % (v/v). Preferably the aqueous
solution is having an icnic strength of 200 mM to 1 M, more preferred of 200 mM
to 800 mM, particularly more preferred of 200 mM to 600 mM and most preferred
of 250 mM to 500 mM. It is preferred that the aqueous solution comprises as
compound A a compound selected from the grcup polyalkylene glycol,
polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol,
glycerol and mixtures thereof, more preferred a compound selected from
polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol and
most preferred ethylene glycol.
[0028] The aqueous solution according to the present invention preferably
does not contain dithiothreitol. Further preferred the aqueous solution according to
the present invention does not contain U-mercapto-ethanol.
[0029] In a further preferred embodiment the pH cf the aqueous solution is
adjusted in the range of 5.6 to 9.6, preferably in the ra nge of 6.0 to 9.0, further
preferred in the range of 6.5 to 8.0 and most preferred in he range of 6.8 to 7.4.
[0030] A particularly preferred embodiment of ihe aqueous solution is
having the capability of reducing unspecific binding, cross-reactivity and disturbing
effects of the matrix. The aqueous solution according to the present invention
particularly has the capability of preventing the low-affiniiy binding with KD values
of up to 10"7 M, compared to standard conditions. Furtfnr preferred the aqueous
solution has the capability of preventing the low-affinity binding with Kd values of
up to 10'7 M and reducing the mid-range affinity binding with Kd values in the
range of between 10"7 M and 10"° M by at least 90 lo compared to standard
conditions. Even further preferred, the aqueous solution has the capability of
preventing the low-affinity binding with KD values of up to 10"T and reducing the
mid-range affinity binding with Kp values in the range of between 10"7 and 10'9 by
at least 90 % compared to standard conditions. As used herein "standard
conditions" are represented by an aqueous solution cc nsisting of 50 mM PBS
(phosphate buffered saline, pH 7.4), 150 mM NaCI, 1 % iw/v) BSA (see also table
1: reference example). The same results were obtained when comparing the
measurements with another standard solution, namey an aqueous solution
consisting of 50 mM PBS (pH 7.4), 100 mM NaCI, 0.05 % (v/v) Tween®20.
[0031] Apart from the effect of reducing the unspecific, low affinity binding
as well as matrix effects, the aqueous solution particularly preferred is having the
capability to increase the binding activity of antibodies, preferably the binding
activity of immobilized antibodies as well as the binding activity between ligands
and receptors. The increase of the binding activity of immobilized antibodies by
using the solution according to the present invention was about 10 % or more.
[0032] The object of the present invention is also solved by a concentrate of
the aqueous solution of the present invention desribed before, preferably a 2 to 10
fold concentrate, more preferred a 3 to 5 fold concentrate.
[0033] Further, t le object of the present invention is solved by the use of the
aqueous solution acccrding to the present invention as a medium for the binding
reaction of a binding pair, wherein a first binding member specifically recognizes
and binds its compler lenlary second binding member. Preferably the aqueous
solution is used as a nedium for the antibody-antigen binding reaction and in an
alternative embodiment the aqueous solution is used as a medium for the
receptor-ligand binding reaction. In other preferred embodiments the aqueous
solution is used as dilution buffer for samples, reagents, ligands, receptors,
antigens, antibodies, "'he aqueous solution may also preferably be used as a
washing buffer in imrnmoassays after the binding reaction was carried out.
[0034] The inve ition further provides a method for reducing unspecific
binding and/or cross- eactivity and/or disturbing effects of matrices during a
specific binding react on of a binding pair, wherein a first binding member
recognises its complementary second binding member, the method comprising the
use of the aqueous sc lution of the present invention as medium for the specific
binding reaction.
[0035] In anothe ¦ aspect of the invention the aqueous solution according to
the present invention c in be provided as a component of a kit. As used herein, the
term "kit" means a co; lection of reagents and associated materials e.g. buffers,
carrier comprising an immobilised binding member and reagents which are
required to perform an assay. Therefore, the present ir ivention provides a kit for
detection by immunoassay of at least one analyte to be tested, wherein the
analyte to be tested is a first binding member of a bincing member pair, wherein
i the first binding member binds specifically to its complementary binding member,
the kit comprising:
a) a vessel containing an aqueous solution of ihe present invention;
b) a carrier comprising the complementary binding member
immobilised thereon to capture the analyte; and
> c) optionally, a reagent which immunologically recognises the analyte
bound to the complementary binding member, wherein the reagent
(antibody) is conjugated to a means of detection; and
d) optionally: reagents which are reactive with said means of detection
to produce a detectable reaction product.
. [0036] A typical kit for example is used as ELISA lit for the detection of, for
example, antibodies in blood serum. In this case the carrier according to b)
comprises as complementary binding member immobilissid thereon, for example, a
virus antigen to capture the analyte. The analyte which ii the antibody in the blood
serum. The reagent according to c) which immunologically recognises the analyte
i then is an anti-antibody which is recognising the capturec antibody.
Best mode for carrying out the invention
[0037] The present invention is characterized in tiat the aqueous solution
for use as medium for the specific binding reaction of a binding member pair,
wherein a first binding member recognizes its complementary second binding
member, comprises:
a) a buffer to control pH;
b) a compound A selected from the group consisting of:
a compound defined by the general formula I
R1-f[CR2l l3]p-0]q-R4, wherein R1 is hydrogen or hydroxy group, R2 for
each un t independently is hydrogen or hydroxy group, R3 is
hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p
is an inte jer of from 2 to 10 and q is an integer of from 1 to 100, with
the proviso that the compound at least carries two hydroxy groups;
po yol;
saccharide;
[0038] c) a r on-ionic detergent.
[0039] The method of the present invention for reducing unspecific binding
and/or cross-reactivity and/or disturbing effects of matrices during a specific
binding reaction of a t inding pair, wherein a first binding member recognises its
complementary seconc binding member, i.e. for a immunoassy, is characterized to
comprise the use of the above aqueous solution.
[0040] Preferably an aqueous solution was found to be useful which
comprises compound A in a concentration in the range of 2 to 7 % (v/v) and a non-
ionic detergent in the range of between 0.2 to 0.5 % (v/v). Preferably the aqueous
solution is having an ic nic strength of 200 mM to 1 M, more preferred of 200 mM
to 800 mM, particularly more preferred of 200 mM to 600 mM and most preferred
of 250 mM to 500 mM. It is preferred that the aqueous solution comprises as
compound A a con pound selected from the group polyalkylene glycol,
polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol,
glycerol and mixtures thereof, more preferred a compound selected from
polypropylene glycol, propylene glycol, polyethylene glycol, ethylene glycol and
most preferred ethylem glycol.
[0041] The present inventions will be explained in more detail in the
following examples. However, the examples are only used for illustration and do
not limit the scope of the present invention.
Accompanying
Description of the/Figures
[0042] Figure 1 shows the amount of detector ant body C6 bound to CRP in
a sandwich assay as absorbance at 450 nm plotted acainst the concentration of
CRP [ng/ml]. The binding reaction of detector antibody C6 to the protein CRP was
performed as outlined under Example 1 under standari conditions and by using
sample buffers according to the present invention, r Sample buffers I to III according to the solution of the present invention reduce the
influence of the matrix effects very well. Sample buffer I shows the best sensitivity.
The low sensitivity of the reference buffer is caused by a strong matrix effect.
[0043] Figure 2 shows the influence of two ilifferent buffers on high
background signals caused by unspecific binding o the polyclonal detector
antibody P2 which binds unspecific to the capture aitibody P3 (according to
Example 2). In this experiment no analyte was present. .'Sample buffer I decreases
significantly the unspecific binding compared to the higli background signals with
reference example buffer!!.
Examples
[0044] Example 1: Reduction of matrix effects
[0045] 100 ul diluted capture antibody C2 (final concentration 1 ug/ml in
PBS-Buffer) was added to each well of a microtiter plnte (C8 StarWell Module,
NUNC) and the plate was covered with a plate sealer The capture antibody is
directed against CRP (c-reactive protein). Then the p ate was incubated for 5
hours at room temperature. The plate sealer was rerr oved and the plate was
washed 4 times with 300 ul washing buffer (10 mM Piosphate, 350 mM NaCI,
0.05 % Tween, pH 7.') per well. Than 200 ul of blocking solution (PBS-Buffer pH
7.4, 1 % BSA) was added to each well. After covering with a plate sealer the plate
was incubated avernic ht at 4 °C. The analyte CRP (c-reactive protein) was diluted
in rabbit serum (0-5 ng/ml) and incubated for 30 min at room temperature. The
biotin-labelled detecto • antibody C6 (directed against CRP) was diluted in different
sample buffers and reference example buffers (see table 1). The final
concentration was 4 jjg/ml in each preparation. The CRP-containing rabbit serum
standards were diluted 1:2 with detector antibody containing sample buffers. Tho
preparations were incL bated for 30 min at room temperature. The plate sealer was
removed and the plate was washed 4 times with 300 ul washing buffer. Oddments
of washing buffer wen: completely removed by taping the plate dry. 100 pi of the
CRP-preparations weia added to the wells. The plate was covered with a plate
sealer and incubated far 4 h at room temperature under gentle shaking. After that
the plate was wash 3d again. 100 ul of diluted NeutrAvidin™'-Horseradish
peroxidase ccnjugatec (final concentration 0,05 ug/ml in PBS-Buffer) was added
to each well. The plat j was incubated for 1 h at room temperature under gentle
shaking. Then the plat a was washed again. Equal volumes of the two solutions of
lmmunoPure©TMB Sibstrat were mixed and 100 pi were added immediately to
each well. The plate vas incubated at room temperature until the desired colour
developed. The colou' changed from clear to brilliant blue. In a final step the
reaction was stoppec by adding 150 pi 2 M H2S04 to each well and the
absorbance was read out at 450 nm with an ELISA plate reader (Molecular
devices). The influence of different buffers on matrix effects are plotted in fig. 1.
Table 1 shows the results of the test. In table 1 the reduction of non-specific
binding, low-affinity binding and matrix effects is indicated in the column "result"
with "+". The number of"+" is indicating the amount of reduction of non-specific
binding, low-affinity binding and matrix effects compared to the reference example
buffer ("-"). Figure 1 stows the amount of detector antibody C6 bound to CRP as
absorbance at 450 nrr plotted against the concentration of CRP [ng/ml]. Sample
buffer I shows the be; t sensitivity. The low sensitivity of the reference buffer is
caused by a strong ma rix effect.
[0046] Example 2: Reduction of an unspecifk binding of a polyclonal
detector antibody
[0047] For the next assay 250 pi diluted captu 'e antibody P3 (polyclonal
rabbit-anti-protease, own preparation, final concentra'ion 1 g/ml in PBS-Buffer)
i was added to each well of a microtiter plate (C8 StarW 2ll Module, NUNC) and the
plate was covered with a plate sealer. The plate was inc ubated for 4 hours at room
temperature- After that the plate sealer was removed a id the plate was washed 4
times with 300 pi washing buffer (10 mM Phosphato, 350 mM NaCI, 0.05 %
Tween®, pH 7.4) per well. Then 200 pi of blocking solitlon (PBS-Buffer pH 7.4, 1
i % BSA) was added to each well. The plate was covered again with a plate sealer
and incubated overnight at 4 °C. The biotin-labelled p Dlyclonal detector antibody
P2 (polyclonal rabbit-anti-protease, own preparation) was diluted in reference
example buffer II or in sample buffer I (see taMe 1) respectively (final
concentration 10 ug/ml in each preparation) and added to the wells (250 pi per
well; fivefold replicates). The plate was incubated for 2 I at room temperature. The
plate sealer was removed and the plate was washed 4 times with 300 pi washing
buffer. Oddments of washing buffer were completely removed by taping the plate
dry. 250 pi of diluted NeutrAvidin71'-Horseradish peroxidase conjugated (final
concentration 0,5 pg/ml in PBS-Buffer) was added to each well. The plate was
incubated for 1 h at room temperature under gentle shaking. Then the plate was
washed again. Equal volumes of the two solutions of lrnmunoPum®TMB Substrat
were mixed and 100 pi were added immediately to ?ach well. The plate was
Incubated at room temperature until the desired cobur develops. The colour
changes from clear to brilliant blue. In a final step the reaction was stopped by
. adding 150 pi 2 M H2SO4 to each well and the absorban ;e was read out at 450 nm
with an ELISA plate reader (Molecular devices). The inlluence of the two different
buffers on high background signals caused by unspecif c binding of the polyclonal
detector antibody P2 which binds unspecifically to th= capture antibody P3 is
plotted in Fig. 2. Figure 2 shows the reduction of background signals due to the
use of sample buffer I according to the present invent on. In this experiment no
analyte was used. Further, as antibody a polyclonal serum was used. A polyclonal
serum comprises many different antibodies directed against a target protein. Many
antibodies will bind with low or lower affinity, while some antibodies will bind with
mid-range affinity anc one or only a few antibodies will bind with high affinity. The
use of the buffer accc rding to the invention prevents the low-affinity binding and at
least reduces the mid-range affinity binding of the respective antibodies as shown
in Figure 2. As a resu t, once an analyte will be added in such an assay the signal-
to noise-ratio will be improved due to the properties of the aqueous solution
according to the prese nt invention.
We claim:
1. An in vitro method for reducing any one or more of the following effects:
effects of heterophilic antibodies, particularly effects due to human anti-
mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and
triglycerides;
during a specific binding reaction of a binding pair, wherein a first binding
member recognises its complementary second binding member, wherein
the method comprises the use of an aqueous solution as medium for the
specific binding reaction which aqueous solution is comprising:
a) a buffer to control pH such as herein described;
b) a compound A selected from the group consisting of:
a compound defined by the general formula I
R1-[[CR2R3]P-O]q,-R4, wherein R1 is hydrogen or hydroxy group, R2 for
each unit independently is hydrogen or hydroxy group, R3 is
hydrogen, methyl group, ethyl group, R4 is hydrogen or alkyl group, p
is an integer of from 2 to 10 and q is an integer of from 1 to 100, with
the proviso that the compound at least carries two hydroxy groups;
polyol;
saccharide such as herein described;
c) a non-ionic detergent.
2. The method as claimed in claim 1, wherein the aqueous solution is
optionally comprising a protein in an amount effective to immunologically
block non-specific antibody binding.
3. The method as claimed in claim 2, wherein the protein is selected from the
group bovine serum albumin, ovalbumin, casein, fetal bovine serum.
4. The method as claimed in claim 2 or 3, wherein the concentration of the
protein is in the range of 0.1 to 2 % (w/v) (preferably in the range of 0.5 to
1.5%(w/v).
5. The method as claimed in any of claims 1 to 4, wherein the aqueous
solution is optionally comprising a salt selected from the group NaCI, KCI,
NH4CI.
6. The method as claimed in any of claims 1 to 5, wherein the aqueous
solution is having an ionic strength of 100 mM to 1.5 M, preferably of 200
mM to 1 M, more preferred of 200 mM to 800 mM, further preferred of 200
mM to 600 mM, most preferred of 250 mM to 500 mM.
7. The method as claimed in any of claims 1 to 6, wherein the buffer is
selected from the group Tris (Tris(hydroxymethyl)-aminomethane, Pipes
(Piperazine-1,4-bis-2-ethane sulfonic acid), Mes (4-Morpholino ethane
sulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid),
phosphate buffer.
8. The method as claimed in any of claims 1 to 7, wherein the compound A is
selected from the group polyalkylene glycol, polypropylene glycol,
propylene glycol, polyethylene glycol, ethylene glycol, monosaccharides,
disaccharides, trisaccharides, saccharose, mannose, trehalose, polyol,
glycerol and mixtures thereof.
9. The method as claimed in any of claims 1 to 8, wherein the concentration of
the compound A is in the range of 0.5 to 25 % (v/v), preferably in the range
of 2.0 to 20 % (v/v), more preferred in the range of 2 to 15 % (v/v), further
more preferred in the range of 2.0 to 10 % (v/v), even more preferred in the
range of 2.0 to 7.0 % (v/v), and most preferred around 5 % (v/v).
10. The method as claimed in any of claims 1 to 9 wherein the non-ionic
detergent is a compound of the general formula selected from the group.
a) a substituted phenyl residue having substituents R1 and R2 (R1-Ph-
R2), wherein R1 is C1-C9 a alkyl group, and R2 is a -O-[CH2-CH2-O]a-H
group, wherein "a" is an integer of 5 to 40, wherein R2 in respect to R1 is in
para, meta or ortho position.
b)

wherein n, x, y and z together is an integer of 5 to 40, R is a fatty acid
residue.
11. The method as claimed in any of claims 1 to 9, wherein the non-ionic
detergent is selected from the group Dodecylpoly(ethyleneglycolether)m,
wherein m is an integer of 5 to 40; 1-O-n-Octyl-ß-D-glucopyranoside (n-
Octylglucoside); Alkylphenolpoly(ethylene-glycolether)m, wherein m is an
integer of 5 to 40, preferably m=11 (Nonidet P40®); 1-O-n-Dodecyl-B-D-
glucopyranosyl (1-4)alpha-D-glucopyranoside; Dodecylpoly-
(ethyleneglycolether)m, wherein m is an integer of 5 to 40, preferably m = 23
(Brij35®); Poly(oxyethylene)(20)-sorbitane mono fatty acid ester, preferably
selected from Poly(oxyethylene)(20)-sorbitane monooleate (Tween®80),
Poly(oxyethylene)(20)-sorbitane monolaurate (Tween®20),
Poly(oxyethylene)(20)-sorbitane monopalmitat (Tween®40),
Poly(oxyethylene)(20)-sorbitane monostearate); Octylphenolpoly(ethylene-
glycolether)m, wherein m is an integer of 5 to 40, preferably m=10 (Triton®X-
100).
12. The method as claimed in any of claims 1 to 11, wherein the concentration
of the non-ionic detergent is in the range of 0.1 to 1.0 % (v/v), preferably in
the range of 0.15 to 1.0 % (v/v), more preferred in the range of 0.2 to 1.0 %
(v/v), further more preferred in the range between 0.2 and 0.8 % (v/v), even
more preferred in the range of 0.25 % to 0.6 % (v/v), and most preferred
about 0.25 % (v/v).
13. The method as claimed in any of claims 1 to 12, wherein the ratio of the
non-ionic detergent to the compound A is from 1:15 to 1:25, preferably
around 1:20.
14. The method as claimed in any of claims 1 to 13, wherein the aqueous
solution does not contain dithiothreitol.
15. The method as claimed in any of claims 1 to 14, wherein the pH is adjusted
in the range of 6.0 to 9.0, more preferred in the range of 6.5 to 8.0, and
most preferred in the range of 6.8 to 7.4.
16. The method as claimed in any of claims 1 to 15, wherein the aqueous
solution is having the capability of reducing unspecific binding, cross-
reactivity, and disturbing effects of the matrices.
17. The method as claimed in any of claims 1 to 16, wherein the aqueous
solution is having the capability of preventing the low-affinity binding with Kd
values of up to 10-7M.
18. The method as claimed in any of claims 1 to 17, wherein the aqueous
solution is having the capability of preventing the low-affinity binding with Kd
values of up to 10-7M and reducing the mid-range affinity binding with KD
values in the range of between 107 M and 10-8 M by at least 90 %.
19. The method as claimed in any of claims 1 to 18, wherein the aqueous
solution is having the capability of preventing the low-affinity binding with KD
values of up to 10-7M and reducing the mid-range affinity binding with KD
values in the range of between 107 and 109 by at least 90 %.
20. The method as claimed in any of claims 1 to 19, wherein the aqueous
solution is having the capability to increase the binding activity (affinity) of
antibodies, preferably the binding activity (affinity) of immobilized
antibodies.
21. The method as claimed in any one of claims 1 to 20, wherein the binding
reaction is an antibody-antigen binding reaction.
22. The method as claimed in any one of claims 1 to 21, wherein the binding
reaction is a receptor-ligand binding reaction.
23. The method as claimed in any one of claims 1 to 22, wherein the aqueous
solution is used as dilution buffer for samples and reagents, preferably
ligands, receptors, antigens, antibodies or as washing buffer.
24. The method as claimed in any one of claims 1 to 23, wherein optionally any
one or more of the following effects are reduced:
unspecific binding, cross-reactivity, disturbing effects of matrices.

The invention discloses an in vitro method for reducing
effects of heterophilic antibodies, particularly effects due to human anti-
mouse antibodies, effects due to rheuma factors, haemoglobin, bilirubin and
triglycerides;
during a specific binding reaction of a binding pair, wherein a first binding
member recognises its complementary second binding member, wherein
the method comprises the use of an aqueous solution as medium for the
specific binding reaction which aqueous solution is comprising:
a) a buffer to control pH ;
b) a compound A selected from the group consisting of:
(i) a compound defined by the general formula I R1-[[CR2R3]p-O]q-R4,
wherein R1, R2, R3, R4, p and and q are as defined in the specification ,
(ii) polyol, and
(iii) saccharide
c) a non-ionic detergent.

AN IN VITRO METHOD FOR REDUCING EFFECTS OF HETEROPHILIC ANTIBODIES DURING SPECIFIC BINDING REACTION OF A BINDING PAIR

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