Title of Invention	"A NOVEL THERMOSTABLE PROTEIN CONJUGATE PROBE"
Abstract	The present invention relates to a novel thermostable probe useful for immunodetection in immunoassays, said probe consisting of antibody and enzyme in the proportion ranging from 1:10 to 2:10.

Full Text

Field of the invention: The present application is derived out of the Indian Application No. 1402/DEL/1998. The present invention relates to immunoassays and development of probes for such assays. Specifically, the invention provides a novel thermostable probe and a process for preparation of the said probe and a method for the use thereof. Background of the invention; Enzymes are complex catalysts that accelerate chemical reaction rates. In addition, to their catalyst activity, enzymes are known for their substrate specificity. Various enzymes labels like horseradish peroxides, alkaline, phosphatase, urease, pencillinase, L-galactosidase have been studied and standardized to conjugate with antibodies for use as probes in immunodetection assays. There are several diagnostic tools for detection of diseases, changes in the blood etc. one such sophisticated and powerful diagnostic tool is ELISA or enzyme-linked immunoassays. The technique is based on detection of proteins in the serum /urine etc with the help of enzyme-linked conjugates. There are several methods available for conjugation of enzyme labels with antibodies. Alkaline Phosphatases can be conjugated by glutaraldehyde method, Horseradish peroxidase can be conjugated by peroxidase method, galactosidase can be conjugated by MBS method. Other enzyme like glucosidase, urease, pencillinase have been used by a few workers. All these enzymes also have good catalytic activity but they yield conjugates which are not very stable at room temperature. These conjugates when packed in immunoassay kits which are susceptible to denaturation at room temperature or, they loose activity in case of frequent power cuts even in refrigerated conditions. In the recent years, some new conjugates with urease/pencillinase have been prepared by conventional methods. These conjugates even though found to be efficient in immunodetection have limited stability. Recently, some new reagents known as homobifunctional and heterobifunctional reagents are being used to prepare protein-protein conjugates for various experimental purposes. They have also been used to prepare conjugates with alkaline phosphatase. The conjugates formed by these reagents have less aggregate formations and hence have improved efficiency in immunodetection. However, the reagents used in the conventional methods of preparation of conjugates are still popular because they are cheap, easily available and easy to use. The conventional enzymes/conjugates have good catalytic but have limited stability at 37 °C. In addition, many conditions of working such as frequent powercuts and poor transportation facility for transport of immunoassay kits, render these enzymes/conjugates inactive. Therefore, there has been a need to identify an active thermostable enzyme as label for preparation of a conjugate which can be used effectively in immunoassays. Amylases are enzymes that hydrolyze starch, a-amylase obtained form B. Lichenformis has a high specific activity of 1000-1200 units/mg and hence is a potential candidate for conjugate labeling. Objects: It is an object of the invention to produce thermostable probe, which can be used for immunodetection in immunoassays. Yet another object is to standardize of the process of linking thermostable amylase with cross-linking agent. It is another object of the invention to develop a process of conjugation of this cross-linked a-amylase with second antibody. Another object is to standardize the use of this probe for immunodetection in immunoassays. A further objective is to work at the sensitivity and effectiveness of use as a probe in immunoassays. Still another object is to confirm the thermostable nature of the formed probe/conjugate. It is also an objective the invention to compare the probe with conventionally available probes in terms of sensitivity in immunodetection and thermostable nature. Statement of invention: Accordingly.xne invention provides a process for the preparation of novel thermostable a-amylase-antibody conjugate, said process comprising the steps of: a) dissolving a-amylase in a buffer of neutral pH to obtain a solution and adding a cross-linking agent such as hereindescribed to the solution, b) incubating the solution for 2 - 24 hrs in a manner such as hereindescribed, c) optionally removing the excess cross-linking agent by dialysis in a buffer of pH 7 to 9.5 and adjusting the pH if desired, d) adding an antibody such as hereindescribed to the solution and incubating the same overnight, e) adding ammonia-donating compounds such as hereindescribed for saturation of unconjugated enzyme, f) changing the buffer and thereby obtaining a-amylase-antibody conjugate which is suspended in neutral pH and storing the amylase-antibody conjugate at 4°C to -20°C. fr grief description of Accompanying Drawings: Fig. 1. • Activity of antibody - amylase conjugates made by glutaraldehyde method in ELISA Fig. 2. Activity of second antibody amylase conjugate made in different buffers in ELISA Fig. 3. Optimum temperature for activity of antibody-amylase conjugate. Fig. 4. Time dependent product formation by second antibody amylase conjugate in immunoassay. Fig. 5. Titration curve of coated IgG by second antibody amylase conjugate in ELISA Fig. 6. Detection of coated antigen by second antibody-amylase conjugate in Direct ELISA. Fig. 7. Antigen detection in competitive Elisa by second antibody-amylase conjugate. Fig. 8. Detection of primary antibody titre by second antibody-amylase conjugate in ELISA. Fig. 9. Optimum incubation time of second antibody-amylase conjugate in Elisa. Fig. 10. Detection of coated IgG by second antibody-HRPO conjugate in Direct Elisa. Fig. 11. Comparison of titration curve of coated antigen as seen with HRPO/Amylase conjugate. Fig. 12 Enhancement of color by alkali after termination of amylase catalysed reaction. Summary: The invention provides a novel thermostable probe and a process for the preparation thereof. The said process involves the linking of thermostable enzyme with crosslinking agent and conjugating the cross-linking enzyme with second antibody and purifying the resultant conjugate from the unconjugated enzyme molecules. The free amino groups in the enzyme, thermostable amylase, and the antibody participate in the conjugation process. Detailed description of the invention: The present invention relates to identification of a novel thermostable probe useful for immunodetection in immunoassays. The invention also provides a novel process for the synthesis of the said probe. The invention further identifies the sensitive substrate that can be utilized for immunodetection using this probe. In accordance with the objectives outlined, the applicants selected a-amylase as the enzyme, having the best potential of acting as a label for the conjugates used in immunodetection assays for the amongst other reasons: The structure of the enzyme amylase is such that it has free amino groups or free carboxyl groups available for conjugation. Sulphdryl groups and carbohydrate groups are absent. Therefore, methods which crosslink free amino groups can be expected to successfully conjugate with the amino group of second antibody. It has been seen that glutaraldehyde method is quite successful in giving crosslinking bridges to the alpha amylase with glutaraldehyde because free amino groups are available on amylase for conjugation. The inability of agents like MBS to conjugate is due to the absence of sulphydral groups on the enzyme. The inability ofperiodate to crosslink is due to the absence of carbohydrate groups. A novel thermostable probe useful for immunodetection in immunoassays, said probe consisting of antibody and enzyme in the proportion ranging from 1:10 to 2:10. In one embodiment, the antibody is a second antibody selected from anti-mouse, goat IgG, anti-rabbit goat IgG, anti-human goat IgG. In another embodiment, the enzyme is ct-amylase extracted from bacterium lichenformis, said enzyme having specific activity of 1000 -1200 units per mg. In yet another embodiment, the free amino groups on the antibody is linked to the free amino groups on the side chains of lysine or arginine of a-amylase through cross-linking bridges. In another embodiment, the probe has a shelf life of 1 to 2 years and is stored at 4°C to 20°C. In still another embodiment, the probe maintains its activity at 37°C to 90°C. In a further embodiment, the probe is as sensitive as HRPO (horse radish peroxidase) label probe in detection of antigens in immunoassays. Accordingly, the applicants used the process detailed hereinbelow to develop a novel thermostable conjugate probe, wherein the activity of the a-amylase is maintained and thermostable nature is thereof is achieved. The process for the preparation of said novel L-amylase conjugates/probes comprises the steps of: (a) dissolving the enzyme a-amylase in a buffer of neutral pH to obtain a sample. (b) adding a cross linking agent to the sample, including the surface of step. (c) incubation for 2 - 24 hrs. (d) removing excess crosslinking agent by dialysis in a buffer having 7- 9.5 pH. (e) adjusting pH of the sample to 9-10, (f) adding antibody to the sample to produce enzyme-antibody conjugates. (g) incubating the sample overnight, (h) adding NH2 - donating compounds for saturation of unconjugated enzyme from the reaction mixture, (i) changing the buffer and suspending in neutral pH and (j) storing the mixture at 4° Cor -20°C. In one embodiment, the crosslinking agent glutaraldehyde is added to the enzyme in neutral buffer. In another embodiment, the incubation may be carried out at 4 - 24° C. In yet another embodiment, the antibodies added to the reaction mixture are selected from anti-mouse, goat IgG, anti-rabbit goat IgG, anti-human goat IgG, preferably second antibody. In yet another embodiment, the NH2 (ammonia) donating compounds added are selected from amino acids, preferably lysine, glycine, Tris buffer and Ammonium chloride. Enzyme used in the reaction was procured from SIGMA, USA. the a-amylase used has specific activity of more than 1000 - 1200 uints/mg. A method of immunodetection assay using the said probe for indirect detection of antigens comprising the steps of :- a) coating specific concentration of the antigen to the ELISA well, b) blocking the wells with excess proteins, c) incubation with different concentrations of antibody, d) washing the sample, e) incubation with second antibody probes for 1 to 24 hours, f) washing the sample, g) adding substrate, h) incubating the sample for 1 hour at 37°C to 60°C, for 24 hrs at 4°C, i) reading directly or after termination with alkali at 405 nm. In one embodiment, the proteins used are selected from the group consisting of 1% BSA (bovine serum albumin), skimmed milk. Substrate of having para-nitro-phenyl malto heptoside and ct-glucosidase at neutral pH undergoes cyclic reaction to form nitro-phenol at37°C-60°C which can be read colorimetrically at 405 NM. Reaction can be read directly or after termination with alkali. Alkali - KOH, N.OH PH 9 - 14 Cone 1 -0.5M The probe is comparable to conventional conjugates (HRPO labeled conjugates). The probe gives very low non specific reaction with the serum. It is a better probe than existing ones as i. it has better thermostability and shelf-life ii equal sensitivity iii gives low blank values. iv. stable color formation which can be read upto 24 hrs. Conditions which promote efficient conjugation: It has been seen that smaller amounts of glutaraldehyde produce better conjugates. If larger amounts of glutaraldehyde are used than shorter period of incubation i.e. 2 hrs at room temperature can also bring about efficient conjugation. This may be because larger amounts of glutaraldehyde or greater time of incubation may promote greater aggregation and form large aggregates, which render the conjugate ineffective. Presence of alkaline pH at the time of conjugating glutaraldehyde treated enzyme with antibody is helpful. Efficiency of antibody from different species to participate in A-conjugate formation: Rabbit, mouse and human second antibodies produce active A-conjugate. Their efficiency in use of immunodetection in Elisa has been established. When compared with HRP conjugate their sensitivity was comparable. In fact low blank values increases sensitivity of the Elisa system. Thermostability factor of A-con jugate Interestingly, the property of thermostability of amylase has been retained on conjugation. The formed conjugates have been shown to retain stability for a month at room temperature. When observed at high dilution some loss of activity was observed but at lower dilution no loss of activity was seen at all at room temperature. Indicating thereby that conjugate can be stored at room temperature for few days. However, it is better to store at 4°C. Its ability to withstand room temperature will make it a desirable reagent to be included in the diagnostic kits which are transported in Indian conditions and withstand powercuts even in the refrigerated condition. In the one step method of conjugation since antibody and amylase are both exposed to free glutaraldehyde there are high chances of antibody itself forming antibody-antibody conjugates. This may also be the reason of the formed one step conjugates giving erratic results and high blank values. Alkali is known to react with nitrophenol and form deep yellow colored complex. This increases the sensitivity of Elisa. Increase in intensity of color at high temperature; The increase in intensity of color at high temperature could be due increased activity of amylase and partially due to nonenzymaltic breakdown of nitrophenylmaltohaptoside to nitrophenol. Efficiency of A-conjugates used in Elisa. The- conjugates are able to detect different concentration of antigen coated and different dilutions of antisera used. The sensitivity of the detection varied with different dilutions of conjugate used. The present invention is described in detail with reference to the drawings and the experiments carried out. The said methods and processes described herein are given by way of examples only and should not be construed to limit the scope of the invention. Various modifications from the invention that would be apparent to there those skilled in the out are also intended to fall within the teachings of the invention. METHOD OF CONJUGATION OF ALPHA AMYLASE WITH SECOND ANTIBODY Modification of two-stap glutaraldehyde method 1. Suspend 3 mg a-amylase in 0.950ml of 0.1M PBS, pH 7.4. 2. Add O.OSOpl of 2.5% glutaraldehyde. 3. Keep shaking overnight at 4°C. 4. Keep for dialysis in 0.1 M PBS, pH 7.4 overnight in dialysis wells. 5. Adjust pH to > 9.0 with 400ul of 0.2M carbonate buffer, pH 9.5. 6. Add 0.3 mg IgG suspended in Tris buffer pH 8.0/.02M carbonate buffer pH 9.5. 7. Keep shaking overnight. 8. Add Ollml of 1M Glycine. Kept shaking for 1 hr at room temperature. 9. Concentrate in centricon, cut out 100 kd. 10. Add 200ul of 0.1M PBS, pH 7.4 with / without 50% glycerol. 11. Keep at 4°C / at -20°C. Need for this type of research The present era has refined several diagnostic tools for detection of diseases, changes in the blood and other fluids. One of the sophisticated and powerful diagnostic tool is ELISA or enzyme linked immunoassay. This technique is based on detection of proteins in the serum with the help of enzyme linked conjugates. These reagents are developed linking enzymes like Horseradish peroxidase, Alkaline Phosphatase, Urease and glucose oxidase. Out of several conjugates available Horseradish Peroxidase and Alkaline Phosphatase are the most popularly used. Even though the use of these conjugates is well established they continue to exhibit certain drawbacks. Both these enzymes are sensitive to high temperature and loose their activity in case of frequent powercuts in refrigerated condition. Thus, we explored into the several thermostable enzymes, with high specific activity, which could be used as enzyme label. Out of these enzymes we found alpha amylase from B. lichenformis having the best potential of being taken up for conjugate labelling. Its properties which has made it as outstanding member of enzyme label are listed below: 1. Alpha amylase from B. Lichenformis is a heat stable enzyme which is active at 90 C. 2. It has specific activity of 500-1000/units (this is comparable to specific activity of Horseradish peroxidase) I 3. It is a small enzyme of molecular weight of 55Kd ( molecular weight of horseradish peroxidase is 44Kd ) 4. It is a cheap enzyme costing 25.40 USD/lOOmg (HRP costs USD 217/ lOOmg, Alkaline Phoshatase costs USD 2142/lOOmg) when purchased from Sigma Chemical Co., USA. 5. Its acivity can be estimated colorimetrically. 6. It can be conjugated easily. 7. It maintains its thermostable nature even after conjugation. 8. It can be used for immunodetection in immunoassay. The details of methodology of how it can be conjugated to second antibody, it can be used in immunoassays and its effeciency in ELISA have been thoroughly investigated. Materials Used Chemicals : Alpha amylase (heat stable) from Bacillus lichenformis, amylase detection kit, glutaraldehyde, p —Nitrophenyl —alpha -D Maltoheptaside, alpha glucosidase from yeast ,centricons, Sephadex G25 were obtained from Sigma, USA. Thermostability and shelf life The compound retains the thermostable nature of the enzyme. It has been observed to retain 40% activity remaining at the end of 2 weeks storage at room temp (approx. 40°) when its activity was monitored in tube immunoassay at 1/2000 dilution ( see - Table 24 as above) . 100% activity was remaining when its activity was monitored at 1:400 dilution in ELISA after one month's storage at 37°C (see - Table 25 as above) . The conjugates were quite active after a years storage at and 2 years when stored at -20° C. Storage conditions of the conjugate The conjugate can be stored in sterile 0.1M PBS, pH 7.4 at 4° C. For long storage conjugates can be stored frozen at -20° with 50% glycerol. Conjugates stored with 50% glycerol have been shown to retain activity for 2 years. Horseradish peroxidase, antihuman goat antibody-HRP conjugate, anti-rabbit goat antibody - HRP conjugate, anti-mouse goat IgG - HRP conjugate, human IgG, rabbit IgG, mouse IgG, TMB substrate for horseradish peroxidase were purchased from Genei, Bangalore. Purification of antihuman and anti-rabbit goat IgG was standardized in the laboratory. Other analytical grade chemicals were obtained from SRL, India. Other accessories: ELISA plates and Dialysis bag, dialysis well, centricon were obtained from Sigma, USA. il.s ol ob-scn aliun.s ;iiu! ix-sults for the fileil patent Our. ot" the several methods tried for conjugation cc alpha -iv L-II;O with antibody the one ,1:1 d two step g lutara idehyde methods •fro observed to bo successful (table 1) Table 1 : The methods which can be used to conjugate alpha amylase with goat antibodv : (Table Removed) Out of the one step and two step glutaraldehyde method the latter one gives better conjugation (table 2; fig 1 ) Table 2 : Activity of alpha amylase - antibody made by one and two step glutaraldenyde method : (Table Remove) The method of conjugation has been optimized. Conjugation in different pH (table 3, fig 2), amount of glutaraldehyde (table, 4) ratio of enzyme and antibody (table 5 & 6) and time of incubation for conjugation has been observed and optimized. Table 3 : Activity of conjugates made in different buffers in two-step glutaraldehyde method. (Table Remove) TABLE 4: Activity of conjugates made by different Concentration of glutaraldehyde in two step method. (Table Removed) Table 5 : Compares the activity of a amylase antibody conjugates formed when different amounts of antigen is provided for conjugation during the conjugating process : (Table Removed) Table 5 and 6 show that enzyme:antibody ratio of 10:1 and 10:2 is sufficient to yield good conjugates. Table 6 : Compares the activity of alpha amylase antibody conjugates formed when different amounts of antibody is provided for conjugation during the conjugation process : (Table Removed) The conjugation was successfully accomplished with antimouse, antihuman and antirabbit goat IgG ( table 8) Table 7 : Shows the time sufficient to complete the conjugation process of a amvlasc with the antibody : (Table Removed) Table 7 shows that good conjugates can be formed even at short durations of -incubations i.e. 2 hrs as compared to 12 hrs incubation. Table 8 : Highlights the second antibodies that can be conjugate to a amylase : (Table Removed) The substrate medium which can sensitively detect activity of amylase is amylase detection substrate medium. Amylase detection substrate medium comprises of 1 mmol/L ETC PNP, 10 ra 7 mol/L MgCl , 50 mmol/L naCl, 25000 U/L a Glucosidase, buffer, pH 7.4. This was purchased from Sigma, USA. The reactions catalysed by a -amylase is given below: 5ET-G7 PNP + Amylase ->2 SET-GS + 2G: PNP + 2ET-G4 + 2G3 PNP + ET-G3 + Ga PNP 2G: PNP + 2G3 PNP alpha - Glucosidase 4PNP + 10 Glucose. Amylase hydrolyses 4,6 Ethylidene G7 PNP (ETG7 PNP) to C2, C3 and C4 PNP fragments, alpha-Glucosidase (1,4 glucan glucohydrolase EC 3.2.1.3) hydrolyses GaPNP and GsPNP to yield p-nitrophenol and glucose. 5 moles of substrate (ETC PNP) is hydrolysed to yield 4 moles of p-nitrophenol. 200 ul of 0.5N KOH can terminate the reaction and deepen the color formed (Table 9, Fig.12). Table 9 : Enhancement of color by termination of the reaction by alkali, in reaction catalysed by amylase conjugate. (Table Remove) The ADSM was stored cor a year at 4°c in the luophilised form each vial was reconstituted wi.tl: 3.5 ml distilled water. 50 ul of this midium was used as substrate. The reconstituted vial is sensitive to light and undergoes rapid deterioration within a o weeks storage at 4° C. Very low blanks were observed when freshly prepared substrate was used in Elisa. Stored substrate solutions gave higher blank values. The components of this medium were used at various concentrations. The colorimetric iramunodetection of conjugate was found most sensitive with the substrate consisting' of p-nitrophenylmaltoheptoside and alpha glucosidase. Tables 10 S 11 show the observation in color development when different ajnounts of p-nitrophenylmaltoheptoside and alpha glucosidase are used in te substrate medium. Table 10 : Shows the activity of amylase-antibody conjugate in presence of different concentration of p-nitrophenolmaltohepatoside and 25 U/ml Glucosidase in the substrate medium : (Table Removed) 25 U/ml of glucosidase and Img/mi p-nitrophenol maltoheptoside is sufficient to give good color. Further increase in concentration of maltoheptoside does not enhance the sensitivity but increases the blank values. Thus, the substrate medium that is standardised can be prepared as a cocktail constituting of alpha-glucosidase 25 units, p-nitrophenylmaltoheptoside Img, mgda, 0.1M PBS, pH 7.4 in 1 ml. Table 11: Shows the activity of amylase-antibody conjugate in Presence of different concentrations of alpha Glucosidase And 1 mg/ml p-nitrophenomaltohepatoside in the substrate medium. (Table Remove) The present product formed is sensitive to the temperature of incubation. (Table 12, Fig. 3). A bell shaped curve is obtained with different temperatures of incubation with substrate. Best specific reaction was observed at 37°C with small nonspecific reaction. Table 12: Enzyme Activity of conjugate when incubated with Substrate at different temperatures for 1 hour. (Table Removed) The product formed is linear with increasing time of incubation (Table 13, Fig. 4) Table 13: To observe the linearity of the enzyme assay in amylase detection substrate medium. Dilution of conjugate used: 1:2000. (Table Removed) The color developed is stable. The plates can be read directly after color development or the reaction can be terminated with 0.5N KOH and read immediately at 405nm. The use of alkali not only terminates a reaction but also enhances the color 3-4 times. After termination of reaction the plates should be read within 1 hr. In case the plates cannot be read immediately then reaction should not be terminated with alkali and should be preserved in dark at 40C. The color has been observed to be stable for period of 24 hrs or more. Establishing the use of second antibody-amylase conjugate in Immunoassay The efficiency of second antibody-amylase conjugate in ELISA was established. Its ability of immunodetection was established by observing its: 1. Sensitivity to detect coated antigen in direct ELISA (Table 14, Figure 5) Table 14:Shows sensitivity of detection of Inununoglobulins by anti-immunoglobulin-amylase conjugates in direct ELISA (Table Remove) Dilutions of conjugate used - 1 : 450 2. Sensitivity to detect coated antigen in indirect ELISA (table 14 A & 15, Fig. 6). Table 15 : Titration of coated antigen by second-antibody amylase conjugate in indirect ELISA. (Table Remove) 3. Sensitivity to detect the presence of antigen in competitive ELISA (table 16, Fig. 7) Table 16 Detection of buffalo LH by second antibody amylase conjugate in competitive ELISA. (Table Removed) 4. senstiveity to detect concentration of antibody in the serum in indirenct elisa (tables 17 & 17A, fig. 8). Table 17 : Detection of'antisera dilution by antibody-amylasa conjugate in indirect Elisa : (Table Removed) 5. Time required by this conjugate to react: with primary antibody ( Table 18, Fig. 9). Table 18: Optimum reaction time of second antibody amylase conjugate with coated IgG in Direct ELISA (Table Removed) Table 17 A: Shows sensitivity of detection of different antisera concentration in different, filaria samples by amylase-antihusian goat IgG (Table Remove) Table 17 A shows that antihuman A-conjugate can detect filaria positive serum effectively. The absorbance obtained was in accordance to the serial dilutions of serum. The antigen blanks were appreciably low. 6. Checkerboard assay in which varying amounts of anrisera and conjugate were used (Table 19). Table 19 : Shows the checkerboard assay with different concentrations of bLH antisera and amylase - Anti-rabbit & goat IgG (Table Remove) It is sensitive enough to coated/competing antigens upto 2ng/well. It can detect antibodies in serum serially diluted. The conjugate can react with primary antibody within 1 hr at 37° C. varying the concentrations of primary antibody and conjugates can effect the development of color in ELJSA 7. The color developed with substrate can remain stable for 24 hrs. or more at 4°C. In case of power failure plates can be stored and read later. It can also produce soluble products when dot blots are developed with this with this conjugate (table 20). Table 20: Efficiency of detection of coated buffalo LH on dot blot by second antibody-amylase conjugate by soluble product formation. (Table Removed) COMPARISION WITH EXISTING CONJUGATES It has been compared with commercially established conjugates like second antibody -HRPO conjugates. It is similar in efficiency to HRPO conjugate as: 1. It can detect coated/competing antigens as low as 2 ng/well conjugates {tables 21 & 22, fig. 10 & 11). Table 21 : Titration curve of coated antigen as observed with second antibody-HRPO conjugate and second antibody-amylase conjugate. (Table Remove) antisera dilution: 1 : 5000 conjugate dilution 1: 2000 (10 mg enzyme: 1 mg antibody antisera-dilution 1:400 conj. Dilution 1: 200 (Img enzyme: 300 ug antibody) Table 22 : Titration curve of coated IgG as detected by second antibody-HRPO conjugate in Direct ELISA (Table Remove) dilution of conjugate used - 1 : 1000 2. It can detect antibodies in serum serially diluted ( table 23 , fig 12). Table 23 : Titration curve of antibody concentrate as observed by HRPO conjugate and amylase conjugate antisera dilution Absorbance 405nm HRPOconj. Amylase Conj. (Table Remove) It can form soluble product which can be read colorimetrically. 4. It is stable for a year at 4°C and for 2 years when stored with 90% glycerol at -20°C It is unique as : 1. It can withstand room temperature 37°C for a month and continue to show 100% activity (tables 24 &: 25). It also exhibits good activity at 60°C when incubated for 2 hrs. Table 24: Stability of conjugate at room temp. (Table Remove) Table 25 : comparing the stability of HRP-Conjugate and amylase-conjugate after 26 days of storage at 37°C (Table Removed) 2. It shows very low blank value as non specific reaction with the serum is minimal. STOICHIOMETRY OF THE CONJUGATION REACTION The conjugation with varying amount of enzyme and antibody suggested that optimum ratio of enzyme : antibody required for conjugation was 10 : 1 and 10 : 2 (Table 26). Table 26 : Shows the stoichiometry of conjugation of amylase With antibody. (Table Removed) This meant that 2-3 molecules of enzyme reacted with antibody. When the conjugate was purified in 100 and 300 Kd centricon it was observed that conjugated enzyme ; antibody product had molecular weight below and above 300 Kd. This suggests that at least 2-3 molecules of enzyme are bound to antibody. WE CLAIM: 1. A novel thermostable probe useful for immunodetection in immunoassays, said probe consisting of antibody such as herein described and an enzyme in a proportion ranging from 1:10 to 2:10. 2. A probe as claimed in claim 1, wherein the antibody is a second antibody selected from anti-mouse, goat IgG, anti-rabbit goat IgG, anti-human goat IgG. 3. A probe as claimed in claim 1, wherein the enzyme is a-amylase extracted from bacterium lichenformis, said enzyme having specific activity of 1000 - 1200 Units per mg. 4. A probe as claimed in claim 1, wherein the free amino groups on the antibody is linked to the free amino groups on the side chains of lysine or arginine of a- amylase through crosslinking bridges. 5. A probe as claimed in claim 1, wherein the probe has a shelf life of 1 to 2 years and is stored at 4°C to -20°C. 6. A probe as claimed in claim 1, wherein the probe maintains its activity at 37°C to 90°C. 7. An in vitro immunodetection assay for indirect detection of antigens comprising the steps of: (a) coating antigen to the ELISA well; (b) blocking the wells with excess proteins; (c) incubation with antibody; (d) washing the sample; (e) incubation with second antibody probes for 1 to 24 hours; (f) washing the sample; (g) adding substrate; (h) incubating the sample for 1 hour at 37°C to 60°C; (i) reading for color directly or after termination with alkali at 405 nm wherein presence of color is indicative of presence of test antigen. 8. An assay as claimed in claim 7, wherein the proteins used are selected from the group consisting of 1% BSA (bovine serum albumin), skimmed milk. 9. A novel thermostable probe useful for immunodetection in immunoassays and an in vitro immunodetection assay for indirect detection of antigens, substantially as herein described with reference to the foregoing examples and accompanying drawings.

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1035-DEL-2002-Abstract-13-05-2008.pdf

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