Title of Invention

A KIT FOR SEMI-QUANTIFICATION OF CRP IN WHOLE BLOOD AND PROCESS RELATED FOR MANUFACTURE OF THE SAME

Abstract

A kit for semiquantification of the level of C- reactive protein (CRP) using whole blood and its process of manufacture involving use of a conjugate reagent comprising crosslinked monovalent Concanvalin A and Fab portion of anti CRP antibody. A process for semiquantification of the level of C- reactive protein (CRP) using whole blood using the kit of the invention. The kit and its process of manufacture enables semiquantification of CRP in whole blood following then process for the same in a faster, simpler and more convenient way than those known in the art. It avoids the separation of blood serum since the whole blood is used for the process using the kit. Moreover the kit has shelf life of 6 months when maintained at 4°C which enables easy operation of the kit without maintenance of any complicated conditions.

Full Text

Field of invention: The present invention relates to a kit for semiquantification of the level of C- reactive protein (CRP) using whole blood and its process of manufacture. The invention also relates to a process for semiquantification of the level of C- reactive protein (CRP) using whole blood using the kit of the invention. Background of Invention C- reactive protein is a homopentameric, classical acute phase plasma protein, synthesised in large amount due to tissue damage or infection. CRP level increases in most cases of inflammatory diseases, allograft rejection, malignancy, necrosis. Hence elevated CRP is a sensitive universal index of acute infection as well as noninfectious inflammation. At present time elevated CRP is used as novel clinical markers of vascular wall inflammation. Traditionally it has been used to clinically monitor infection and autoimmune disorder. Many immunochemical methods for the estimation of CRP in the human blood have been devised like capillary tube immunoprecipitation, single radial immunodiffusion, passive latex agglutination, rocket immunoelectrophoresis, nephalometric assay, imrnunofiuorornetric assay, radioimmuno assay, and ELISA using blood serum All the above methods used blood serum for determination of CRP level. They involved complicated processes of separation of the serum from the cells. Moreover they had to be done immediately and involved specially trained technical staff to handle the processing of blood and estimation of CRP levels. Objects of the Invention Thus the basic object of the present invention is to provide for a kit to measure the level of CRP in blood samples using whole blood which would avoid the prior art problems in estimation involving blood serum as discussed above. Another object of the present invention is to provide for a simple process of manufacture of a kit to measure the level of CRP in blood samples using whole blood. Yet another object of the present invention is to provide for a process of estimation of C-reactive protein (CRP) using whole blood which would be fast and convenient to carry out. A further object is to provide for a simpler method of semiquantification of CRP in the blood. Another object is to provide for a kit for semiquantification of CRP in the blood which would have a shelf life of about 6 months at about 4° C. Summary of Invention Thus according to one aspect of the present invention there is provided a kit for use in semi quantification of CRP present in whole blood sample comprising : i) conjugate reagent; ii) buffer; iii) multiple well haemagglutination plate; iv) standard curve. According to another aspect of the present invention there is provided a process for manufacture of kit comprising providing i) the conjugate reagent; ii) the buffer iii) the multiple well haemagglutination plate iv) the standard curve According to further aspect of the present invention there is provided a process for semi quantification of CRP in whole blood using the kit of invention comprising: i) incubating patient's blood with various concentrations of the conjugate for agglutination of CRP ii) semi quantification of CRP from dilution of conjugate using the standard curve. According to preferred aspect of present invention the process for semi quantification of CRP in whole blood using the kit of invention comprises the following steps of: i) taking 5-50µl, preferably 3-10 µl of blood to be tested in 6 different wells of 96 well haemagglutination plate of the kit; ii) adding 50µl of buffer to the first well to serve as control; iii) adding conjugate reagent to the other wells in an amount of 35 to 50 µl; iv) making the volume of each well made up to 55µl with buffer; v) incubating for 30-45 minutes till button formation in control well; vi) noting conjugate concentration at which complete agglutination occurs. Detailed Description of Invention The kit and its process of manufacture enables semiquantification of CRP in whole blood following the process of invention for the same is a simpler way than those known in the art. It avoids the separation of blood serum since the whole blood is used for the process using the kit of the invention. Moreover the kit of invention has shelf life of 6 months when maintained at 4°C which enables easy operation of the kit without maintenance of any complicated conditions. The conjugate reagent of the kit of the present invention comprises crosslinked monovalent Concanvalin A and Fab portion of anti CRP antibody present in a molar ratio of 3:1 to 1:3, preferablyl: 1 in which the monovalent Concanvalin A is a limited trypsin digestion product of tetravalent Concanvalin A and the Fab portion of anti-CRP antibody is preferably obtained from goat serum. This conjugate reagent is adapted to agglutinise CRP present in blood to be tested. The multiple well haemagglutination plate of the said kit comprises 'U' bottom wells which are preferably 96 in number. The buffer used in the kit is phosphate buffer saline having a pH of 7.4 The standard curve provided with the kit comprises the end points of agglutination between various concentrations of conjugate reagent from a batch of conjugate reagent obtained from a particular lot of goat anti-CRP antibody and CRP from sample blood plotted with CRP concentration as X- axis and conjugate concentration as Y-axis. According to another aspect the process for manufacture of kit comprises preparation of the conjugate reagent by mixing monovalent Concanvalin A and Fab portion of anti CRP antibody in a molar ratio of 3:1 to 1:3, preferably 1:1 in PBS and conjugating with EDC followed by incubation for 18 hours in dark room with continuous stirring and dialyzing and centrifuging the conjugate product. The Concanvalin A provided in the conjugate reagent is prepared by limited tryptic digestion of tetravalent Con A bounded to malatmyl-affinity matrices, elution by 0.1 M to 0.5M , preferably 0.2 M glucose followed by purification of monovalent Con A by gel filtration. On the other hand the Fab fragment of anti-CRP antibody provided in the conjugate reagent is prepared by purification of goat serum containing medium titre of anti-CRP antibody by ammonium sulphate fractionation and ion exchange chromatography followed by papain digestion of flow through part of chromatography containing high titre of anti-CRP separating the Fab part which is then purified by protein affinity chromatography or by gel permeation chromatography. The kit is calibrated by mixing various known concentrations of the said conjugate reagent and CRP from test blood along with fixed amount of washed RBC to note the end of agglutination between conjugate reagent and CRP. Such end points of agglutination between various concentrations of conjugate and CRP are plotted using CRP concentration as X- axis and Conjugate concentration as Y-axis to form the standard curve provided with the kit. The process for semi quantification of CRP in whole blood is based on agglutination of the whole of patient's blood with the conjugate reagent and noting the concentration of conjugate reagent at which agglutinisation occurred. From the standard curve provided with the kit the amount of CRP present in patient's blood is then detected. The level of CRP detected by this process lies between 0.5 to 60 jag/ml. Thus the amount of CRP in patient's blood is determined from the standard curve or from concentration of conjugate required for agglutination. The invention shall now be discussed with reference to non limiting examples Example 1: Process of preparation of kit As discussed above the kit of the invention comprises: conjugate reagent; buffer; multiple well haemagglutination plate; standard curve. The conjugate used in the kit is prepared by mixing monovalent Concanvalin A and Fab portion of anti CRP antibody in a molar ratio of 1:1 in PBS and conjugating with EDC followed by incubation for 18 hours in dark room with continuous stirring. The conjugated product was extensively dialysed and centrifuged at 5000 rpm for 10 minutes to remove the particulate materials and the reagent is ready. The Concanvalin A provided in the conjugate reagent is prepared by limited tryptic digestion of teravalent Con A bounded to malatmyl-affinity matrices, elution by 0.2 M glucose followed by purification of monovalent Con A by gel filtration. The Fab fragment of anti-CRP antibody provided in the conjugate reagent is prepared by purification of goat serum containing medium titre of anti-CRP antibody by ammonium sulphate fractionation and ion exchange chromatography followed by papain digestion of flow through part of chromatography containing high titre of anti-CRP separating the Fab part which is then purified by affinity chromatography or by gel permeation chromatography. The multiple well haemagglutination plate of the said kit comprises 'U' bottom wells which are preferably 96 in number. The buffer used in the kit is phosphate buffer saline having a pH of 7.4 The kit is calibrated by mixing various known concentrations of the said conjugate reagent and CRP from test blood : conjugate reagent (3.48 to 4.87 mg/ml), CRP (0.48 to 62.50 µg/ml) along with fixed amount of washed RBC (5µl) to note the end of agglutination between conjugate reagent and CRP. Such end points of agglutination between various concentrations of conjugate and CRP are plotted using CRP concentration as X- axis and Conjugate concentration as Y-axis to form the standard curve provided with the kit. Example 2 The process for semi quantification of CRP in whole blood is based on agglutination of the whole of patient's blood with the conjugate reagent and noting the concentration of conjugate reagent at which agglutinisation occurred. From the standard curve provided with the kit the amount of CRP present in patient's blood is then detected. i) Calibration: 1-5 µl of washed RBC from healthy donor is taken and mixed with different concentration of CRP and various dilutions of conjugate reagent in a total reaction volume of 100 µl in 96 wells of the kit. The conjugate reagent used is prepared as in example 1. The mixture is incubated for 45-60 minutes. For each concentration of CRP and 5µl of washed RBC the end point dilution was measured where agglutination just ended.: Table 1 provides the conjugate concentration which agglutinise various CRP concentrations Table 1 The values from Table 1 were plotted and a standard curve was obtained with CRP concentration as X- axis and conjugate concentration as Y-axis. ii) Determination of CRP in patient's blood 5µ1 of patient's blood sample was taken in 6 different wells of 96 well haemagglutination plate of the kit. 50µl of buffer is added to the first well to serve as control. Conjugate reagent added to the other wells in an amount of 35 to 50 µl Volume of each well made up to 55µ1 with buffer Incubated for 30-45 minutes till button formation in control well Conjugate concentration is noted at which complete agglutination occurs which is 2 to 8 mg/ml. From the dilution of conjugate the CRP concentration is determined using the standard curve provided. Thus the process of semiquantification of CRP using the kit of the invention is a simple method in which whole blood is used. It is a faster method without the complication of separation of serum from whole blood. Thus the invention is directed to provide a ready kit for such semiquantification of CRP. We Claim : 1. A kit for semi quantification of CRP present in whole blood sample comprising : i) conjugate reagent comprising crosslinked monovalent Concanvalin A and Fab portion of anti CRP antibody; ii) buffer; iii) multiple well haemagglutination plate comprising 'U' bottom wells; and iv) standard curve comprising the end points of agglutination between various concentrations of conjugate reagent from a batch and CRP from sample blood plotted with CRP concentration as X- axis and conjugate concentration as Y-axis. 2. The kit as claimed in claim 1 wherein conjugate reagent comprises monovalent Concanvalin A and Fab portion of anti CRP antibody in a molar ratio of 3:1 to 1:3, preferably 1:1. 3. The kit as claimed in claim 1 or 2, wherein the monovalent Concanvalin A is a limited trypsin digestion product of tetravalent Concanvalin A. 4. The kit as claimed in any preceding claim wherein the Fab portion of anti-CRP antibody is preferably obtained from goat serum. 5. The kit as claimed in any preceding claim wherein the said conjugate reagent is adapted to agglutinise CRP present in blood to be tested. 6. The kit as claimed in any preceding claim wherein the multiple well haemagglutination plate comprises 96 wells. 7. The kit as claimed in any preceding claim wherein the buffer is phosphate buffer saline. 8. The kit as claimed in any preceding claim wherein the buffer has a pH of 7.4 9. A process for manufacture of kit comprising providing i) conjugate reagent comprising crosslinked monovalent Concanvalin A and Fab portion of anti CRP antibody; ii) buffer; iii) multiple well haemagglutination plate comprising 'U' bottom wells; and iv) standard curve comprising the end points of agglutination between various concentrations of conjugate reagent from a batch and CRP from sample blood plotted with CRP concentration as X- axis and conjugate concentration as Y-axis. 10. The process for manufacture of kit as claimed in claim 9 wherein the conjugate reagent provided is prepared by mixing monovalent Concanvalin A and Fab portion of anti CRP antibody in a molar ratio of 3:1 to 1:3, preferably 1:1 in PBS and conjugating with EDC followed by incubation for 18 hours in dark room with continuous stirring and dialysing and centrifuging the conjugate product. 11. The process for manufacture of kit as claimed in claims 9 or 10 wherein the Concanvalin A provided in the conjugate reagent is prepared by limited tryptic digestion of tctravalent Con A bounded to malatmyl-affinity matrices, elution by 0.1 M to 0.5 M , preferably 0.2 M glucose followed by purification of monovalent Con A by gel filtration. 12. The process for manufacture of kit as claimed in claims 9 - 11, wherein the Fab fragment of anti-CRP antibody provided in the conjugate reagent is prepared by purification of goat serum containing medium titre of anti-CRP antibody by ammonium sulphate fractionation and ion exchange chromatography followed by papain digestion of flow through part of chromatography containing high titre of anti-CRP separating the Fab part which is then purified by protein affinity chromatography or by gel permeation chromatography. 13. The process for manufacture of kit as claimed in claims 9 12 wherein the calibration of the kit is carried out by mixing various known concentrations of the said conjugate reagent and CRP from test blood along with fixed amount of washed RBC to note the end of agglutination between conjugate reagent and CRP. 14. The process for manufacture of kit as claimed in claim 13 wherein the end points of agglutination between various concentrations of conjugate and CRP are plotted using CRP concentration as X-axis and Conjugate concentration as Y-axis to form the standard curve provided with the kit. 15. A process for semi quantification of CRP in whole blood using the kit as claimed in claims 1 to 8 wherein the whole of blood to be tested is taken and agglutinised with the conjugate reagent noting the concentration of conjugate reagent at which agglutinisation occurred. 16. A process for semi quantification of CRP in whole blood as claimed in claim 15 comprising: i) taking 5-50µ1, preferably 3-10 µl of blood to be tested in 6 different wells of 96 well haemagglutination plate of the kit; ii) adding 50µl of buffer to the first well to serve as control; iii) adding conjugate reagent to the other wells in an amount of 35 to 50 µl; iv) making the volume of each well made up to 55µl with buffer; v) incubating for 30-45 minutes till button formation in control well; vi) noting conjugate concentration at which complete agglutination occurs. 17. A process for semi quantification of CRP in whole blood as claimed in claims 15- 16 wherein the amount of CRP in patient's blood is determined from the standard curve using concentration of conjugate required for agglutination. A kit tor semiquantification of the level of C- reactive protein (CRP) using whole blood and its process of manufacture involving use of a conjugate reagent comprising crosslinked monovalent Concanvalin A and Fab portion of anti CRP antibody. A process for semiquantification of the level of C- reactive protein (CRP) using whole blood using the kit of the invention. The kit and its process of manufacture enables semiquantification of CRP in whole blood following the process for the same in a faster, simpler and more convenient way than those known in the art. It avoids the separation of blood serum since the whole blood is used for the process using the kit. Moreover the kit has shelf life of 6 months when maintained at 4°C which enables easy- operation of the kit without maintenance of any complicated conditions.

Documents:

727-CAL-2002-FORM-27.pdf

727-cal-2002-granted-abstract.pdf

727-cal-2002-granted-claims.pdf

727-cal-2002-granted-correspondence.pdf

727-cal-2002-granted-description (complete).pdf

727-cal-2002-granted-examination report.pdf

727-cal-2002-granted-form 1.pdf

727-cal-2002-granted-form 18.pdf

727-cal-2002-granted-form 2.pdf

727-cal-2002-granted-form 3.pdf

727-cal-2002-granted-pa.pdf

727-cal-2002-granted-reply to examination report.pdf