Highly sensitive immunoassay for rapid quantification of meningococcal capsular polysaccharide antigens

Abstract

The present disclosure relates to the field of immunoassays for Gram negative bacteria, in particular N. meningitidis. The disclosure provides a simple and affordable immunoassay to quantitate polysaccharides in meningococcal vaccines for the evaluation of antigen content and lot-to-lot manufacturing consistency. The inventors have found a Sandwich ELISA that can be applicable for the quantitation and identification of N. meningitidis serogroup X polysaccharide in a multivalent meningococcal polysaccharide-protein conjugate vaccine as well as in a multivalent meningococcal plain polysaccharide vaccine. Said assay employs a polyclonal antibody as capture antibody and a novel monoclonal antibody against serogroup X polysaccharide as detection antibody. Further the assay is rapid, robust and reproducible.

Claims

1. A method for quantification of conjugated N. meningitidis serogroup X capsular polysaccharide in a sample using an immunological assay, the method comprising: providing a solid support coated with a capture polyclonal antibody; providing a sample including N. meningitidis serogroup X capsular polysaccharide-TT conjugate and unconjugated N. meningtitidis serogroup X capsular polysaccharide, wherein the sample is lyophilized and resuspended in phosphate buffer saline having pH 7.20.2 comprising KCl at a concentration from 75 mM to 140 mM followed by further dilution or the sample is liquid and directly diluted with phosphate buffer saline having pH 7.20.2 comprising KCl at a concentration from 75 mM to 140 mM; applying the sample to the solid support coated with capture polyclonal antibody; removing unbound sample; providing a detection monoclonal antibody; applying the detection monoclonal antibody to the solid support; removing unbound detection monoclonal antibody; applying labeled secondary antibody with a label; and detecting the label; wherein the assay does not show any interference due to presence of sucrose and glucose excipients.

2. The method according to claim 1, wherein the capture polyclonal antibody is obtained from rabbit antisera and the detection monoclonal antibody is obtained from fusion of mice splenocyte cells and mouse myeloma cells.

3. The method according to claim 1, wherein dilution of capture polyclonal capture antibody is between 1:5000 to 1:15000 in carbonate buffer, pH 9.6; dilution of the detection monoclonal antibody is between 1:3000 to 1:10000 in phosphate-buffered saline (PBS), pH 7.2; and dilution of the labeled secondary antibody is between 1:5000 to 1:15000 in phosphate-buffered saline (PBS), pH 7.2.

4. The method according to claim 1, wherein said detection monoclonal antibody does not show cross-reactivity with N. meningitidis serogroup polysaccharides A, C, W and Y.

5. The method according to claim 1, wherein limit of detection (LOD) is 0.4 ng/ml for N. meningitidis X.

6. The method according to claim 1, wherein the solid support is selected from the group consisting of polyvinyl chloride (PVC), polystyrene, agarose and sepharose.

7. The method according to claim 6, wherein the solid support is polystyrene.

8. The method according to claim 6, wherein the support is formed as a microtitration plate, tube or bead.

9. The method according to claim 8, wherein the microtitration plate is an activated immuno-assay plate.

10. The method according to claim 1, wherein the label is selected from the group consisting of an enzyme label, fluorescent label, a radioactive label and biotin.

11. The method according to claim 10, wherein the enzyme label is selected from the group consisting of a single enzyme, an oligomeric form of an enzyme, and an enzyme/antienzyme complex.

12. The method according to claim 11, wherein the enzyme label is coupled to an alternative detection system.

13. The method according to claim 11, wherein the enzyme label is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, beta-galactosidase and urease.

14. The method according to claim 11, wherein the label enzyme is horse radish peroxidase.

15. The method according to claim 1, wherein the sample comprises a monovalent N. meningitidis serogroup X capsular polysaccharide.

16. The method according to claim 15, wherein the sample comprises at least one additional N. meningitidis serogroup capsular polysaccharide selected from the group consisting of N. meningitidis serogroup A, N. meningitidis serogroup C, N. meningitidis serogroup W and N. meningitidis serogroup Y.

17. The method according to claim 15, wherein the sample comprises at least one additional N. meningitidis serogroup capsular polysaccharide-protein conjugate selected from the group consisting of N. meningitidis serogroup A, N. meningitidis serogroup C, N. meningitidis serogroup W and N. meningitidis serogroup Y.

18. The method according to claim 1, wherein the sample comprises at least one non N. meningitidis antigen selected from the group consisting of S. pneumoniae and H. influenzae.

19. The method according to claim 1, wherein the sample includes Neisseria meningitidis X polysaccharide from N. meningitidis X8210 strain and tetanus toxoid protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Sandwich ELISA's accuracy study for Men X Serogroup;

(2) FIG. 2: Sandwich ELISA's accuracy study for Men A Serogroup;

(3) FIG. 3: Sandwich ELISA's accuracy study for Men C Serogroup;

(4) FIG. 4: Sandwich ELISA's accuracy study for Men Y Serogroup;

(5) FIG. 5: Sandwich ELISA's accuracy study for Men W Serogroup;

(6) FIG. 6: Assay variation for Sandwich ELISA's for all serogroups Men X, A, C, Y and W;

(7) FIG. 7: Sandwich ELISA's Sensitivity study for Men X Serogroup;

(8) FIG. 8: Sandwich ELISA's Sensitivity study for Men A Serogroup;

(9) FIG. 9: Sandwich ELISA's Sensitivity study for Men C Serogroup;

(10) FIG. 10: Sandwich ELISA's Sensitivity study for Men Y Serogroup; and

(11) FIG. 11: Sandwich ELISA's Sensitivity study for Men W Serogroup.

DETAILED DESCRIPTION

(12) As required, detailed embodiments are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary and that various and alternative forms are possible. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

(13) The present disclosure relates to a Sandwich ELISA for Men X polysaccharide quantification in multivalent polysaccharide-protein conjugate vaccine/plain polysaccharide vaccine samples comprising of: 1. Coating of Immunoplates by serogroup specific antiserum (Men A, C, Y, W & X). (Dilution is 1:5000 to 1:15000). 2. Incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hour. 3. Washing of Immunoplates for 3 to 5 times by wash buffer: WFI+0.05% Tween 20). 4. Addition of blocking buffer containing Phosphate buffer saline, 2 to 7% FBS and 0.05% Tween 20. 5. Incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 6. Washing of Immunoplates for 3 to 5 times with wash buffer. 7. Addition of appropriately serially diluted polyvalent (Pentavalent/Quadrivalent) sample and standard. 8. Incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 9. Washing of Immunoplates for 3 to 5 times with wash buffer. 10. Addition of detection antibody solutions (Monoclonal Antibodies: 1:3000 to 1:10000). 11. Incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 12. Washing of Immunoplates for 3 to 5 times with wash buffer. 13. Addition of a secondary antibody preferably HRP-Conjugate antibody (Dilution is 1:5000 to 1:15000). 14. Incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 15. Addition of 100 L substrate (TMB) in all the wells. 16. Incubation at RT for 15 to 30 minutes. 17. Final addition of 100 L 2 N Sulphuric acid. 18. Reading the OD at 450 nm with reference at 630 nm.

(14) A method for the detection and/or quantification of N. meningitidis serogroup X capsular polysaccharide in a sample is provided. The method includes providing a solid support coated with polyclonal antibody; providing a sample; applying the sample to the polyclonal antibody coated solid support; removing unbound sample; providing monoclonal antibody; applying the monoclonal antibody to the solid support; removing unbound monoclonal antibody; applying labeled secondary antibody; and detecting the label. The capture polyclonal antibody and detection monoclonal antibody recognize two distinct non-overlapping epitopes of N. meningitidis Serogroup X capsular polysaccharide.

(15) An important aspect of instant disclosure is that said sample can be lyophilized or liquid. Preferably, i) lyophilized sample can be resuspended in phosphate buffer saline having pH 7.20.2 comprising of 75-140 mM KCl followed by further dilution and ii) liquid sample can directly be diluted with phosphate buffer saline having pH 7.20.2 comprising of 75-140 mM KCl.

(16) A second embodiment of the instant disclosure is that said Sandwich ELISA does not show any interference due to presence of excipients like sucrose and glucose.

(17) A third embodiment of the instant disclosure is that said detection monoclonal antibody against serogroup X 8210 shows no cross-reactivity with A, C, W, Y.

(18) A first aspect of the third embodiment The accuracy of the Sandwich ELISA for quantifying N. meningitidis group X in a pentavalent conjugate vaccine (A-TT, X-TT, C-CRM197, W-CRM197, Y-CRM197) is about 100%.

(19) A second aspect of the third embodiment of the instant disclosure is that said assay shows LOD of about 0.4 ng/ml and LOQ of about 1.6 ng/ml for serogroup X polysaccharide.

(20) A fourth embodiment of the instant disclosure is that said solid support can be selected from PVC, polystyrene, agarose and sepharose most preferably polystyrene wherein support may be formed as a microtitration plate, tube or bead.

(21) An object of the instant disclosure is that the microtitration plates are activated immunoassay plates.

(22) A fifth embodiment of present disclosure is that said capture antibody is a Rabbit Polyclonal antibody or antiserum against N. meningitidis serogroup polysaccharides selected from but not limited to A C W Y and X.

(23) A sixth embodiment of present disclosure is that said detection antibody is a Mouse Monoclonal antibody against N. meningitidis serogroup polysaccharides selected from but not limited to A C W Y and X.

(24) A further embodiment of the present disclosure is that preparation of said detection monoclonal antibody against serogroup X 8210 can comprise of following steps: 1. Obtaining splenocytes from mice immunized with Men X conjugate. 2. Fusing splenocytes with SP2/O cells (mouse myeloma cells) 3. Selecting fused cells using Hypoxanthine Aminopterin Thymidine medium. 4. Screening supernatant of fused cells for presence of anti-Men X antibody by using bead based assay and rate nephelometry 5. Making limiting dilution of positive clones. 6. Propagating single anti-Men X antibody secreting clone to 6 well plate. 7. Subjecting clone to a second limiting dilution. 8. Selecting Anti-Men X antibody secreting clone by bead based assay. 9. Propagating selected clone to 2 Liters for production of Anti-Men X Monoclonal antibody. Purifying monoclonal antibody by protein A chromatography.

(25) A still further object of the instant disclosure is that

(26) i) preferred dilution of polyclonal capture antibody can be between 1:5000 to 1:15000 in carbonate buffer, pH 9.6.

(27) ii) preferred dilution of the secondary monoclonal antibody can be between 1:3000 to 1:10000 in PBS, pH 7.2; and

(28) iii preferred dilution of the antibody to the secondary antibody (preferably HRP-Conjugate antibody) can be between 1:5000 to 1:15000 in PBS, pH 7.2.

(29) Yet another aspect of the instant disclosure is that blocking buffer can comprise of Phosphate buffered saline pH 7.2, 2 to 7% FBS and 0.05% Tween 20.

(30) The labelled secondary antibody preferably HRP-Conjugate antibody of present disclosure can be labeled directly wherein direct labels typically include enzyme labels, fluorescent labels, radioactive labels and biotin; preferably enzyme labeled.

(31) The enzymes used for labeling can either be a single enzyme, an oligomeric form of the enzyme, or an enzyme/antienzyme complex may be used.

(32) Typically the label can be an enzyme selected from alkaline phosphatase, horse radish peroxidase, -galactosidase and urease; a radioisotope selected from 125 I and 131 I; a fluorescent label selected from a fluorochrome, FITC and TRITC, or biotin; preferably horse radish peroxidase (HRP).

EXAMPLES

(33) In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure.

Example 1

(34) Total Phosphorus Estimation in Multivalent Vaccine by Chen Method or Ames Method

(35) TABLE-US-00001 TABLE 1 Total Batch Phosphorus Product Dose No. (g/mL) Meningococcal 1 Dose 234E4001 0.805 ACYWX 5 Dose 235E4001 3.883 Polysaccharide 1 Dose 234E4002 0.829 Conjugate 5 Dose 235E4002 4.683 vaccine 1 Dose 234E5001 0.904 5 Dose 235E5001 4.740

(36) Thus it was found that individual polysaccharide concentration for Men A and Men X cannot be calculated by chemical methods like chen method or Ames method

Example 2

(37) Preparation of MenX-TT Conjugate

(38) Polysaccharide Preparation

(39) MenX-TT conjugate was prepared by cyanylation and carbodiimide chemistry. The native polysaccharide was size-reduced by mechanical means. The size-reduced PS (120-150 kD on SEC-HPLC) was diafiltered & concentrated on 10 kD cuttoff membrane. To 20 mg/ml PS, freshly prepared solution of CPPT (also known as CPPT dissolved 114 mg/ml in acetonitrile) was added and the pH was shifted to 9.5 with NaOH solution and incubated for 2-4 min. Adipic acid dihydrazide (ADH) was dissolved 100 mg/ml in water and added to the PS solution. The reaction was stirred for 3 hrs at 25 C. The reaction was quenched by addition of excess glycine solution. The reaction mixture was diafiltered in MES buffer to remove residuals and unreacted components. The sample was stored at 2-8 C. until use. The PS content was measured by phosphorous assay and extent of derivatization was measured by TNBS assay.

(40) Carrier Protein (TT) Preparation:

(41) The carrier Protein preparation process was similar as described above.

(42) Conjugation of Men X to TT:

(43) The ADH-derivatized PS, purified TT and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) were mixed in a ratio of 1:1.5:0.7 by weight under stirring condition. The reaction progress was monitored on HPLC. The reaction was quenched after 3 hrs by raising pH with 10 mM PB+EDTA pH 8.0 buffer.

(44) Conjugate Purification:

(45) The conjugate was purified on 300 kD cut off membrane by passing 30 volumes of 100 mM PBS and followed by 30 volume 10 mM Tris pH 7.2. The purified bulk conjugate was analyzed for total and unbound (free) PS, total and protein and residual 4-PPY in the conjugate bulk.

(46) TABLE-US-00002 TABLE 2 Total PS Total Protein Test Sample mg/ml mg/ml % Free PS Men X-TT Purified 1.08 2.44 7.87 Conjugate

Example 3

(47) Development of Monoclonal Antibodies Against Serogroup X of Neisseria meningitidis

(48) Splenocytes isolated from mice immunized with Men X conjugate were fused with SP2/O cells (mouse myeloma cells). The fused cells were selected using HAT (Hypoxanthine Aminopterin Thymidine) medium. The supernatant of respective fused cells was screened to check the presence of anti-Men X antibody by in-house analytical methods like bead based assay and rate nephelometry. Limiting dilution of the positive clones were done. The single anti-Men X antibody secreting clone was propagated to 6 well plate. The clone was subjected to second limiting dilution. Anti-Men X antibody secreting clone was selected by bead based assay. Selected clone was further propagated to 2 Liters for production of Anti-Men X Mab. The produced MAb was purified using protein A resin by affinity chromatography.

Example 4

(49) Characterization of In-House Developed Monoclonal Antibody Against Meningococcal Polysaccharide Serogroup X (MEN XLD2P1A12P1CP2D10)

(50) TABLE-US-00003 TABLE 3 Sr. No. Tests Specifications Result Remark 1 Specificity of MAb Bead Based assay Reactive to Complies Complies Serogroup X of MenPs, <10% cross reactive to other MenPs.sup.# Nephelometry Reactive to Complies Complies Serogroup X of MenPs, <10% cross reactive to other MenPs.sup.## 2. Protein Report value 1.0 Complies Concentration (mg/ml) 3. Analysis of Antibody by SDS- PAGE Reducing sample Two bands of heavy Complies Complies treatment and light chain should be observed Non-reducing Antibody band of Complies Complies sample treatment approximately 150 KD should be observed .sup.#Cross reactivity was checked against Meningococcal Capsular Polysaccharides from serogroup A, C, W, Y and X. Other antigens were Tetanus toxoid and CRM197. .sup.##Cross reactivity was checked against Meningococcal Capsular Polysaccharides from serogroup A, C, W, Y and X.

Example 5

(51) Dilution of Antibodies

(52) Capture antibody was commercially procured from BD Biosciences. The dilution for capture antibody was made from the stock and was optimized. Similar approach was utilized for dilution of in-house monoclonal antibodies i.e. binding antibody against A C W Y & X.

(53) The dilution for polyclonal capture antibody was between 1:5000 to 1:15000 in carbonate buffer, pH 9.6. The dilution for detection monoclonal antibody was between 1:3000 to 1:10000 in PBS, pH 7.2. The dilution for secondary antibody (preferably HRP-Conjugate antibody) was between 1:5000 to 1:15000 in PBS, pH 7.2.

Example 6

(54) Sample Preparation Pre-Sandwich ELISA

(55) Sample in lyophilized form was resuspended in phosphate buffer saline (PBS) (pH 7.2) having 107 mM KCl followed by further dilution. Whereas for liquid samples, direct dilution with PBS (pH 7.2) having 107 mM KCl was carried out.

(56) Three vials of vaccine were taken for testing. Each vial was resuspended into 1 mL PBS and mixed. Appropriate volume of sample was taken in test tube and desired volume of PBS was added to dilute the sample. Sample was serially diluted so that the optical density of sample would fall into the standard curve. Three dilution prepared from individual three vials were used to have sample in triplicate in the assay.

Example 7

(57) Protocol for Sandwich ELISA

(58) 1. The immunoplates were coated by serogroup specific antiserum (Men A, C, Y, W & X). (Dilution is 1:5000 to 1:15000). 2. Said plates were incubated at a temperature between 30 to 40 C. for a duration of about 1 to 2 hour. 3. Immunoplates were washed for 3 to 5 times by wash buffer: WFI+0.05% Tween 20). 4. Blocking buffer containing Phosphate buffer saline, 2 to 7% FBS and 0.05% Tween 20 was added. 5. Mixture was subjected to incubation at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 6. Immunoplates were washed for 3 to 5 times with wash buffer. 7. Serially diluted polyvalent (Pentavalent/Quadrivalent) sample and standard were added. 8. The mixture was incubated at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 9. Immunoplates were washed for 3 to 5 times with wash buffer. 10. The detection antibody solutions (Monoclonal Antibodies: 1:3000 to 1:10000) were added. 11. The mixture was incubated at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 12. Immunoplates were washed for 3 to 5 times with wash buffer. 13. Secondary antibody preferably HRP-Conjugate antibody (Dilution is 1:5000 to 1:15000) was added. 14. The mixture was incubated at a temperature between 30 to 40 C. for a duration of about 1 to 2 hours. 15. 100 L substrate (TMB) was added in all the wells. 16. Mixture was incubated at RT for 15 to 30 minutes. 17. 100 L 2 N Sulphuric acid was finally added. 18. OD was read at 450 nm wherein reference was 630 nm.

Example 8

(59) Accuracy, Variation and Sensitivity of Novel Sandwich ELISA Method

(60) TABLE-US-00004 TABLE 4 Accuracy study: Percentage Recovery (Refer FIGS. 1 to 5) Concen- tration Expected in vaccine Con- Obtained Vial Spike centration Concentration Recovery Serogroup g/vial) (g) (g/vial) (g/vial) (%) X 30.44 9.14 39.36 40.14 102 A 30.44 12.00 38.55 38.55 100 C 26.35 9.37 38.85 42.38 109 Y 33.10 13.63 51.90 51.48 99 W 27.21 11.40 37.90 38.12 101 Note: All values for the assay in g/Vial

(61) TABLE-US-00005 TABLE 5 Assay Variation(Refer FIG. 6) Sero- Assay Assay Assay Standard % Coefficient group 1 2 3 Average Deviation Variation X 24.98 32.48 33.79 30.42 4.75 15.63 A 28.58 38.19 24.16 30.46 6.98 22.92 C 25.42 27.59 26.10 26.37 1.11 4.21 Y 30.50 31.31 37.44 33.08 3.79 11.47 W 24.73 28.58 28.33 27.21 2.15 7.92

(62) TABLE-US-00006 TABLE 6 Optical density details Average Average Optical Optical Concentration Average Optical Density of sample of least Density of least last standard Serogroup Blank standard dilution (g/mL) X 0.071 0.191 0.178 0.0004 A 0.103 0.352 0.388 0.002 C 0 . . . 135 0.267 0.287 0.002 Y 0.201 0.543 0.609 0.008 W 0.234 0.359 0.386 0.008

(63) The sensitivity of the assay was assessed based on the above data

(64) TABLE-US-00007 TABLE 7 Sensitivity (Refer FIGS. 7 to 11) Sensitivity of the Serogroup method X 0.4 nanogram/mL A 2.0 nanograms/mL C 2.0 nanograms/mL Y 8.0 nanograms/mL W 8.0 nanograms/mL

(65) TABLE-US-00008 TABLE 8 Limit of Detection (LOD) and Limit of Quantitation (LOQ) Limit of detection Limit of quantitation Serogroup (LOD) (LOQ) X 0.4 nanogram/mL 1.6 nanogram/mL A 2.0 nanograms/mL 8.0 nanograms/mL C 2.0 nanograms/mL 8.0 nanograms/mL Y 8.0 nanograms/mL 31 nanograms/mL W 8.0 nanograms/mL 31 nanograms/mL Note: The values obtained based on 4-Parametric curve analysis. The quantitation range is selected in a linear region of the curve from the standard curve.

(66) Said Sandwich ELISA of instant disclosure carried out using i) a novel monoclonal antibody against N. meningitidis serogroup X Polysaccharide, ii) specific dilutions of polyclonal capture antibody, detection monoclonal antibody and secondary antibody and iii) a prior sample treatment step, surprisingly showed a) optimal specificity, sensitivity for quantifying serogroup X polysaccharide in a multivalent ACWYX vaccine wherein LOD was 0.4 ng/ml and LOQ was 1.6 ng/ml for Men X b) did not show any interference due to excipient unlike the HPAEC-PAD and other chemical methods.

(67) While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the disclosure. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the disclosure.