COMPETITIVE ENZYME IMMUNOASSAY FOR ASSESSING TOTAL ANTIGEN CONTENT OF ALUMINUM-ADSORBED ANTIGENS

Abstract: The present invention describes a competitive EIA for assessing total antigen content of aluminum adsorbed antigens. The competitive enzyme immunoassay method of the present invention provides a means for reproducibly quantitating antigens adsorbed to aluminum hydroxide without desorption or pretreatment steps.

Description: COMPETITIVE ENZYME IMMUNOASSAY FOR ASSESSING TOTAL ANTIGEN CONTENT OF ALUMINUM-ADSORBED ANTIGENS BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the field of quantitative determination of antigen content in aluminum-adsorbed antigen vaccine compositions. Summary of the Related Art Vaccines are comprised of antigens intended to produce a desired protective immune response. Adjuvants have been incorporated in many vaccines to enhance the immune response to vaπous antigens. Some adjuv ant types include oil emulsions (Freund's adjuvant), aluminum salts and synthetic compounds (poly[dι(carboxylatophenoxy)]phosphazene) Currently, the only adjuvants approved for human use are the aluminum containing adjuvants ( 1 ). These are broken down into two types: aluminum hydroxide and aluminum phosphate (3). Aluminum hydroxide adjuvant is crystalline aluminum oxv hydroxide with the mineralogical name of boehmite Aluminum phosphate adjuvant is amorphous aluminum hydroxyphosphate Researchers hav e shown that aluminum hydroxide adjuvant has a high point of zero charge (PZC) and is a good absorbent for acidic proteins, as compared to aluminum phosphate which has a low PZC and thus is better able to absorb more basic proteins (4). Regulatory authorities require that vaccines be tested to quantitate the antigen content in the final vaccine product. The addition of adjuvants with high absorptiv ity such as those containing aluminum complicates such quantitation by making the antigen unavailable for detection. One approach to the quantitation of antigens bound to aluminum is to desorb or elute the alum away from the antigen pπor to performing the quantitation assay For example, phosphate anion has been found to desorb ovalbumin (5), porcine parvovirus (6). and FMDV-antigen (7) from Al(OH) 3 adjuvant In the study by Katz, 66% of total protein could be recovered after desorption of the antigen using phosphate anion. The protein antigens were also tested for activity and was shown to be active. Another approach to quantitation of antigens adsorbed to aluminum entails elution of the antigens from aluminum-containing adjuvants by treatment with vanous surfactants (8) Unfortunately, the surfactant most effective for elutmg protein antigens from aluminum adjuvants provides oniy a 50° o protein elution (8) Furthermore, surfactants are knoun to interfere with immunological complexes. Indeed, this interference phenomenon is exploited routinely in immunoassays by incorporating surfactants into washing buffers (9) A related approach, descπbed by Scattergood and Grabner in U S Patent \ τ o 4. 894.444. entails dissolution of the antigen-aluminum complexes by dissolving the aluminum awa from the antigen with an alkali metal salt of mono, di or tπcarboxylic acid This method requires careful selection of dissociation conditions, both to ensure complete dissociation of aluminum from the antigen, and to avoid inactivation or denaturation of the antigen of interest Thus, such a method may not be used w ith labile antigens Also, the method involv es sev eral steps, including careful adjustment of the pH of the dissociation solution, follow ed by careful monitoπng of the reaction over a peπod of from 1 to 3 hours, neutralizing the dissociation solution, then recoveπng the dissociated antigen by dialysis or ultrafiltration. To date, there has not been a method to reproducibly quantitate antigens adsorbed to aluminum hydroxide without desorption All of the present methods are quite labor intensi e. requiπng a lengthy elution times, followed by dialysis and concentration steps. Such methods are unsatisfactory for routine commercial use. Thus, a need exists for impro ed methods capable of precisely and accurately quantitating antigens that have been adsorbed to aluminum hydroxide. SUMMARY OF THE INVENTION The present invention provides an immunoassay for direct quantitation of antigen within vaccine compositions containing aluminum salts. The method of the present invention provides a simple, accurate and precise way to quantitate antigens in vaccine compositions containing aluminum salts without having to first remov e the aluminum from the vaccine composition. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 displays a graph representing assay specificity determined by compaπson of assay controls. Figure 2 displays a graph representing assav selectivity determined bv testing vaπous absorbed proteins. DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for directly quantitating antigen within v accine compositions, wherein the antigen has been adsorbed to aluminum salts. Bv "direct" is meant that the antigen within the vaccine composition can be quantitated without first remov ing the aluminum from the antigen, i.e.. without pretreatment. elution or dissociation of aluminum salt from the antigen. The method of the present inv ention can be applied to any number of potential antigens within v accine compositions. Such antigens mav be prepared by anv means, including puπfication or partial puπfication from the native state or by recombinant methods Similarly antibodies to antigens can be prepared by standard immunological techniques Such antibodies may be monoclonal, polyclonal, or monospecific polvclonal antibodies, or combinations thereof The method ot the present inv ention may be used to quantitate antigens in a vaccine composition containing combinations of one or more antigens The immunoassay method of the present invention can be used to quantitate individual antigens within such combination vaccines Antigen adsorbed to aluminum salt is quantitated according to the invention by a competitiv e immunoassay procedure Antigen that has not be adsorbed to aluminum salt is used to coat reaction plates λntigen that has been adsorbed to an aluminum salt is allow ed to react with antibody to the antigen, resulting in the formation ot antigen-antibody complexes Such antigen-antibody complexes are prepared using reference standard antigen as w ell as vaccine antigen The antigen-antibody complexes are then added to the reaction plates and allo ed to incubate The reaction plates are then w ashed to remove unbound antigen-antibody complexes and the amount of antibody bound to the reaction plates is determined The amount ot antibody bound to the reaction plate is inv ersely proportional to the amount of antigen present in the antigen-antibody complexes A standard curv e is prepared using the results obtained from the reference standard antigen, and the amount of antigen present in the vaccine composition is determined with reference to the standard curv e In the immunoassay method ot the present invention, reaction v essels are first coated with an antigen of interest Antigens of interest include any antigens present ithin vaccine formulations For example Psp or PsaA ot 5 pne momae OspA. OspC or Dpb λ of B bwgdoi iei i toxoids ot diphtheπa or tetanus toxin, Ό name but a few Reaction e ^ el could include, by way of example and not limitation, wells within multi-w ell reaction plates or individual test tubes The reaction vessels are prepared by adsorbing antigen to each site, using methods well known to those skilled in the art It is important to note that the antigen that is to be applied to the reaction site is not adsorbed to an aluminum salt "Reference" and "test" samples are then prepared Reference samples are samples in which the quantity of antigen present in the sample is known, whereas test samples are those in which the quantity of antigen in the sample is to be determined In a preferred embodiment of the present invention, the reference antigen is adsorbed with 520 g of aluminum hydroxide per ml pπor to prepaπng reference samples Reference and test samples are prepared at a plurality of dilutions within the range of detection of standard immunoassays {e g . ng to g range) and added to antibody specific to the antigen In a preferred embodiment, monoclonal antibody is used These reaction samples, containing antigen-aluminum and antibody , are transferred to the reaction v essels and incubated tor sev eral hours Incubation results in the formation of complexes ot antigen-aluminum plus antibody Following incubation, the reaction vessels are washed sev eral times to remove unreacted complexes At this point, the amount of antibody that has bound to the reaction vessel is determined The amount of antibody that has bound to the reaction vessel can be determined by any number of methods well known to those skilled in the art The antibody can be labeled, e g . with a radioisotope such as I 1 " or with an enzymatic label, at any point pπor to addition of the antibody to the antigen The antibodv could also be labeled with a hgand such as av idin or biotin. and its presence then could be determined indirectiv by reaction w ith labeled anti-hgand (e _ ? • biotm tor avidin. or ersa ) Alternativ ely , the amount of antibody bound to the reaction v essel can readily be determined indirectly by reaction with a labeled secondary antibody, i e . an antibody that is capable of binding specifically to the first antibody For example, if mouse monoclonal antibodies are used in the reaction sample step, then binding of mouse monoclonal antibodies to the reaction vessels can be determined following a reaction with goat anti-mouse antibody which is labeled, e.g , with a radioisotope, an enzyme, a fluorescent dye or a visible dye. Some of the antibody in the reaction solution binds to the antigen that was applied to the reaction vessel Antigen within the reaction solution competes w ith antigen on the reaction vessel Therefore, the more antigen there is in the reaction solution, the less antibody w ill bind to the reaction vessel Thus, the amount of antibody that binds to the reaction vessel is inv ersely proportional to the amount of antigen that was present in the reaction solution Hav ing determined the amount of antibody that has been bound to each reaction v essel, a standard cur e is prepared in which the amount of antibody bound is plotted vs. the amount of antigen present in each reference sample. The results obtained with dilutions of the test sample are compared to this standard curv e, and the amount of antigen present within the test sample is readily determined with reference to the standard curve The following non-limiting examples further illustrate the present invention. EXAMPLES Reagents and Conditions: Chemicals" Bovine serum albumin (BS Λ). alkaline phosphatase substrate ( λBTS). and TRIZMA base were from Sigma (St. Louis. MI. USA) Al(OH)-; gel (alhydrogel ) was from Superfos (\ edbaek. Denmark) Tw een-20 w as from BioRad (Hercules. CA. USA) \aHCC . Na 2 CCA and MgCl; • 6H 2 0 were from JTBaker (Philhpsburg, NJ, USA) NaCl. \a : HP0 4 . KH : P0 4 . NaOH w ere from V R (Pittsburgh. PA, USA) Antibodies: Monoclonal antibodies were made by immunizing Balb/c female mice three times intrapeπtoneally at approximately one month intervals and one time intravenously three days prior to spleen harvesting. Spleen cells were fused using the PEG cell fusion method descπbed by Lane ( 1 1 ) to the muπne myeloma cell line P3X63Ag8.653 (ATCC CR 1580). Hybridoma cells were selected using HAT media (hypoxanthine, armnopteπn, and thymidine) and screened by ELISA for IgG antibodies to the specific protein of interest Alkaline phosphatase comugated goat anti-mouse IgG antibodies were from Cappel (IC\. Aurora. Ohio, USA). Buffers Filters (0.22μm pore size) and Milh Q plus water puπfication sy stem for puπfied w ater w ere from Milhpore (Boston. MA, USA) Phosphate buffered saline ( PBS) at 0.01 M phosphate and 0.85° o NaCl with a pH 7.2 ± 0 1 was prepared by the PMC Bioserv ices Department Coating buffer for the immunoassay was prepared from NaHCO-, and \a;C0 3 to make a final concentration of 0.015 M NaCO-. and 0.035 M NaHCO-, at pH 9 6 ± 0 1 Blocking buffer was 2.0% BSA in PBS. Washing buffer was 0.05% Tween-20 in PBS Diluent used for sample dilutions was 1 0% BSA in PBS. Alkaline phosphatase substrate buffer w as 0.05M NaC0 3 . 1.0 mM MgCl; at pH of 9.8 ± 0.1. Enzymatic stopping solution is 5 N NaOH TRIS buffered saline (TBS) buffers at 1.0 M and 25 mM TRIS with 0.85% NaCl at pH 7 0 __= 0 1 were prepared by the PMC Product Dev elopment Department Antigens Vanous strains of recombinant pneumococcal surface protein, proteins ( 1, 2. 3. 4. 5), w ere prepared by the P 1C Product Dev elopment Department. These antigens were prepared under GLP GMP conditions The recombinant protein was absorbed to 520 ug of aluminum/mL of 25 m\I TBS by batch incubation The aluminum w as in the form of aluminum hydroxide (alhyrdogel. Supertos™) Example 1 Competitive EIA Procedure Following a four plate randomization scheme, reference and vaccine test samples were prepared and tested Initially, four Immulon IV assay plates (Dynatech, Chantilly, VA. USA) were coated with non-adsorbed PspA antigen diluted in coating buffer by adding 100 μL of the diluted antigen to each well of the plate The assay coated plates were then sealed with a plate sealer (Dynatech. Chantilly, \ \. L SA) and ere incubated for 16-24 hr at 4 Q C Simultaneously. an assay dilution plate was prepared \ aπous concentrations of the reference and v accine test samples were prepared The reference, recombinant protein also absorbed to 520 μg ot aluminum/mL of 25 mM TBS. was diluted to an initial concentration with assay diluent and seπally diluted 2-fold down the column ot the dilution plate The adsorbed v accine test sample was also diluted to the same initial concentration as the reference mateπal and seπally diluted 2- fold down the column of the dilution plate. Both the reference and the vaccine test sample w ere prepared in duplicate on each of the four assay dilution plates The diluted reference and v accine test samples w ere incubated overnight at 4°C with an equal volume ot an appropπately diluted, antigen specific, monoclonal antibody Positiv e control consisted of the appropπately diluted. antigen specific, monoclonal antibody only The negative control (or background control) consisted of assay diluent only Both were incubated in replicates at a single concentration in the assay dilution plate The next day . the assay coated plates were remov ed from 4°C and w ashed six times with w ashing butter Then, the reference monoclonal mixtures and the accine test sample monoclonal mixtures were transferred from the assay dilution plate to the washed assay coated plate This was repeated tour times, corresponding to each set ot the tour plates due to the randomization scheme The assay coated plates were then sealed and incubated for 2 hr at 37 ± 2°C The plates were washed six times with w ashing buffer and then incubated with appropπately diluted alkaline phosphatase conjugated goat anti-mouse IgG for 1 5 hr at 37 ± 2°C The alkaline phosphatase conjugated goat anti-mouse IgG was diluted to an appropπate concentration with assay diluent After which, the plates were washed with a seπes of washes one final time Next, an appropπate concentration of alkaline phosphatase substrate (p- Nitrophenyl pho phate or p-NPP) diluted with substrate butter w as added to all of the wells The enzymatic reaction was stopped after 60-90 min with 10 μL ot 5\ NaOH and the bound specific monoclonal was detected by monitoπng the resulting enzyme product at 405 nm Absorbance measurements w ere performed on a SpectraMax™ microtiter plate reader (Molecular Dev ices. Menlo Park. CA. USA) The antibody bound to the assay coated plate is inversely proportional to the concentration of antigen present in the reference or the v accine test sample Example 2 Quantitation of Psp 4 Adsorbed to Aluminum droxide Recombinant pneumococcal surface protein (Psp A) w as expressed and adsorbed to aluminum hydroxide. The protein w as formulated with 520 ug mL aluminum hydroxide to 10. 20, 50, and 100 μg mL total protein, as detected by Lo ry These samples are referred to as vaccine test samples from this point forth The vaccine test samples were tested for antigen content by competitive EIA using an aluminum hydroxide adsorbed reference mateπal The reference and v accine test samples w ere diluted to the same initial starting concentration ot 1 0 μg/mL The assay s were performed as prev iously descπbed in Example 1 To assess the assay for precision, accuracy and parallelism, a seπes of four independent runs using a tour plate randomization scheme w as performed The antigenic content was calculated tor the tour v accine test samples by interpolating a concentration from the reference and then correcting for dilution The calculated antigenic content was then statistically analyzed tor precision, accuracy, and parallelism Precision is defined as the closeness of agreement, or the degree of scatter, among a seπes of measurements obtained from multiple sampling of the same homogeneous sample under prescπbed conditions In the analysis performed, precision was calculated by transforming the data into the natural log and using equation 1 Equation 1 Precision = (e " - l) x l00% Accuracy was also assessed and reported as percent recoverv This is defined as the average of the interpolated, dilution-corrected concentrations of the vaccine test sample relative to that of the total protein value determined by Lowry The percent recovery is calculated by equation 2 below Equation 2 % Recovery = 100% \ (Calculated antigen content Lowry value) Lastly, parallelism was determined using the dilution effect equation depicted by equation 3 Equation 3 Dilution Effect = 100 χ 2' " " " ' - 1 . where b, is the slope of the test sample and b, is the slope ot the reterence The percent dilution effect provides an indication ot parallelism between the dilution seπes ot the reference as it compares to the dilution seπes of the vaccine test sample(s) The effect of dilution can be estimated as the percent dev tation at 2-fold dilution of the test article. using the slopes of the reference and the vaccine test sample profiles. The assay which exhibits perfect parallelism has a dilution effect value of 0%. Table 1 represents the statistical summary of the calculated antigen concentration values obtained for the four vaccine test samples tested. Table 1 Summary of results from 4 independent runs using the four plate randomization scheme with dilution points for the reference and each vaccine test sample, n-128 Precision: Precision was assessed for the 10. 20. 50. and 100 us L formulated vaccine test samples over different days with different assay buffers. Table 1 above lists the average precision value as the percent relativ e standard deviation and shows that the % RSD obtained over the different days ranged from 13.3 to 19.7%. Howev er, table 2 further defines the precision for each day w ith precision alues range from 6.35 - 20.9%. Table 2 Precision values obtained over four different days. Accuracy: Results generated by the competitive EIA method were compared to dilution corrected Lowry values. Since the material being tested is adsorbed to aluminum hydroxide, it is difficult to measure total protein content after protein absorption. Also, the Lowry is providing a total protein concentration value which is not specific to the antigen in question. How ev er, the dilution coπected Lowry values of the pre-adsorbed material is the only v alue av ailable for comparison at the time of assay design. Table 3 summanzes the accuracy values obtain for each of the days independently and as an o erall measure. Table 3 Accuracy values obtained during testing The accuracy values range from 80.7 - 109% and are consider acceptable for an immunological procedure. Parallelism: To assess parallelism, the log of the OD v alues are plotted against the log of the protein concentration. The slopes of the reference and the v accine test samples are compared for percent bias using equation 3 above. Table 4 contains the percent dilution effect obtained for each run and over all the runs. Table 4 Percent Dilution Effect (percent bias) for each ine test sample as a measure ot parallelism. Since 0% dilution effect represents perfect parallelism, the parallelism results shown in table 4 are excellent, ranging from -2.60 to 2.93 % bias over a concentration range of 0 ns - l 0 μg'mL. Specificity Selectiv ity To deteπrune if the aluminum hydroxide interferes in the assay, a vaπety of expeπments were performed. One expeπment tested the nonspecific binding of the monoclonal antibody to aluminum hydroxide duπng the incubation step. Figure 1 depicts the results There is no difference between the 'blank' samples, buffer only, and the 'control' samples, those containing aluminum hydroxide without adsorbed protein. In a separate experiment, vaπous proteins were adsorbed to the aluminum hydroxide and tested in the assay Only the vaccine test sample coπesponding the specific monoclonal in the assay produced a positive response (figure 2) All others produced unchanging OD v alues independent of dilution, exhibiting a similar profile to the blank depicted in figure 1 \ third expeπment was performed to determine the effect of aluminum hy droxide concentration on the calculated antigenicity values There are no apparent differences in the competitive EIA calculated values for samples containing different amounts ot aluminum hydroxide but a constant amount of protein. Repeatability Not only are the conditions repeatable for different operators on different day with different instruments but two other independent antigens hav e also produced similar precision. accuracy, hneaπty. and specificity results using the same concept descπbed here. REFERENCES 1 Edelman, R. Vaccine adjuvants Rev Infect Dis 1980. 2, 370-83 2 W orld Health Organization Manual for the Production and Control of Toxoid BLG LNDP 77.2. Rev 1. WHO. Geneva. Sw iterland. 1977 3. Shirodkar. S. Hutchinson. RL. Perry, DL. White, JL. and Hem. SL. Aluminum compounds used as adjuvants in vaccines. Pharm Res 1 90. 7. 1282-88 4. Al-Shakhshir, R, Regnier, F, White. JL. and Hem, SL Effect of protein adsorption on the surface charge characteπstics of aluminum-containing adjuvants. Vaccine. 1994, 12. 472-74. 5. Rinella, JV, Jr., White, JL, and Hem, SL. Effects of anions on model aluminum adjuv ant containing vaccines. J Colloid Interface Sci. 1995, 172, 121-30. 6 Katz, J Desorption of porcine parvovirus from aluminum hydroxide adjuvant with subsequent viral immunoassay or hemagglutination assay Vetennan Research Communications 1987, 11, 83-92. 7. Rinella, JV, Workman, RF, Hermodson. MA, White. JL. and Hem. SL Elutabi ty of proteins from aluminum-containing vaccine adjuvants by treatment w ith surfactants J Colloid Interface Sa. 1998. 197. 48-56. S Visser, N. and Woortmeyer. R Quantification, characteπzation and safetytestmg of foot-and- mouth disease virus antigens eluted from alhydrogel v accines. Biol Stand 1981. 50. 277-83. 9. Harlow, E and Lane, D. Using Antibodies: A Laboratory Manual. Cold Spπng Harbor Laboratory Press. Cold Spring Harbor, NY. 1999. 10. Houen. G and Koch. C. A non-denatuπng enzyme linked immunosorbent assay with protein preadsorbed onto aluminum hydroxide. J Immunol Methods. 1997. 200. 99- 105. 1. Lane, R.D. A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybπdomas. J Immunol Methods 1985, 81. 223-228.

Claims: We Claim: 1. A competitive immunoassay method for quantitating antigen in a v accine composition containing aluminum salts, the method comprising: a) binding antigen that has not been adsorbed to an aluminum salt to a plurality of identical reaction vessels; b) adsorbing a known quantity of reference antigen to an aluminum salt to fonn a reference standard: c) prepaπng dilutions of test samples and reference standard of antigen in the presence of antibody to the antigen to form a seπes of individual reaction mixtures: d) adding each reaction mixture to an individual reaction vessel and incubating for a peπod of sev eral hours to allow for the formation of antigen-antibody complexes. e ) remov ing reaction mixtures from the reaction vessels and washing the reaction essels to eliminate antigen-antibody complexes in solution; and f) determining the amount of antibody which has bound to each reaction v essel. 2. The method of claim 1 , wherein the aluminum salt is aluminum hydroxide. 3. The method of claim 1 , wherein the antigen is PspA. 4. The method of claim 1. herein the antibody is monoclonal antibody to PspA.

Inventors: DOUGHERTY CRISTY S, DOUGHERTY, CRISTY, S.
Applicants: AVENTIS PASTEUR, DOUGHERTY CRISTY S, DOUGHERTY, CRISTY, S.
Application year: 2001
Main classification: G01N33
IPC codes: G01N33