FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Method for Evaluation of Multiple Antibodies or Multiple Antigens in a
Single Sample"
Serum Institute of India Ltd., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

Method for evaluation of multiple antibodies or multiple antigens in a single sample.
BACKGROUND OF THE INVENTION
Evaluation of the vaccine efficacy requires estimation of post vaccination antibody titers in vaccinated individuals. The quantitation of antibody titers to various antigens is required after immunization with a multivalent vaccine formulation e.g. Pneumococcal conjugated/unconjugated vaccines, DPTHepBHib vaccine etc.
The quantitation of serotype specific polysaccharide antigen, as in-process analysis is required during manufacturing of multivalent vaccine e.g. Pneumococcal conjugated/unconjugated vaccines, DPTHepBHib vaccine etc .Several microbes e.g. Streptococcus sp. , Leptospira sp. etc have more than one serotypes which have similar antigenic structures.
For antibody titration and antigen detection, ELISA has been conventionally utilized. ELISA methods provide the benefit of relatively high sensitivity, but have the disadvantage of taking a relatively long time to execute to obtain maximum sensitivity. ELISA tests also have other disadvantages such as instability of the linked enzyme, relatively expensive substrates and requiring multiple steps to execute, all of which lead to relatively high costs for ELISA tests. Performing ELISAs for multiple antigens or multiple antibodies in vaccine evaluation is a time consuming and laborious job.

There is a need to develop more efficient immunoassays.
US 7659085 claims assays that are useful for the rapid and simultaneous detection of multiple different antigens and antibodies. The assays included fluorescent labels of multiple wavelengths or intensities, which were used to label the antigens and antibodies directly and to label beads coated with molecules specific for the antigen or antibody. Wherein the detection of a fluorescence shift indicated the presence or identity of the antigen or antibody in the sample.
Raymond E. Biagini et al discusses 25-plex PnPS fluorescent covalent microsphere immunoassay (FCMIA) for measuring all 23 components of PPV23 simultaneously as well as C-PS and a nonvaccine PnPs. Preadsorption of serum with C-PS allowed for the measurement of PnPS-specific antibodies without the contribution of anti-C-PS antibodies. See " Method for Simultaneous Measurement of Antibodies to 23 Pneumococcal Capsular Polysaccharides " Clin Diagn Lab Immunol. 2003 September; 10(5): 744-750.
A multiplex microbead immunoassay (MMIA) has been previously described for the simultaneous detection of antibodies to all six viruses in single serum samples of Monkeys, based on individually identifiable, fluorescent microbead sets, where each bead set was coated with antigens from a purified preparation of a specific virus.See Imran H. Khan et al

"Simultaneous Detection of Antibodies to Six Nonhuman-Primate Viruses by Multiplex Microbead Immunoassay", Clinical and Vaccine Immunology, Jan. 2006, p. 45-52
Genevieve G. Fouda et al has previously disclosed SAT (Suspension Array Technology) multiplex system for measuring antibodies against nine malarial vaccine candidate antigens wherein various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Cameroonian adults were screened by SAT in mono-and multiplex formats. See "Multiplex Assay for Simultaneous Measurement of Antibodies to Multiple Plasmodium falciparum Antigens"Clin Vaccine Immunol. 2006 December; 13(12): 1307-1313.
Gageldonk et al has previously discussed a rapid and simple microsphere-based multiplex assay (pentaplex) for the Quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA),Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin, to increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials.See "Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus"Journal of Immunological Methods,Volume 335, Issues 1-2, 1 June 2008, Pages 79-89..
Rocio et al has described a direct binding electro

chemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well. See "Optimization and Validation of a Multiplex, Electro chemiluminescence-Based Detection Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti pneumococcal Antibodies in Human Serum" Clinical and Vaccine Immunology, March 2009, p. 387-396, Vol. 16, No. 3
Groen J et al describes development of Plexus™ Parasitic Multi-Analyte Diagnostics assay based on the Luminex xMAP™ system see^A multiplex microsphere-based assay for the simultaneous detection of C. parvum, E. histolytica and G. Iambiia antigen in human faecal sample" Abstract number: 1733_57,17 th European Congress of Clinical Microbiology and Infectious Diseases ,ICC, Munich, Germany, 31 Mar - 04 Apr 2007.
Several methods of conjugating polysaccharides to microspheres have been reported earlier. Zielen et al discusses use of EDC to couple pneumococcal polysaccharides to the beads with amino groups ;J Immunol Methods; 1996; 193 pgs.1-7.Conjugation of polysaccharides to microspheres using the poly-L-Lysine method has been reported earlier by Pickering et al(2002).Further a method of coupling a polysaccharide to a microsphere or biomolecule comprising the step of reacting an activated polysaccharide with a microsphere (primarily coupled with a linker like ADH) or biomolecule has also been described, see Biagini et al S. Clinical and Diagnostic Laboratory Immunology

Vol.10 2003.No.5 pgs.744-750. US 2005/0118199 claims a method for conjugating polysaccharides to microspheres or biomolecules by using DMTMM [4-{4,6-dimethoxy[1,3,5] triazin-2-yl)-4-methyl-morpholinium Chloride.
A major disadvantage of the multiplex luminex technology, however, it is the requirement that PnPs be covalently attached to the microspheres. This is accomplished by chemical alteration of polysaccharide e.g. by introduction of primary amino groups into the polysaccharide structure and subsequently coupling the amino-modified polysaccharides to the carboxyl groups attached to the microspheres. Chemical alteration of the polysaccharide structure raises the possibility that critical type-specific epitopes may be altered or destroyed or that new epitopes might be introduced. For example, Biagini and coworkers could not demonstrate homologous inhibition for 9 of 24 serotypes coupled to Luminex microspheres by the periodate method. This suggests that the type-specific epitopes were destroyed during the coupling procedure.
According to the available literature ,coupling procedures may be difficult to standardize for all 23 serotypes and to control from lot to lot. Heterologous inhibition of less than 30% for nine pneumococcal serotypes has been disclosed by Gouri et al " Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes;Journal of immunological

methods;2005;296;135-147.
Prior art fails to address any variations in assay results due to cross reactivity when utilized for all the 23 serotypes.
Surprisingly we have found that when a mixture of antigens is coupled to the beads by using different conjugation chemistries instead of a single chemistry, a uniform correlation is obtained.
Based on the type of chemistry used for the conjugation of each antigen to the beads, minor or major changes in the epitopic configuration of the antigen can occur. This possible change in epitopic structures can lead to cross-reactivity with relevant epitopes in other similar antigens or also the sufficient homologous reactivity may not be detected for various antigens. We have found that , a best method of conjugation of the various candidate antigens to the beads may be optimized which can give a maximum homologous reaction and minimum heterologous reaction during the multiplexed assay.
We have developed a novel multiplex system based on individually identifiable, carboxylated polystyrene microsphere sets, where each microsphere set can be conjugated with a defined candidate antigen. The conjugated bead sets can be mixed to enable the detection of multiple antibodies or multiple antigens in one sample.
SUMMARY OF THE INVENTION

The invention relates to a multiplex assay for simultaneous detection of multiple antibodies or multiple antigens in single sample, wherein carboxylated polystyrene microsphere sets are conjugated with a defined candidate antigens by an optimized conjugation method and are mixed to enable the detection of multiple antibodies or multiple antigens, respectively.The instant method results in less than 20% heterologous inhibition.
DESCRIPTION OF THE DRAWINGS
Fig. 1 Comparison of IgG titre and concentration by
multiplexed assay (serotype 14)
Fig. 2 Comparison of IgG titre and concentration by
multiplexed assay (serotype 19A)
Fig. 3 Comparison of multiplexed assay with conventional
ELISA(serotype 1)
Fig. 4 Comparison of multiplexed assay with conventional
ELISA(serotype 23)
Fig. 5 Antigen identification and quantification(serotype 4)
Fig. 6 Antigen identification and quantification(serotype 14)
Fig. 7 Assay specificity for antigen identification and
quantification(serotype 5)
Fig. 8 Assay specificity for antigen identification and
quantification(serotype 14)
Fig. 9 Assay specificity for antigen identification and
quantification(serotype 19F)
Definitions:

According to the instant invention said "microsphere" is a small spherical, or roughly spherical, particle having a diameter less than about 1000 micrometers {e.g., less than about 100 micrometers, optionally less than about 10 micrometers/ ; comprises a polymer selected from the group consisting of a polystyrene, a polyester, a polyether, a polyolefin, a polyalkylene oxide, a polyamide, a polyacrylate, a polymethacrylate and a polyurethane, or a mixture thereof and can optionally have various surface chemistries selected from a group of free carboxylic acid, amine, or hydrazide groups, among many others.
Many such microspheres for use in conjugates and methods of the present invention are commercially available. They may be purchased from Luminex Corporation {Austin, Tex.) (xMAP™). xMAP™ microspheres are 5.6 um in diameter and composed of polystyrene, divinylbenzene and methacrylic acid, which provides surface carboxyiate functionality for covalent attachment of polysaccharides and biomolecules. The microspheres may be dyed with red- and/or infrared-emitting fluorochromes. By proportioning the concentrations of each fluorochrome, spectrally addressable microsphere sets may be obtained. When the microsphere sets are mixed and analyzed using the Luminex instrument, each set can be identified and classified by a distinct fluorescence signature pattern.
The term bacterial polysaccharide refers to polysaccharide

isolated from a bacterium selected from the group consisting
of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia
trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae,
Staphylococcus spp., Staphylococcus aureus, Streptococcus
spp., Streptococcus pyogenes, Streptococcus pneumoniae,
Streptococcus viridans, Enterococcusfaecalis, Neisseria
meningitidis, Neisseria gonorrhoeae, Bacillus anthracis,
Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella
pestis, Pseudomonas aeruginosa, Campylobacter spp.,
Campylobacter jejuni, Clostridium spp., Clostridium difficile,
Mycobacterium spp., Mycobacterium tuberculosis, Treponema
spp./ Borrelia spp., Borrelia burgdorferi, Leptospira spp.,
Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella
pertussis, Bordetella parapertussis, Bordetella
bronchiseptics, Hemophilus influenzae, Escherichia coli, Shigella spp., Erlichia spp., and Rickettsia spp. Detailed Description:
The method of the instant invention for simultaneous detection of multiple antibodies in single sample ,comprises following steps:
a) conjugating microspheres to serotype specific polysaccharide by an optimal method;
b) Adding the conjugated microspheres to microwell plate;
c) Adding standard and test serum sample to microwell plate;
d) Incubating the mixture for atleast lhr at 37°C with

shaking;
e) Adding Phycoerythrin-conjugated anti-analyte antibody as fluorescent reporter.
f) Incubating the mixture for 30 min at 37°C with shaking;
g) Measuring fluorescence emission of reporter and thereby calculating antibody concentration.
Also said method of the instant invention for simultaneous detection of multiple antigens in single sample ,comprises following steps:
a) Preadsorption of standard and unknown polysaccharide containing samples to monoclonal antibodies or known sera.
b) conjugating microspheres to serotype specific polysaccharide by an optimal method;

c) Adding the conjugated microspheres to microwell plate;
d) Adding preadsorbed mixture to microwell plate;

e) Incubating the mixture for Ihr at 37°C with shaking;
f) Adding Phycoerythrin-conjugated anti-analyte antibody as fluorescent reporter.
g) Incubating the mixture for atleast 30 mim at 37 C with
shaking;
h) Measuring fluorescence emission of reporter and thereby calculating antibody concentration.
The said candidate antigen coupled with microsphere ,can be a capsular polysaccharide derived from any of the serotypes of Streptococcus pneumoniae selected from: 1, 2, 3, 4,5,6A , 6B, IF, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20,

22F, 23F, and 33F.
A preferred embodiment of this invention is that the multiplex assay of the instant invention is utilized for quantitation of antibody or antigen wherein minimum heterologous inhibition (cross-reactivity) of less than 20% for all serotypes and homologous inhibition greater than 8 0% can be obtained, wherein the polysaccharide coupled microspheres have been prepared by an optimal method without altering the critical epitopes of polysaccharide.
One aspect of the instant invention is that conjugation of candidate antigens to beads can be done by a chemistry suitable for individual capsular polysaccharide, said chemistry can be selected from a group of Poly-L-Lysine, EDC-ADHf DMTMM-NH2 and DMTMM-COOH.
In each of the embodiments of the invention, fluorescence may be measured through the use of a flow cytometer or a bead array reader. For example, a BioPlex-100, a BioPlex-200, a Luminex-100, or a Luminex-200 bead array reader may be used.
The BioPlex reader is designed for immunosorption experiments, where it reads fluorescence from phycoerythrin-conjugated anti-analyte antibodies attached to the 5.6-um plastic beads coated with analyte-capturing antibodies. The beads flow through the capillary fluorescence chamber.
In general, this scheme is similar to a most basic flow cytometry experiment, where calibrated micro beads play the

role of the cells. However, there is an important difference. The beads used in the BioPlex are coded via their intrinsic red/infrared fluorescence in such a way that the ratio of the red and infrared components determines the code number of the bead. This allows discrimination of signals from a multitude (up to 500) of beads with different codes, which provides for the exceptional multiplexing capacity of the Luminex technology.
Following are the advantageous features of the instant invention:
1. This method of antibody or antigen quantitation,particularly with respect to pneumococcal polysaccharides is rapid, taking no longer than a few hours (normally, 1.5-3 hrs) ,whereas conventional ELISA requires 48 hrs.
2. The method is appropriate for a) multiple antibody quantitation from sera and in-process polysaccharide samples, b) multiple antigen detection by competitive inhibition.
3. The assay is inexpensive, using, for example, the rugged BioPlex or similar bead array platform.
Examples:
Example 1: Antibody quantitation by utilizing a single
"antigen mircosphere conjugation method" as disclosed in prior
art.
Conjugated microspheres were prepared by utilizing PLL

chemistry and the 14-plex assay was put for evaluation specificity in the assay. The serum sample was adsorbed with homologous and heterologous pneumococcal polysaccharides and then incubated with 14-plex microspheres. The percentage inhibition of antibody signal after adsorption as compared to unadsorbed control was determined in the assay .
Table 1:

Homologous inhibition Heterologous inhibition(cross reactivity)

The results indicate that there was more than 80% homologous inhibition whereas, heterologous inhibition which is an indication of cross reactivity was found to be more than 30% .
Example 2: Preferred chemistries for coupling of various

antigens to carboxylated microspheres
A single chemistry results in more than 30% heterologous inhibition (refer example 1) which is not desired. Hence four different chemistries{Poly-L-Lysine, EDC-ADH, DMTMM-NH2 and DMTMM-COOH) were used for coupling of each of pneumococcal polysaccharides to the carboxylated microspheres to identify the optimal method that would result in least cross-reaction and maximum homologous reaction .
Methods for Coupling of polysaccharide to the carboxylated microspheres:
Various methods of coupling of the polysaccharides to the microspheres were investigated. Briefly, PLL-method involved the activation of polysaccharide with cyanuric chloride followed by polysaccharide modification using poly-L-Lysine(PLL). The NH2 group of lysine on the modified polysaccharide was then used for conjugation with COOH- group on the microspheres using EDC and sulpho-NHS. DMTMM-COOH method involved the activation of polysaccharide using DMTMM and conjugation with COOH- group on the activated microspheres. DMTMM-NH2 method involved modification of microsphere using adipic acid dihydrazide(ADH) to generate NH2 group followed by conjugation with reactive- group on the polysaccharide using DMTMM. EDC-ADH method involved modification of microsphere using adipic acid dihydrazide(ADH) to generate NH2 group followed by conjugation with reactive-group on the polysaccharide using EDC.

Each of the bead set was used for specificity check using inhibition assay and homologous and heterologous inhibitions were determined for each method. Theteafter , a mixture of beads coupled with method most suitable for a particular polysaccharide was used in the specificity assay and following results were obtained.
Table2:

1 4 5 6B 7F 9V 14 18C 19A 19F 23F
Type 1 1:100 97 -4 2 -10 -9 -3 0 " -6 -11 -8 -5
Type 1 1:400 98 -1 7 -6 -3 -3 -1 -5 -1 -1 3
Type 4 1:100 3 91 7 -2 1 4 0 6 1 3 7
Type 4 1:400 10 88 6 2 2 1 1 3 -1 3 4
Type 5 1:100 5 4 99 -2 -2 0 0 2 -3 1 -2
Type 5 1:400 11 6 99 2 -3 2 -1 -1 -2 0 7
Type 6B 1:100 4 13 2 95 -4 2 0 1 -7 -2 0
Type 6B 1:400 10 19 1 95 -3 2 -1 1 -2 1 4
Type 7F 1:100 4 6 10 1 91 0 -1 2 0 2 5
Type 7F 1:400 2 5 -3 5 92 1 1 -1 2 7 4
Type 9V 1:100 -2 10 5 -4 0 90 0 0 -3 0 -10
Type 9V 1:400 7 12 -2 2 7 91 3 -1 0 2 1
Type 14 1:100 1 8 5 -1 1 0 96 5 -4 -3 5
Type 14 1:400 1 2 -4 -2 -6 -2 99 5 -4 -3 -10
Type 18C 1:100 i 8 8 1 4 4 0 87 -2 3 7
Type 18C 1:400 6 5 0 3 5 3 0 80 1 5 -1
Type 19A 1:100 2 6 12 0 1 4 0 8 93 36 10
Type 19A 1:400 4 2 -2 -2 1 7 1 3 91 45 -5
Type 19F 1:100 7 4 17 5 6 4 1 4 76 95 -9
Type 19F 1:400 8 5 14 5 11 5 5 3 76 93 3
Type 23F 1:100 2 5 9 -4 -2 2 1 ' 1 -4 2 93
Type 23F 1:400 2 2 -1 3 -5 0 1 ' 3 0 3 92
The results indicate a minimum heterologous inhibition of less than 20% for all serotypes and more than 80% homologous inhibition.
Table 3:Preferred methods were evaluated for 23 serotypes:

Serotype Preferred methods
1 DN or EA or DC
2 PLL or DN
3 DC or PLL
16

4
EA or DN or DC
5 PLL or DN or DC
6A DN or PLL
6B DC or DN or EA
7F PLL or EA or DC
8 DCorDN
9N PLLorEA
9V DC or PLL or DN
10A EAorPLL
11A PLL or DN
12F PLLorEA
14 PLL or DN or DC
15B EAorDC
17F EA or PLL
18C EAorDNorPLL
19A DN or EAorDC
19F DC or PLL or DN
20 EA or DC
23F DN or EA or DC
33F PLL or DN
DC : DMTMM-COOH DN: DMTMM-NH2
EA : EDC-ADH PLL: Poly-L-Lysine
Example 3: Multiplexed Quantitation of IgGs in pneumococcal quality control sera
Using the above combination the assay accuracy was confirmed by performing monoplex assays for various serotypes and a multiplexed assay and the percent coefficient of variation(Percent CV) were calculated between the concentrations of IgG in two of the quality control sera obtained from the two assay formats. The antibody quantitation comprised of following steps : 50ul of diluted suspension of microspheres coupled with serotype specific antigen was added in wells of filter plate (4000 microspheres/well); aspiration was done and 50 ul of different dilutions of standard serum and test serum were added; the plate was incubated for 60

min, at 37°C with shaking; washing the plate three times with wash buffer and 50 ul of diluted. PE conjugate added to wells. Plate was incubated for 30 min, 37°C with shaking; plate was washed 3-times and 130ul sheath fluid was added;plate was read in BioPlex- 200 system ;standard curve was prepared from median fluorescence value(MFI) of various standards and quantity of IgG in unknown sample was calculated from the standard curve equation.
Table 4 :Comparison of monoplex vs multiplexed assay (%CV)

Percent CV
Serotype HQC - 768 HQC - 770
1 3.6 10.7
4 6.2 3.3
5 4.1 4.3
6B 9.3 10.8
7F 7.4 12.4
9V 3.3 4.5
14 8.9 10.5
18C 3.8 5.5
19A 8.8 11.3
19F 8.3 6.2
23F 6.3 11.9
The results indicate that the variation with respect to
monoplex and multiplex assay format was within 15 % for all
the serotypes.
Example 4: Comparison of IgG titre and concentration by-
multiplexed assay
The multiplexed assay was also qualified by comparison of IgG
concentration and titer results for various serotypes. The IgG

concentrations and antibody titers obtained by standard formula (reciprocal of serum dilution showing MFI above the defined cut-off) had good amount of correlation as evidenced in the following figures with a correlation coefficient of more than 0.93.
Example 5: Comparison of multiplexed assay with conventional
ELISA
The multiplexed assay was verified by comparison of IgG concentration and titer results when compared with those obtained from conventional ELISA.
The results indicate that the IgG concentrations and antibody titers obtained from two assay format(Multiplex & ELISA) had good amount of correlation as evidenced in the above given figures with a correlation coefficient of more than 0.95. Further ELISA method was found to be time consuming and required approximately 48 hrs for completion, whereas the multiplex assay format required only 3 hrs for completion.
Example 6: Screening of mouse hybridoma by multiplexed assay
The multiplexed assay was used for screening of mouse hybridomas secreting IgGs against various pneumococcal serotypes and CRM197.

Table 5:

The results presented in the above table for checking the specificity of purified monoclonal antibody from the hybridoma culture supernatant after limiting dilution, show a high amount of specificity of the multiplexed assay except minor cross-reactivity of Type 1 monoclonal antibody with serotype 20.
Example 7: Antigen identification and quantification

Similar to the antibody identification and quantitation(as disclosed in above examples) in sera, the multiplexed assay was used in the antigen identification and quantitation by utilizing a competitive inhibition assay. The method comprised of the following steps: 100µl of different dilutions of polysaccharide standards and lOOµl of different dilutions of samples were incubated with lOOul of optimized dilution of respective monoclonal/ polyclonal antibody in dark at 37°C for 1 hour in shaker incubator at 150rpm; filter plate was prewetted with 50ul of luminex buffer; 50ul of polysaccharide coated beads (4000beads/50ul/well) were added into the each well of filter plate;plate was vacuum filtered; 50ul of preadsorbed monoclonal/ polyclonal antibody was added into the respective well of 96 well filter plate in duplicate; plate was incubated in dark at 37 Deg C for 1 hour in shaker incubator at 150rpm; further steps were similar to that for antibody quantitation method; plate was read in the Bio-plex suspension array reader; standard curve was prepared and unknown polysaccharide was calculated based on standard curve equation. The following are the representative standard curves for 2 serotypes.
Example 8: Assay specificity for Antigen identification and quantification
The beads coupled with pneumococcal polysaccharides with preferred method of coupling were tested for specificity in the inhibition assay with homologous and heterologous

polysaccharides and the representative results are presented below.
The results indicated that there was only homologous inhibition observed, whereas, least amount of reduction in MFI was observed due to heterologous inhibition confirming the specificity of the assay.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are/therefore intended to be embraced therein.

We claim
1. A method for simultaneously detecting the presence of multiple anti-polysaccharide antibodies in a single test sample comprising the steps of (a)preparing a microsphere -polysaccharide conjugate; (b)mixing of polysaccharide coupled microspheres ;(c)contacting a sample containing said anti-polysaccharide antibodies with mixture of polysaccharide coupled microspheres; and (d) measuring the amount of anti-polysaccharide antibody bound to said microsphere-polysaccharide conjugate.
2. A method for simultaneously detecting the presence of multiple polysaccharide antigens in a single test sample comprising the steps of: (a) Preadsorption of standard and unknown polysaccharide containing samples to monoclonal antibodies or known sera ;(b)preparing a microsphere polysaccharide conjugate; (c}mixing of polysaccharide coupled microspheres ; (d)contacting a pre-adsorbed sample containing said anti-polysaccharide antibody with mixture of polysaccharide coupled microspheres; and e) measuring the amount of anti-polysaccharide antibody bound to said microsphere-polysaccharide conjugate.
3. The method according to claim 1 or 2, wherein specificity
studies of the method indicate greater than 8 0% homologous
inhibition and less than 20% heterologous inhibition for all
serogroups without alteration of critical serotype specific
polysaccharide epitopes.

4. The method according to claim 3, wherein the minimum
heterologous inhibition is obtained by coupling microsphere to
polysaccharide by an optimal serotype specific method selected
from a group of Poly-L-Lysine, CDAP, EDC-ADH, DMTMM-NH2 and
DMTMM-COGH.
5. The method of claim 1 or 2 , wherein said polysaccharide is
a bacterial polysaccharide.
6. The method of claim 5, wherein said bacterial
polysaccharide is isolated from a bacterium selected from the
group consisting of Helicobacter pylori, Chlamydia pneumoniae,
Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma
pneumoniae, Staphylococcus spp., Staphylococcus aureus,
Streptococcus spp., Streptococcus pyogenes, Streptococcus
pneumoniae, Streptococcus viridans, Enterococcusfaecalis,
Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus
anthracis, Salmonella spp., Salmonella typhi, Vibrio cholera,
Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter
spp., Campylobacter jejuni, Clostridium spp., Clostridium
difficile, Mycobacterium spp., Mycobacterium tuberculosis,
Treponema spp., Borrelia spp., Borrelia burgdorferi,
Leptospira spp., Hemophilus ducreyi, Corynebacterium
diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Erlichia spp., and Rickettsia spp.
7. The method of claim 6 wherein said bacterial polysaccharide is a capsular polysaccharide isolated from Streptococcus

pneumoniae.
8. The method of claim 7, wherein said capsular polysaccharide
is of a serotype selected from the group consisting of: 1, 2,
3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,
18C, 19A, 19F, 20, 22F, 23F, and 33F.
9. The method of claim 5, wherein said N.meningitidis
polysaccharide is of a serotype selected from the group
consisting of Men A,C,Y and W135.
10. The method of claim 1 or 2 ,wherein said microsphere
comprises' a polymer selected from the group consisting of a
polystyrene, a polyester, a polyether, a polyolefin, a
polyalkylene oxide, a polyamide, a polyacrylate, a
polymethacrylate and a polyurethane, or a mixture thereof.
11. A method of claim 1, wherein altered anti-polysaccharide antibody levels in bodily tissue or fluid are measured for detecting a disease, disorder or condition .
12. A method for assessing the efficacy of a vaccine which vaccine alters anti-polysaccharide antibody levels in a mammal comprising the steps of: (a) administering an effective amount of said vaccine to said mammal; (b) allowing said mammal to develop anti-polysaccharide antibodies; (c) measuring the amount of anti-polysaccharide antibody bound to said microsphere-polysaccharide conjugate.
13. A method of claim -2, wherein polysaccharide antigens

levels of vaccine in-process samples are measured.
14. The method according to any of the previous claims, , wherein the fluorescence can be measured using a bead array reader.