A Multiplexed Fluorescent Microsphere Immunoassay for Antibodies to ...

Microbiology and Infectious Disease / MULTIPLEXED LUMINEX ASSAY FOR PNEUMOCOCCAL ANTIBODIES

A Multiplexed Fluorescent Microsphere Immunoassay for Antibodies to Pneumococcal Capsular Polysaccharides Jerry W. Pickering, PhD,1 Thomas B. Martins,1 Ryan W. Greer,1 M. Carl Schroder, MT(ASCP), MPA,1 Mark E. Astill, MS,1 Christine M. Litwin, MD,1,2 Stephen W. Hildreth, DrPH,3 and Harry R. Hill, MD1,2 Key Words: Pneumococcal polysaccharide; Pneumococcal vaccine; Luminex; ELISA; Enzyme-linked immunosorbent assay

Abstract We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F, 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

© American Society for Clinical Pathology

Since 1985, 23-valent Streptococcus pneumoniae polysaccharide vaccines have been recommended for adults and children older than 2 years of age.1 Infants and children younger than approximately 2 years of age do not produce a protective antibody response to purified pneumococcal polysaccharide vaccines, but do respond to the same polysaccharides when the polysaccharides are conjugated to T-dependent protein carriers. 1-3 This observation has led to the development of a new generation of pneumococcal-conjugated vaccines for infants and toddlers. The first of these to receive approval by the US Food and Drug Administration is Prevnar (Wyeth Lederle Vaccines, West Henrietta, NY). Prevnar contains purified capsular polysaccharides from 7 S pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F), coupled to a nontoxic variant of diphtheria toxin, CRM197.1-3 In efficacy studies, Prevnar was shown to reduce invasive pneumococcal disease by more than 93% and pneumococcal pneumonia by 73%.4 Prevnar also reduces the number of episodes of otitis media caused by serotypes present in the vaccine by 57%.5 Evaluation of immune responses to vaccines requires precise and serotype-specific serologic measurements. Precision is achieved by standardization of reference reagents and methods across multiple laboratories.6,7 Enzyme-linked immunosorbent assay (ELISA) is the method most often used for pneumococcal vaccine antibody testing and has largely replaced earlier radioimmunoassay technology.5-10 ELISA technology is well suited for screening large numbers of samples against a single analyte. However, testing antibody responses to pneumococcal vaccines by ELISA requires a separate assay for each serotype contained in the vaccine. Furthermore, the dynamic range of the current Am J Clin Pathol 2002;117:589-596

589

Pickering et al / MULTIPLEXED LUMINEX ASSAY FOR PNEUMOCOCCAL ANTIBODIES

ELISAs is limited so that repeated testing after additional dilutions often is required. These factors contribute to sample size considerations for clinical trials. Additional assays must also be done to assess possible interference with other childhood vaccines that are administered simultaneously. As more serotypes are added to the vaccine formulas, immunogenicity testing by ELISA becomes ever more laborious and time-consuming. Luminex multiple analyte profiling technology (Luminex, Austin, TX) is a flow cytometric system that allows a single sample to be tested simultaneously for several different analytes. Our goal was to determine whether the Luminex system could replace ELISA to provide higher throughput and greater dynamic range on a smaller sample volume for pneumococcal antibody testing. We describe a Luminex immunoassay that simultaneously measured concentrations of antibody to 14 pneumococcal capsular polysaccharide serotypes.

Materials and Methods Reagents Pneumococcal polysaccharide (PnPs) antigens were purchased from American Type Culture Collection (ATCC), Manassas, VA. Pneumococcal C polysaccharide (C-Ps) was obtained from Statens Serum Institute, Copenhagen, Denmark. Pneumococcal ELISA absorbent (PnA), a crude cell wall polysaccharide preparation, was provided by Wyeth Lederle Vaccines, West Henrietta, NY. N-Hydroxysulfosuccinimide (Sulfo-NHS) and 1-ethyl-3 (3-dimethylamino-propyl) carbodiimide-HCl (EDC) were obtained from Pierce, Rockford, IL. Cyanuric chloride, polyL-lysine (PLL), diethanolamine, polyoxyethylene 23 lauryl ether (Brij 35), tris(hydroxymethyl)aminomethane (Tris) buffer, polyoxyethylene sorbitan monolaurate (Tween 20), and magnesium chloride were from Sigma, St Louis, MO. Pneumococcal Antisera The US human antipneumococcal standard reference serum, lot 89SF-2, was obtained from the Center for Biological Evaluation and Review, US Food and Drug Administration, Bethesda, MD.6,11 A panel of 50 pneumococcal quality control serum samples, which consists of samples obtained before and after administration of pneumococcal vaccine samples, was obtained from the National Institute for Biological Standards and Control, Potters Bar, England. The quality control serum panel was provided by David Goldblatt, Institute of Child Health, London, England, and is intended to be used for standardizing pneumococcal ELISAs. After establishing comparability of our ELISA method to the established 590

Am J Clin Pathol 2002;117:589-596

assay, we subsequently used these serum samples to compare the Luminex assay with the ELISA. Other serum samples used in this study were submitted to ARUP Laboratories, Salt Lake City, UT, for pneumococcal antibody testing. Serum samples containing various concentrations of antibodies to pneumococcal polysaccharides were pooled to prepare controls and a secondary standard for the Luminex assay. Serum samples obtained after administration of 23-valent pneumococcal polysaccharide vaccine were used for competitive inhibition-of-binding studies. Coupling of Pneumococcal Polysaccharides to Luminex Microspheres PnPs were coupled to PLL by slight modifications of the cyanuric chloride method described by Gray.12 PnPs were reconstituted in pyrogen-free water (NANOpure, Barnstead/Thermolyne, Dubuque, IA) at 5 mg/mL. Reconstituted PnPs antigens were mixed with an equal volume of a 0.01-mol/L concentration of sodium hydroxide with a 0.001% solution of phenolphthalein. The solution was added to cyanuric chloride (5 mg/mL) and mixed briefly. PLL was added to a concentration of 5 µg/mg of PnPs. The mixture was mixed again briefly and incubated overnight at 2°C to 8°C. Each PnPs/PLL conjugate was passed through a Sephadex G-25 (PD-10, Amersham Pharmacia Biotech, Piscataway, NJ) desalting column and collected in 3.5 mL of a 0.05-mol/L concentration of phosphate and a 0.15-mol/L concentration of sodium chloride, pH 7.3 (PBS). Each PnPs/PLL conjugate was coupled to a carboxylated Luminex microsphere using a 2-step carbodiimide reaction. 13 Carboxylated microspheres were activated for 20 minutes at 6.25 × 106/mL in PBS, pH 6.1, with 5 mg/mL of EDC and 5 mg/mL Sulfo-NHS. Activated microspheres were washed with PBS, pH 7.3, by centrifugation and incubated with PnPs/PLL at 1.25 × 10 8 microspheres per milliliter of PnPs/PLL conjugate. Following a 1-hour incubation at room temperature, microspheres were washed and stored in PBS, pH 7.3, with a solution of 0.1% bovine serum albumin and 0.05% sodium azide. ELISA for Anti-PnPs The ELISA method for determination of antibodies to PnPs was performed essentially as described by Quataert et al. 6,7 PnPs antigens were diluted in PBS, pH 7.2, and absorbed to medium binding 96-well microtiter plates (Costar 9017) by incubation at 37°C for 5 hours. Test serum samples and the 89SF-2-reference serum samples were diluted in PBS with a solution of 0.05% Tween 20, 1 µg/mL of PnA and 2 µg/mL of serotype 22F polysaccharide. PnA absorbent is a preparation of soluble capsular components including C polysaccharide prepared from a capsule-negative © American Society for Clinical Pathology

Microbiology and Infectious Disease / ORIGINAL ARTICLE

Luminex Multiplexed Assay for Pneumococcal Antibodies A 2-step indirect procedure was used for the multiplexed Luminex assay for antibodies to pneumococcal polysaccharides. Pneumococcal standards and patient serum samples were diluted in PBS, pH 7.2, with a 0.05% solution of Tween 20 (PBST) and 2 µg/mL of PnA. Some assays also contained PnPs serotype 22F in the sample diluent at a final concentration of 2 µg/mL as a second absorbent. Serial dilutions of standard were included in each assay. Unknown serum and control samples were diluted 1:100. Each dilution of standard, control, and unknown serum samples was mixed with a set of PnPs-coupled Luminex microspheres in 96-well filtration plates (Millipore multiscreen, Millipore, Bedford, MA) and incubated for 20 minutes at room temperature with shaking. Microspheres were collected by vacuum filtration and washed with PBST. R-phycoerythrin–conjugated antihuman IgG (gamma-chain specific) (Jackson Immunoresearch) was added to each well. Following a second 20minute incubation and wash step, microspheres were resuspended in PBST, transferred to a 96-well round bottom plate, and read in a Luminex 100 analyzer equipped with an XY platform. Data reduction was accomplished with StatLIA software (Brendan Scientific, Grosse Point Farms, MI). Competitive Inhibition-of-Binding Studies Serum samples from subjects after immunization with the vaccine were diluted 1:200 in PBST with 2 µg/mL of PnA. Some experiments also contained 4 µg/mL of serotype 22F polysaccharide as a second absorbent. Serum dilutions were preincubated with each of the purified pneumococcal serotype polysaccharides and C-Ps at final concentration of 50 µg/mL. Following a 1-hour incubation at room temperature, a 100-µL aliquot was mixed with 100 µL of PnPscoupled microspheres and assayed as described in the preceding section. © American Society for Clinical Pathology

Results We prepared a reference standard for our multiplexed assay by pooling human serum samples with high antibody levels to PnPs. Eight 4-fold dilutions of our reference standard were tested in the multiplexed Luminex assay ❚Figure 1❚. All 14 serotypes had similar parallel slopes and were linear over approximately seven 4-fold dilutions of our reference standard. Antibody concentrations for PnPs serotypes have been assigned to the international reference standard, 89-S, using a well-established ELISA procedure that included PnA.6,7 We used the Luminex assay to assign anti-PnPs IgG concentrations to our pooled in-house standard by comparison with the 89-S reference standard. The Luminex assay also included PnA. Antibody concentrations for unknown serum samples then were determined by using our pooled secondary standard. Antibody levels in serum samples obtained from 3 individuals before and after receipt of pneumococcal polysaccharide vaccine are shown in ❚Table 1❚. The vaccine response to each of the 14 serotypes varied considerably from individual to individual, but in most cases there was an excellent rise in IgG antibody concentration after receipt of the vaccine. Antibody concentrations in paired serum samples (before and after receipt of vaccine) determined by the Luminex assay are shown in ❚Figure 2A❚ for an individual who received the 7-valent Prevnar conjugate vaccine and in ❚Figure 2B❚ for an individual who received 23-valent polysaccharide vaccine.

100,000 1 3 10,000

Mean Fluorescence Intensity

strain of S pneumoniae.6 Two-fold dilutions of the 89SF-2 standard and dilutions of test serum samples were added to prewashed PnPs-coated microtiter plates. Following a 2-hour incubation at room temperature, plates were washed with Tris-buffered saline, pH 7.2, with a 0.1% solution of Brij 35 and alkaline phosphatase–conjugated goat antihuman IgG (gamma-chain specific) (Jackson Immunoresearch, West Grove, PA) was added. After a second 2-hour incubation at room temperature, plates were washed again, and 100 µL of a 5 mg/mL solution of p-nitrophenyl phosphate in a 1-mol/L concentration of diethanolamine, pH 9.8, with a 5-mmol/L concentration of magnesium chloride was added to each well. The substrate reaction was stopped after a 2-hour incubation at room temperature by addition of 100 µL of 3N sodium hydroxide.

4 5 6B 7F

1,000

8 9N 9V 12F 14

100

18C 19F

10

23F

1 1:20

1:80

1:320

1:1,280

1:5,120

1:20,480

1:81,920

Blank

Dilution of Reference Serum

❚Figure 1❚ Titration of the pooled pneumococcal antibody standard serum against 14 pneumococcal polysaccharide serotypes. The mean fluorescence intensity (MFI) for each dilution of the standard was determined simultaneously for all 14 pneumococcal polysaccharide serotypes with the Luminex 100 analyzer (see text for proprietary information). The curves were constructed by plotting the log MFI against the dilution factor for the standard serum.

Am J Clin Pathol 2002;117:589-596

591


❚Table 1❚ Immunoglobulin G Concentrations in Serum Samples Before and After Pneumococcal Vaccine for 14 Pneumococcal Polysaccharide Serotypes Determined by the Multiplexed Luminex Assay* Serotype Serum Sample No.

1

3

4

5

6B

7

8

9N

9V

12F

14

18C

19F

23F

Before After

1.06 4.12

1.54 31.29

4.76 229.70

4.15 31.45

1.93 60.02

1.26 23.89

1.38 8.67

3.68 68.71

3.07 32.48

0.80 30.53

24.65 229.40

2.22 79.68

0.74 5.43

1.94 364.80

Before After

1.42 47.81

0.13 2.25

0.39 6.76

1.40 10.38

0.54 27.84

0.24 3.51

0.32 15.71

0.05 2.41

0.68 4.14

0.29 1.13

0.08 27.64

0.59 16.14

0.36 4.89

0.05 0.17

Before After

0.38 2.84

0.71 6.35

0.05 1.35

0.60 23.70

0.27 8.24

0.39 10.79

0.28 16.45

0.07 6.32

0.81 7.05

0.33 2.12

0.31 79.21

0.72 17.37

0.24 11.15

8.07 20.85

1 2 3

*

IgG concentrations are given as micrograms per milliliter. For proprietary information, see the text.

Competitive Inhibition-of-Binding Studies To assess the specificity of the individual PnPs assays, we measured inhibition of binding of human pneumococcal antiserum to PnPs-coupled microspheres. We preincubated 1:200 dilutions of serum from adults after pneumococcal vaccination (Figure 2B) with 50 µg/mL of each purified PnPs and with C-Ps at 50 µg/mL as a control. We then tested the preabsorbed serum dilutions for reactivity to the pooled PnPs-coupled microspheres in the Luminex assay. Results of the absorption experiments with a high antibody–containing serum for serotype 14 are shown in

❚Figure 3A❚. Type 14 PnPs inhibited 98% of the binding to the microspheres coupled with the serotype 14 PnPs, while inhibition by the heterologous PnPs serotypes was less than 10% of the binding. This serum sample with high antibody content also showed excellent specificity for serotypes 3, 4, 6B, 7F, 18C, and 23F with more than 97% specificity. Absorption of the antibody was less than 10% with most of the heterologous serotypes. The specificity of the serum samples to serotypes 1 and 19F was lower than for the aforementioned serotypes. For these serotypes, inhibition of binding by the homologous serotypes was 92% and 87%, respectively. Inhibition by

B

A

229

230

100

70

365

90 60

80 70

IgG µg/mL

IgG µg/mL

50 40 30

60 50 40 30

20

20 10

10

0

0 1

3

4

5

6B

7F

8

9N

Serotype

9V 12F 14 18C 19F 23F

1

3

4

5

6B 7F

8

9N 9V 12F 14 18C 19F 23F

Serotype

❚Figure 2❚ Luminex results for serum samples from before (white bars) and 1 month after (black bars) receipt by an individual of the 7-valent pneumococcal-conjugate vaccine (A) (the asterisks indicate serotypes in the vaccine) and an individual who received 23valent polysaccharide vaccine (B). Following administration of the 7-valent conjugate vaccine, significantly increased antibody concentrations for 5 of 7 vaccine serotypes can be seen. There was little or no response to serotypes 4 and 18C and to serotypes not present in the vaccine. In B, after administration of the 23-valent vaccine, the subject responded to most of the 14 serotypes, but there were weak but still protective responses to serotypes 1, 8, and 19F. For proprietary information, see the text.

592

Am J Clin Pathol 2002;117:589-596



A 16,000

MFI

12,000

0 None 1

Discussion The Luminex multiple analyte profiling technology has a number of potential advantages over ELISA for pneumococcal vaccine testing. Rather than performing separate individual assays for each PnPs serotype in the vaccine, antibody concentrations for all serotypes can be determined simultaneously from a single serum dilution. This not only greatly reduces time and labor, but also conserves sample volumes, which, in the case of pediatric samples, might be in limited © American Society for Clinical Pathology

3

4

5

6B 7F

8 9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

B 1,000 800 600 400 200 0 None 1

3

4

5

6B 7F

8 9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

C

MFI

Comparison of Luminex With ELISA Last, we compared the multiplexed Luminex assay with a standard ELISA method used for pneumococcal vaccine immunogenicity testing.2-4,6,7 Fifty serum samples obtained before and after administration of pneumococcal polysaccharide vaccine, representing a range of pneumococcal antibody concentrations, were tested by both methods. ELISA results for 9 PnPs serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F) were compared with the multiplexed Luminex assay results for the same serotypes. The 9 serotypes were tested simultaneously in the Luminex assay (along with the 5 additional serotypes) and individually by ELISA. For this comparison, both PnA and serotype 22F at final concentrations of 1 and 2 µg/mL, respectively, were added to the sample diluent of both assays as absorbents. Antibody concentrations for all 50 serum samples determined by both methods were analyzed using linear regression analysis. The slope and correlation coefficients determined from the linear regression analysis are summarized in ❚Table 2❚. A graphic display of the linear regression analysis for serotypes 1, 6B, 18C, and 23F is shown in ❚Figure 5❚. The Luminex assay had excellent correlation with ELISA for serotypes 6B, 14, and 23F with correlation coefficients (R2) of approximately 0.95. The correlation between Luminex and ELISA for 6 other serotypes (1, 4, 5, 9V, 18C, and 19F) was also good, with R2 values of 0.85 or higher.

8,000 4,000

MFI

heterologous serotypes, however, was high, ranging between 30% and 60% ❚Figure 3B❚ and ❚Figure 3C❚. We next investigated whether inclusion of serotype 22F polysaccharide in the sample diluent as an absorbent might improve the specificity of the Luminex assays. We repeated the aforementioned inhibition-of-binding studies but added serotype 22F polysaccharide at a final concentration of 2 µg/mL in addition to PnA in the sample diluent. The addition of 22F dramatically reduced the high level of heterologous inhibition that was seen with some serotypes. As shown in ❚Figure 4❚, inhibition of binding of the serum to serotypes 1 and 19F was reduced to less than 10% to 12% for most of the heterologous serotypes. Serotype 14, which showed good specificity without 22F, was not affected.

350 300 250 200 150 100 50 0 None 1

3

4

5

6B 7F

8

9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

❚Figure 3❚ Competitive inhibition of binding of a postpneumococcal vaccine serum to pneumococcal polysaccharide–coupled Luminex microspheres. The serum was diluted in sample diluent containing 1 µg/mL of pneumococcal absorbent. Each of the 14 pneumococcal polysaccharides (PnPs) and C-polysaccharide (C-Ps) were added separately to the diluted serum at 50 µg/mL. The mean fluorescence intensity (MFI) of binding of the inhibited serum to PnPs-coupled microspheres is shown for serotypes 14 (A), 1 (B), and 19F (C). For proprietary information, see the text.

supply. For example, with Luminex, a single dilution of 10 µL or less of serum is sufficient for all 14 assays. By contrast, more than 100 µL of serum sample would be required to run 14 ELISAs using our current automated format. The Luminex assay described herein has a much greater dynamic range than ELISA. ELISA is linear over a 10- to 12-fold dilution range,11,14 while the Luminex assay was linear over a 32-fold dilution range (Figure 1). With ELISA, a number of dilutions of unknown samples must be tested to ensure that optical density values fall within the limited range of the standard curve. The Luminex system with its greater dynamic range should greatly reduce the Am J Clin Pathol 2002;117:589-596

593


A 25,000

MFI

20,000 15,000 10,000 5,000 0 None 1

3

4

5

6B 7F

8 9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

MFI

B 700 600 500 400 300 200 100 0 None 1

3

4

5

6B 7F

8 9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

C 300 250

MFI

200 150 100 50 0 None 1

3

4

5

6B 7F

8 9N 9V 12F 14 18C 19F 23F C-Ps

Inhibitor

❚Figure 4❚ Assay conditions are the same as described for Figure 3 except that serotype 22F was added to the sample diluent at 2 µg/mL in addition to pneumococcal absorbent. Results are shown for serotypes 14 (A), 1 (B), and 19F (C). C-Ps, C-polysaccharide; MFI, mean fluorescence intensity.

number of sample dilutions required. Finally, the reaction kinetics of the particle-based Luminex system is more rapid than the solid-phase ELISA system. Incubation times and total assay times, therefore, are much less with the Luminex system, thus allowing many more assays to be completed within an allotted time.

To covalently attach PnPs to Luminex microspheres, some chemical modification of the polysaccharides was required. Conjugation of PnPs antigens to PLL has been used to facilitate binding of PnPs antigens to low binding microtiter plates.12,14,15 We adapted and extended this technique to allow us to covalently attach PnPs antigens to carboxylated Luminex microspheres. Once PnPs antigens were successfully coupled to Luminex microspheres, we sought to confirm that the serotype-specific epitopes remained intact. To establish the serotype specificity of each individual PnPs assay, we did competitive binding studies with serum samples from adults after pneumococcal vaccination and demonstrated more than 94% inhibition of binding by homologous serotypes for all 14 serotypes. With only a few exceptions, inhibition of binding by heterologous serotypes was less than 15% for all 14 serotypes. These results indicate that the serotype-specific epitopes remained intact after PnPs antigens were coupled to the microspheres. PnPs antigens available from ATCC contain from 1% to 30% by weight of C-Ps.16 C-Ps is covalently linked to the type-specific PnPs.17 All individuals have antibodies to C-Ps before immunization with pneumococcal vaccines.16,18,19 Antibodies to C-Ps are not opsonic and, therefore, are presumed not to confer protective immunity.18 Immunization with pneumococcal vaccines induces serotype-specific anticapsular antibodies that are opsonic and provide protection against S pneumoniae infection.18 The more recent protocols for pneumococcal antibody testing by ELISA incorporate absorption with C-Ps to remove the nonspecific C-Ps antibodies.9,10,20 Several groups of investigators, however, have reported antibodies in serum samples from before and after administration of vaccine directed to common antigens other than C-Ps. 8,20-23 Soininen and coworkers22 measured inhibition of binding in an enzyme immunoassay to serotypes 6B, 14, 19F, and 23F. They found cross-reactivity of 50% or more to serotypes 6B, 19F, and 23F in serum samples from nonimmunized infants and adults and, to a lesser extent, in postvaccination serum samples from adults. Yu and coworkers23 reported that 50% of adult prevaccination serum samples and 10% of adult postvaccination serum samples were cross-reactive to serotypes 4, 9V, 18C, 19F, and 23F. Up to 50% of the reactivity to these serotypes

❚Table 2❚ Summary of Slope and Correlation Coefficient (R2) Values for Linear Regression Analysis of Pneumococcal Antibody Concentrations Determined by Luminex and Enzyme-Linked Immunosorbent Assay Serotype

R2 Slope

594

1

4

5

6B

9V

14

18C

19F

23F

0.855 0.946

0.888 0.966

0.874 1.082

0.946 1.010

0.858 1.110

0.964 1.171

0.854 1.130

0.927 1.135

0.965 0.982

Am J Clin Pathol 2002;117:589-596



A

B

10.00

10.00

Luminex

100.00

Luminex

100.00

1.00 0.10 0.01 0.01

1.00 0.10 0.01

0.10

1.00

10.00

100.00

0.01

0.10

ELISA

10.00

100.00

10.00

100.00

D

C

100.00

10.00

10.00

Luminex

100.00

Luminex

1.00

ELISA

1.00 0.10 0.01 0.01

1.00 0.10 0.01

0.10

1.00

10.00

100.00

ELISA

0.01

0.10

1.00

ELISA

❚Figure 5❚ Comparison of IgG concentrations with pneumococcal polysaccharide (PnPs) serotypes determined by the multiplexed Luminex assay and enzyme-linked immunosor-bent assay. Adult serum samples (n = 50) from before and after pneumococcal vaccine administration were measured by both methods as described in the “Materials and Methods” section. Antibody concentrations in micrograms per milliliter after log transformation are shown for PnPs serotypes 1 (A; y = 0.9458x + 0.1466; R2 = 0.8554), 6B (B; y = 1.008x + 0.0092; R2 = 0.9463), 18C (C; y = 1.1304x – 0.1724; R2 = 0.8549), and 23F (D; y = 0.9820x – 0.0077; R2 = 0.9648). For proprietary information, see the text.

was nonspecific. Both of the aforementioned groups of investigators attributed the cross-reactivity to antibodies against impurities other than C-Ps in the PnPs antigen preparations. Concepcion and Frasch8 recently reported that most of the 24 pairs of adult serum samples from before and after pneumococcal vaccination they studied contained crossreacting antibodies. These cross-reacting antibodies were not removed by absorption with C-Ps alone. They observed heterologous inhibition of binding in ELISA to serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F but not to serotype 14. In some prevaccination serum samples, the heterologous inhibition was nearly as high as the homologous inhibition. These investigators have shown that the non–type-specific antibodies are not opsonic. Absorption with a heterologous serotype such as 22F in addition to C-Ps further removes non–serotype-specific antibodies and improves the correlation between ELISA and opsonophagocytosis assay titers.8 As shown in Table 1, the responses to 23-valent polysaccharide vaccines among the individuals we tested were quite variable. Individuals had strong IgG responses to some serotypes as evidenced by a high concentration of antibody that © American Society for Clinical Pathology

was highly specific as shown for serotype 14 in Figure 3A. All serum samples, however, also had lower antibody IgG concentrations to some serotypes. These lower-concentration antibodies were less specific. As shown in Figures 3B and 3C, more than 50% of the antibody in this serum to serotypes 1 and 19F was not specific. As reported by others, these nonspecific antibodies were not removed by absorption with C-Ps. In our studies, the addition of up to 50 µg/mL of additional C-Ps did not remove the cross-reacting antibodies. Inclusion of serotype 22F as an absorbent, however, greatly improved the serotype specificity of these low titer serum samples (Figure 4). We believe that the Luminex assay reported herein is an attractive alternative to ELISA for pneumococcal antibody testing. The procedure is rapid and sensitive and permits simultaneous testing of serotypes of interest. The assay shows good correlation with an established ELISA method used in recent pneumococcal vaccine efficacy studies and demonstrates good postvaccine responses. In lieu of more highly purified PnPs antigens, pneumococcal antibody testing in general might be improved by preabsorption of serum samples with serotype 22F. Am J Clin Pathol 2002;117:589-596

595


From 1Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City; 2Department of Pathology, Pediatrics, and Medicine, University of Utah School of Medicine, Salt Lake City; and 3Wyeth Lederle Vaccines and Pediatrics, West Henrietta, NY. Address reprint requests to Dr Pickering: ARUP Institute, 500 Chipeta Way, Salt Lake City, UT 84108.

References 1. Overturf GD and the Committee on Infectious Diseases. Technical report: Prevention of pneumococcal infections, including the use of pneumococcal conjugate and polysaccharide vaccines and antibiotic prophylaxis (RE9960). Pediatrics. 2000;106(2 pt 1):367-376. 2. Rennels MB, Edwards KM, Keyserling HL, et al. Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in the United States. Pediatrics. 1998;101:604-611. 3. Shinefield HR, Black S, Ray P, et al. Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers. Pediatr Infect Dis J. 1999;18:757-763. 4. Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr Infect Dis J. 2000;19:187-195. 5. Eskola J, Kilpi T, Palmu A, et al. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Engl J Med. 2001;344:403-409. 6. Quataert SA, Kirch CS, Wiedl LJQ, et al. Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S. Clin Diagn Lab Immunol. 1995;2:590-597. 7. Quataert SA, Martin D, Anderson P, et al. A multi-laboratory evaluation of an enzyme-linked immunoassay quantitating human antibodies to Streptococcus pneumoniae polysaccharides. Immunol Invest. 2001;30:191-207. 8. Concepcion NF, Frasch CE. Pneumococcal type 22F polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 2001;8:266-272. 9. Koskela M, Leinonen M. Comparison of ELISA and RIA for measurement of pneumococcal antibodies before and after vaccination with 14-valent pneumococcal capsular polysaccharide vaccine. J Clin Pathol. 1981;34:93-98. 10. Siber GR, Priehs C, Madore DA. Standardization of antibody assays for measuring the response to pneumococcal infection and immunization. Pediatr Infect Dis J. 1989;8(1 suppl):S84-S91.

596

Am J Clin Pathol 2002;117:589-596

11. Concepcion N, Frasch CE. Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization. Clin Diagn Lab Immunol. 1998;5:199-204. 12. Gray BM. ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes. J Immunol Methods. 1997;28:187-192. 13. Staros JV, Wright RW, Swingle DM. Enhancement by Nhydroxysulfosuccinimide of water-soluble carbodiimidemediated coupling reactions. Anal Biochem. 1986;156:220222. 14. Messina JP, Hickox PG, Lepow ML, et al. Modification of a direct enzyme-linked immunosorbent assay for the detection of immunoglobulin G and M antibodies to pneumococcal capsular polysaccharide. J Clin Microbiol. 1985;21:390-394. 15. Chudwin DS, Artrip SG, Schiffman G. Immunoglobulin G class and subclass antibodies to pneumococcal capsular polysaccharides. Clin Immunol Immunopathol. 1987;44:114121. 16. Koskela M. Serum antibodies to pneumococcal C polysaccharide in children: response to acute pneumococcal otitis media or to vaccination. Pediatr Infect Dis J. 1987;6:519526. 17. Sorensen UB, Henrichsen J, Chen HC, et al. Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. Microb Pathog. 1990;8:325-334. 18. Musher DM, Luchi MJ, Watson DA, et al. Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and the effect of adsorption of serum with non–type-specific cell wall polysaccharide. J Infect Dis.1990;161:728-735. 19. Musher DM, Watson DA, Baughn RE. Does naturally acquired IgG antibody to cell wall polysaccharide protect human subjects against pneumococcal infection? J Infect Dis. 1990;161:736-740. 20. Goldblatt D, Levinsky RJ, Turner MW. Role of cell wall polysaccharide in the assessment of IgG antibodies to the capsular polysaccharide of Streptococcus pneumoniae in childhood. J Infect Dis. 1992;166:632-634. 21. Coughlin RT, White AC, Anderson CA, et al. Characterization of pneumococcal specific antibodies in healthy unvaccinated adults. Vaccine. 1998;16:1761-1767. 22. Soininen A, van den Dobbelsteen G, Oomen L, et al. Are enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific? Clin Diagn Lab Immunol. 2000;7:468-476. 23. Yu X, Sun Y, Frasch C, et al. Pneumococcal capsular polysaccharide preparations may contain non–Cpolysaccharide contaminants that are immunogenic. Clin Diagn Lab Immunol. 1999;6:519-524.
