Kinetics of IgM and IgA Antibody Response to 23-Valent Pneumococcal Polysaccharide Vaccination in Healthy Subjects
A poor antibody response of IgM and IgA antibodies upon vaccination with pneumococcal polysaccharides (PnPS) is discussed as independent risk factors for bronchiectasis in patients with antibody deficiency syndrome (ADS) receiving immunoglobulin replacement therapy. However, the kinetics of the specific IgM and IgA response to vaccination with multivalent pneumococcal polysaccharides requires a more detailed knowledge. In this study we aimed i) to develop a standardised multivalent PnPS-IgM and IgA-ELISA, and ii) to compare the sensitivity of the multivalent to the serotype specific antibody response, and iii) to determine the kinetics of the anti-PnPS IgM and IgA antibodies in healthy subjects.
We immunised n = 20 healthy adults with a 23-valent PnPS vaccine (Pneumovax®). The kinetics of the 23-valent antibody response was assessed for 1 year with newly developed ELISAs for IgM and IgA isotypes, along with serotype specific responses.
The IgA and IgM antibody response peaked at 2 and 3 weeks, respectively. IgM antibody levels remained at a plateau (above 80 % of peak response) for 3 months. After one year, specific antibody levels were still at about 30 % of the peak response. The 23-valent antibody response yielded significantly higher responder rates than assessment of single serotypes.
Testing the IgM and IgA immune response to polysaccharide vaccination with a multivalent PnPS ELISA may be a feasible tool for assessment of the immune function in patient groups who receive IgG replacement therapy.
KeywordsPneumococcal polysaccharides vaccination PCP-IgM PCP-IgA 23 PCP ELISA PCP antibody kinetics
S. pneumoniae is one of the most frequent causes of bacterial respiratory infections in both healthy subjects and patients with structural lung disease.[1, 2] Anti-pneumococcal defence mechanisms critically rely on the formation of antibodies directed against pneumococcal polysaccharides (PnPS) in a T cell independent process. The ability of the immune system to respond to unconjugated PnPS vaccine (Pneumovax®) has frequently been used to evaluate the T cell independent IgG antibody formation in a large number of studies.[4, 5, 6]. However, the role of the systemic PnPS IgG response in discriminating patients at risk for chronic bacterial infections at the respiratory mucosa remained inconclusive.
Both IgM and IgA are the predominate immunoglobulin isotypes in the upper and bronchial airways . Thus, measurement of IgM or IgA production in response to PnPS and PnPS vaccines, may be more meaningful to evaluate the ability of the immune system for the prevention of severe respiratory infection with encapsulated bacteria. The clinical relevance of PnPS specific IgM antibodies has been suggested in patients with common variable immunodeficiency (CVID) with evidence of chronic lung disease due to bacterial infection who have been shown to possess lower levels of PnPS specific antibody levels than those without .
However, interpretation of the PnPS-specific IgM and IgA responses to assess B-cell function is complex for several reasons. First, a large number of serotypes varying in epidemiology with time and region leads to a substantial variety of individual pre-immunisation levels. Second, B-cells may be hyporesponsive due to a previous contact, contributing to an even larger inter-individual variation of serotype-specific responses. Studies to assess the B-cell function by their response to PnPS antigens, therefore, may not be easily comparable, and even more so, if not the same set of serotypes is used. Third, data on the kinetics of the PnPS-specific IgM and IgA response is scarce [4, 9] It is unclear at which interval after vaccination the measurement is suitable as particularly the IgM response may be short lived. Again, comparison of studies using different intervals may lead to skewed results. Finally, data of the PnPS specific IgM and IgA response in healthy and disease is limited, making it difficult to follow a standardised procedure in future studies. A standardised single-run procedure based on a broadset of pneumococcus serotypes to measure the complete response to the vaccine antigens would be desirable.
Here we report the development of a standardised 23 valent PnPS-IgM and IgA ELISA which was subsequently used to quantify the kinetics of both, the PnPS-IgM and IgA antibody response to a 23 valent PnPS vaccine (Pneumovax®) in healthy subjects.
Our findings suggest that the measurement of Pneumovax® specific PnPS-IgM and IgA antibodies in the 23 PnPS-IgM and PnPS-IgA ELISAs is more sensitive for evaluating the T-cell independent immune responses than the measurement of selected pneumococcal serotypes. This may particularly apply to patients with CVID.
Healthy individuals (n = 20, ages 22–50 years, 10 male, 10 female) without previous conjugated or unconjugated pneumococcus vaccination were immunised with the 23-valent PnPS vaccine Pneumovax® (23PnPS, Merck and Co., Inc., West Point, PA, Lederle Pearl River, NY, USA) and tested for their antibody response. Blood samples were obtained prior to and at day 4, 7, 11, 14, 21, 28, 42, 90, 180 and 360 post vaccination. Samples were stored at 4 °C over night and centrifugated at 1100 g at room temperature for 15 min. Serum samples were stored in aliquots at −80 °C. Each sample was thawed once and tested for IgM, IgA, IgG and IgG2 immunoglobulins raised against 23PnPS. IgM immunoglobulin titres for pneumococcal serotypes 1, 4, 14 and 19 F were determined on days 0 and 14. PnPS-IgA antibody titres for serotypes 1 and 3 were measured on days 0 and 28 and total IgG and IgM on days 0, 7 and 42.
PnPS-IgG and PnPS-IgG2 antibody levels were determined by the commercially available 23 PnPS Pneumococcal Capsular Polysaccharide IgG VaccZyme™ (MK012) and IgG2 (MK013) ELISA, The Binding Site Group Ltd. (TBS), Birmingham, UK. The assays were performed according to the instructions of the manufacturer.
The PnPS-IgM ELISA based on 23 serotypes (23 PnPS-IgM ELISA) was performed with purified peroxidase labeled rabbit anti-human IgM conjugate. The measuring range of calibrators 0 to 5 were determined as 0, 3.33, 10, 30, 90, 270 U/ml for samples diluted 1:100. For the 23 PnPS-IgA ELISA purified peroxidase labeled rabbit anti-human IgA conjugate was used. To establish a calibration curve an IgA cut-off provided from TBS was diluted 1:100, 1:200, 1:400, 1:800 and 1:1,600. The measuring range of these calibrators was determined as 100, 50, 25, 12.5 and 6.25 U/ml. Calibrators, sample diluent, conjugate and substrate were provided by TBS.
To eliminate cross reactive antibodies and minimise the competition with IgG antibody binding the 23 PnPS-IgM and IgA ELISA were established by optimising the concentrations of cell wall polysaccharide (CWPS, Staten Serum Institut, 3459, distributed in Germany by Medco Diagnostica GmbH, München) and IgG removal agent (TBS, Birmingham, UK).
To optimise the concentration of CWPS and IgG removal agent serum samples before and 14 days post-Pneumovax® vaccination from 5 subjects and a pool of all samples post-vaccination (day 14) were diluted with sample diluent containing different concentrations of CWPS and levels of 23 PnPS IgM antibodies were measured. A significantly high portion of anti pneumococcal response is attributable to the presence of CWPS antibodies and not to specific anti-pneumococcal antibodies. These CWPS antibodies confer limited protection against pneumococcal infection .
As an IgG removal agent an anti-human antibody raised against the Fc portion of human IgG antibodies was used. Serum samples were diluted 1:8 with different concentrations of the IgG removal agent, dispensed into sample diluent and incubated for 30 min at room temperature. After centrifugating at 20,000 g at room temperature for 15 min, serum was separated from the precipitate and diluted 1:500 into the sample diluent. The samples were assayed for 23-PnPS IgM, IgA and IgG titres according to the protocol described below.
To determine an optimal dilution of the human serum samples for the PnPS IgM and IgA ELISAs, a pool of 5 human serum samples was made and measured at different dilutions. To ensure the appropriate dilution for low and high antibody levels the dilution experiment was performed with samples pre and post (14 days) vaccination. The samples obtained pre-vaccination were diluted 1:100 and the samples post-vaccination 1:500 for both IgM and IgA ELISA. However, in two subjects both sets of samples were diluted 1:100 due to a low response to Pneumovax® vaccination.
Final PnPS-IgM and IgA ELISA Protocol
The assays were performed according to the instructions of the manufacturer of the PnPS-IgG ELISA VaccZyme™ (TBS). Briefly 100 μl of 1:500 diluted samples were dispensed into the appropriated wells coated with 23 PnPS and incubated for 30 min at room temperature. After that the plate was washed 3 times with 250–350 μl/well of washing buffer, 100 μl of rabbit anti-human IgM or IgA conjugate was dispensed in each well and incubated for a further 30 min at room temperature. After washing 3 times again with 250 μl–350 μl per well, 100 μl of substrate were applied to each well. After incubation for 30 min at room temperature, 100 μl stop solution were added to each well. The optical density was read at 450 nm on a microplate reader.
Precision of the 23 PnPS-IgM ELISA. Data are expressed as U/ml
Intra assay precision
Inter assay precision
Precision of the 23 PnPS-IgA ELISA. Data are expressed as U/ml
Intra assay precision
Inter assay precision
Conversion of Units and μg/ml
Data were calculated as Units per ml. Therefore the calibrators were plotted against the OD on a linear calibration graph. Units of the samples were calculated linear with Excel. To compare the serotype specific response to the 23 PnPS response data were converted into μg/ml. A standard PnPS-IgM serum was developed by loading a known dilution with a known amount of human IgM antibodies (IgM human serum, Sigma-Aldrich®, St. Louis, USA) onto an ELISA plate coated with purified goat anti-human IgM antibodies (UNLB-goat anti-human antibody, Southern Biotech, distributed in Germany by Biozol Diagnostica Vertrieb GmbH, Germany). At the same time human serum samples of unknown concentration (diluted 1:100 and 1:500), 6 PnPS-IgM calibrators, one high and low control and Lot 89-SF International Reference serum( Dr. Milan S. Blake, Food and drug administration, USA) were run in duplicate on a plate coated with 23 PnPS. The calibration curve was plotted automatically with Excel by plotting the IgM-antibody concentration on the abscissas against the OD on the axis of ordinate for each calibrator. To avoid variations this was repeated 4 times on different days and the mean value was determined.
The plates were coated with 2 ng/ml of purified goat-anti human IgM which was diluted in PBS. 100 μl of this dilution was dispensed into each well and incubated overnight at 4 °C. The wells were washed 3 times with 250 μl/well of washing-buffer, blocked with 200 μl PBS/3%BSA per well and incubated overnight at 4 °C. After further 3 washes, 100 μl of serial dilutions of human IgM standard serum (starting at 400 ng/ml) were dispensed per well and incubated overnight at 4 °C. The next day the plates were washed 3 times and 100 μl of 1:4000 diluted goat-anti-human IgM antibodies (goat anti-human IgM-HRP, Biozol, Eching, Germany) were dispensed into each well. After incubating for 1 h at room temperature it was washed 3 times and 100 μl o-phenylene diamine dihychloride (OPD, FIRMA) were dispensed into each well. This reaction was terminated with the addition of 100 μl 0,5 M H2SO4 and the optical density recorded at 450 nm in the microplate reader.
Serotype Specific Measurement
The determination of the serotype specific PnPS antibody levels was accomplished following the WHO standard protocol . PnPS-IgM antibodies against serotypes 1,4,14 and 19 F were determined in serum samples of all subjects pre vaccination and on day 14 post vaccination. PnPS-IgA antobdies against serotypes 1 and 3 were tested pre and 28 days post vaccination. These serotypes represent the most frequently tested pneumococcal serotypes in other studies [4, 5, 6, 8, 12] moreover these serotypes appear in the pneumococcal vaccines Prevenar 13® (PCV13), Synflorix® (PHiD-CV) and Pneumovax®.
Each serotype antigen was diluted in sterile PBS. The multi screen plates (multiScreen HTS IP Durapore PVDF, Millipore, Molsheim, France) were coated with 100 μl per well of each serotype (2 μg/mL) and incubated overnight at 4 °C. After being washed 3 times with 250 μl/well of TBS washing buffer, 50 μl of the same samples mentioned above were dispensed into each well and stored at 4 °C over night. Serial dilution of Lot 89-SF International Reference serum was performed in PBS and CWPS/22 F buffer (5 μg/ml) starting at 1:400 dilution. The samples were diluted to 1:6000 for measurement. After washing 3 times with TBS, washing buffer 100 μl of TBS purified peroxidase labeled rabbit anti-human IgM conjugate was dispensed into each well and incubated for 30 min at room temperature. After a final 3 times washing, 100 μl of TBS substrate was applied to each well and incubated at room temperature for a further 30 min. The reaction was terminated with cmthe addition of 100 μl of TBS stop-solution. The optical density was measured at 450 nm with a microplate reader.
89-SF reference serum was run on each plate as well as 6 calibrators and a high and low control.
In order for all assays to be valid calibrators and controls were included in each run. For each calibrator, control and sample the mean OD of the duplicate reading was calculated. To avoid inter-assay variations pre- and post-vaccination samples were all run simultaneously. The calibration curve was plotted automatically with Excel by plotting the OD of each calibrator on the Y axis against the known antibody concentration in the calibrator material on the X-axis. For samples diluted further than the 1:100 dilution recommended by the manufacturer the results were multiplied by this factor.
Definition of Vaccination Responses
Standardised evaluations of the PnPS IgM antibody response are still missing in literature. In this study we defined a positive response according to the definition of Carsetti et al. where a positive immune response is described as an at least two fold rise of serotype specific PnPS IgM antibodies . This definition was also adopted in an unpublished study of our partner laboratory (Isabella Quinti, La Sapienza University, Rome).
The Gaussian distribution of the data was tested by Kolmogorow-Smirnow test. Data are given as mean and standard error of mean (SEM). The inter- and intra-assay precisions were calculated as %CV which was determined as the quotient of standard deviation and mean value with Excel. The quotients of correlation are given as Pearson. Serotype specific assays and 23-PnPS ELISA assays were compared by performing the simple linear regression analysis. Units of the samples were calculated linear with Excel. The level of significance and r squared were calculated with GraphPad Prism, GraphPad Software. A p-value less than 0.05 was considered statistically significant. Values shown as mean values followed the Gaussian distribution.
Development of the 23 PnPS-IgM Antibody ELISA
Determination of the CWPS Concentration
As the response in 30 % of subjects to vaccination with S. pneumonia is attributable to CWPS antibodies and not to specific anti-PCP antibodies we optimised the concentration of CWPS in the sample diluent. Therefore we measured levels of 23 PnPS IgM antibodies in samples diluted with sample diluent containing different concentrations of CWPS pre and post vaccination.
Determination of the Concentration of IgG Removal Agent
To avoid the potential interference of PnPS-IgG antibodies likely to have high affinity with binding of PnPS-IgM antibodies with potentially lower affinity we investigated whether the addition of the IgG removal agent would improve the levels of detected IgM and IgA antibodies.
Reproducibility of PnPS-IgM and IgA ELISAs
In the PnPS-IgM and IgA ELISAs, serum samples with an antibody concentration within the range of the calibration curve were measured in 4 wells of one plate to determine the intra-assay precision. To determine the inter-assay precision serum samples were measured on different plates on different days (see Tables I and II).
Data of Tables I–II demonstrate the high stabilities and reproducibilities of the PnPS-IgM and IgA ELISAs. In order for these assays to be valid a calibrator set and controls were included in each run and all components were loaded in duplicate. For each calibrator, control and sample, the mean OD of the duplicate reading was calculated and the coefficient of variation for each duplicate OD was less than 15 %. To further minimise inter-assay variations pre- and post-vaccination samples from one subject were all run simultaneously in the same assay.
Validation of the PnPS-IgM and IgA ELISAs and the PnPS-IgM and IgA Responses in Healthy Subjects
The kinetics of PnPS antibody formation in response to Pneumovax® vaccination were investigated in 20 healthy subjects. Samples were obtained on day 0, 4, 7, 11, 14, 21, 28, 42, 90, 180 and 360 post vaccination and assayed for IgM, IgA, IgG and IgG2 pneumococcal specific immunoglobulin production in response to 23PnPS. IgM immunoglobulin titres for pneumococcal serotypes 1, 4, 14 and 19 F were determined on day 0 and 14, serotype specific IgA antibodies for serotype 1 and 3 on day 0 and 4 weeks post vaccination. Total IgG and IgM were measured on day 0, 7 and 42.
Kinetics of PnPS-IgM Specific Antibody Production in Normal Healthy Subjects
Sixty percent (n = 12) of healthy subjects had no detectable PnPS-IgM antibody titre before vaccination but showed a significant mean increase to 622 U/ml 4 weeks post vaccination. 40 % of healthy subjects had an average PnPS IgM concentration of 88 U/ml before vaccination with a 10 fold increase 4 weeks post vaccination (Supplementary Fig. 1a). An average increase for all subjects of 20 folds 4 weeks post vaccination was achieved. PnPS-IgM antibodies ranges from 0 to 204 U/ml pre vaccination, from 145 to 1333 U/ml 4 weeks post vaccination and from 0 to 824 U/ml one year post vaccination (Fig. 3a). From 20 healthy subjects one non-responder possessing no PnPS-IgM antibody titre post vaccination was observed. In all other subjects a strong response was observed independently from a high or low titre pre vaccination.
Kinetics of PnPS-IgA Specific Antibody Production in Normal Healthy Subjects
The kinetics of the IgA response to Pneumovax® shows its peak 2 weeks post-vaccination with a 13 fold increase of PnPS-IgA antibodies. The antibody level decreased to almost 200 U/ml at day 360 post vaccination (Fig. 3b).
Only 10 % (n = 2) had no detectable PnPS-IgA antibody titre before vaccination. One of these subjects did not develop any PnPS-IgA immune response (same subject who did not build any PnPS-IgM antibodies). In all other subjects a strong response was observed independently from a high or low titre pre vaccination. (Supplementary Fig. 1b) PnPS-IgA antibodies ranges from 0 to 279 U/ml pre vaccination, from 91 to 1402 U/ml 4 weeks post vaccination and from 0 to 527 U/ml one year post vaccination (Fig. 3b).
Kinetics of PnPS-IgG Specific Antibody Production in Normal Healthy Subjects
The IgG response to Pneumovax® showed a mean 12 fold increase in titre 11 days post vaccination (Fig. 3c). The concentration remained stable in level for one year post vaccination. The SEM demonstrated high variation in the IgG concentrations between healthy subjects. PnPS-IgG antibodies ranges from 12 to 296 mg/l pre vaccination, from 45 to 2879 mg/l 4 weeks post vaccination and from 118 to 3813 mg/l one year post vaccination (Fig. 3c).
The antibody kinetics of the PnPS-IgG2 response was also measured. (data not shown) The kinetic profile was similar to that of the PnPS IgG antibodies suggesting that some of the IgG response to Pneumovax® involved in part the production of IgG2 subclass antibodies.
Comparison Between 23 PnPS ELISA and Serotype Specific Measurement
PnPS IgM Antibodies
Comparison of the sensitivity between 23PnPS IgM ELISA and serotype specific measurement. Seroconversion was defined as an at least two fold increase of the antibody concentration
23 PnPS ELISA
1 + 4
1 + 14
1 + 19 F
4 + 19 F
14 + 19 F
1 + 4 + 14
1 + 4 + 19 F
Seroconverter (% of all subjects)
PnPS IgA Antibodies
Comparison of the sensitivity between 23PnPS IgA ELISA and serotype specific measurement. Seroconversion was defined as an at least two fold increase of the antibody concentration
23 PnPS ELISA
Seroconverter (%of all subjects)
Using the principles of the PnPS-IgG ELISA, we have developed and validated a 23 PnPS-IgM and IgA ELISA which showed consistent data for stability and reproducibility and is easy to perform.
The kinetics of PnPS-IgM and IgA antibody production after Pneumovax® vaccination showed that the period of time to detect these antibodies is not limited to 4 weeks post vaccination , and in fact increased levels of both PnPS-IgM and IgA antibodies were still detectable for one year post vaccination. At this point the PnPS-IgM antibody level declined to about one third, the PnPS-IgA antibody level to half of the initial concentration of PnPS-IgM and PnPS-IgA antibodies, respectively. The PnPS-IgM and PnPS-IgA titres were considerably stable in healthy subjects. The level of PnPS-IgM antibodies reached its maximum 3 to 4 weeks post vaccination, the PnPS-IgA antibodies 2 weeks post vaccination. Healthy Subjects with a high pre-vaccination PnPS-IgM antibody titre also developed a positive immune response which suggests that they were not passing a refractory period even though they had a positive PnPS-IgM titre pre vaccination. However, until now only the refractory period for PnPS-IgG antibody response to Pneumococcal polysaccharide vaccine was analysed . In this study we did not observe any refractory period for PnPS-IgM and IgA antibodies.
The immune response to PnPS-IgA and -IgM antibodies appeared more uniform in the variation of levels as compared to the PnPS-IgG antibody response. The variation of values measured by the 23 PnPS-IgM and IgA antibody ELISA was smaller, shown by the low SEM, compared to the PnPS-IgG antibody responses. This may help to make a more meaningful clinical interpretation of the results concerning responders and non responders. It remains to be determined whether the IgG response correlates to the presence of memory B-cells which may vary between the individuals. Vice versa, a T cell independent B cell response without IgM and IgA memory B cells may be more uniform between the subjects of this study.
The immune response measured by the 23 PnPS-IgM ELISA showed a quantitative higher and more sensitive result than the results measured in the serotype specific ELISAs. All subjects with a serotype specific antibody response also exhibited a response detectable with the 23 PnPS-IgM ELISA. However, a major proportion of the 23 PnPS-IgM responders showed no or a weak response if analysed by specific serotypes. Thus, the detection of seroconversion strongly depends on the selection of serotypes analysed, which carries the considerable risk that the use of selected serotypes may not detect either a PnPS-IgM or IgA response. As the immune response to single serotypes do not correlate significantly with a positive immune response to the 23 PnPS IgM ELISA, it seems to be impossible to infer the result from a single serotype response to that obtained on a 23 PnPS IgM ELISA response. The results of the PnPS-IgA measurement show that all subjects showed a positive immune response when measured in the 23 PnPS-IgA ELISA and for serotype 1. Other studies are needed to determine PnPS-IgA antibodies of more serotypes to show whether a positive response of all subjects correlates also with other serotypes.
We conclude that assessment of the antibody response towards Pneumovax® using a 23 PnPS-IgM and IgA ELISA is more sensitive than if only a limited number of single serotypes are tested. For assessment of the ability of an individual to mount an antibody response against polysaccharides, a multivalent assay appears more suitable than the use of a multitude of serotype specific assays still bearing the risk of an inadequate serotype selection. Using a multivalent assay a consistent definition of an immune response would be definable as well as a better comparability of different studies.
A decreased amount of IgM and/or class switched memory B cells in the peripheral blood has been proposed as a marker of risk for developing structural lung disease in patients with ADS, in particular CVID [8, 15]. However, the data is not consistent . A functional test may be more meaningful clinically as the phenotype of a cell is not necessarily representative for its full function in primary immunodeficiency. Assessment of antibody formation upon vaccination is frequently used as a diagnostic tool in patients with suspected of immunodeficiency. However, the IgG antibody response to Pneumovax® yielded conflicting results in patients with susceptibility to infection. Moreover, quantification of an IgG antibody response can be problematic in patients who receive immunoglobulin replacement, as the assay cannot discriminate between the lent and proprietary immune response. In addition, IgG antibody levels in such patients vary over time during replacement therapy, rendering it even more difficult to discriminate between a efficient and deficient vaccine response. Since the production of IgM and IgA isotypes is independent of immunoglobulin replacement therapy, these isotypes appear to be particularly suitable for assessing the function of the humoral immune system during replacement therapy. The assessment of IgM and IgA antibodies may be particularly meaningful for the assessment of risk of pulmonary morbidity in such patients as these two isotypes are prominent on the surface of the upper and bronchial airways. The fact that IgG antibodies do not reach these areas in such patients particularly at risk of acquiring infection with encapsulated bacteria is in line with the observation of the limited effect IgG replacement therapy has on the prevention of pulmonary disease in patients with antibody deficiency, such as CVID and XLA. The ability of a patient to mount antibodies against bacterial polysaccharides such as PnPS may, therefore, be a more meaningful parameter for assessment of the pulmonary risk in ADS patients. A present study (Quinti et al.) using the newly developed 23 valent IgM- and IgA-ELISA demonstrates this ELISA promises to be a useful tool in these studies.
In summary, we have characterised the IgM and IgA response upon polysaccharide vaccination as a long lasting and stable response with newly developed 23 PnPS IgM and IgA ELISAs. These tests may provide reliable markers to assess the risk of pulmonary morbidity, as it can be easily applied in patients along immunoglobulin replacement and assesses the isotypes that are present on the airway surface.
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2011) under grant agreement no201549 (EURO-PADnet)
Conflict of Interest
None of the authors has any potential financial conflict of interest related to this manuscript.
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