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Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens.

Infections caused by Streptococcus pneumoniae are an important cause of mortality and morbidity, particularly among children and older adults and immunocompromised patients (1). Also of major concern is emerging resistance of S. pneumoniae to antibiotic drugs (2). Phagocytes, complement, and specific antibodies to the capsular polysaccharides (caps-PS) are important in the host defense against S. pneumoniae (3).

Pneumococcal caps-PS are polymers composed of repeating units of 2 to 5 different saccharide moieties. It has long been known that anti-caps-PS antibodies provide protection against invasive pneumococcal infection (3). Reports have appeared of patients suffering from recurrent infections with encapsulated microorganisms with normal antibody concentrations, but also with a specific deficiency in the production of anti-caps-PS antibodies (4, 5). Wasserman et al. estimated that 5%-10% of children referred for evaluation of recurrent infection suffer from this syndrome (6). Javier et al. found this disorder in 23% of patients who underwent immunologic evaluation for recurrent infection (7).

The deficient antibody response to caps-PS is determined by measurement of the anti-caps-PS antibody concentrations after immunization with the 23-valent vaccine (Pneumovax (6). Assays in which the total antibody response is measured are commercially available, but these assays have some disadvantages. These ELISAs do not use all the absorption steps to remove nonfunctional antibodies. Antibodies to the cell wall polysaccharides (C-PS) are not functional, and they interfere with the determination of serotype-specific antibodies. Absorption of sera with C-PS reduces the mean concentrations of anti-caps-PS antibodies by 15%-84% and is incorporated in most assays (8, 9). Absorption with 22F, which is thought to remove cross-reactive antibodies (10), is not used in current commercially available assays. In addition, the diversity in immunogenicity between the different serotypes may lead to domination of the overall antibody response by high antibodies to a single serotype, despite a possible deficiency in the antibody response to serotypes that are less immunogenic (11). Reference intervals for an assay that is based on the detection of antibodies to a mixture of 23 serotypes have recently been published (12). For children <4 years of age, the lower reference limit was less than the detection limit of the assay.

Several investigators have evaluated the immune response to individual pneumococcal polysaccharide serotypes (13-21), but some issues remain unsolved. Because the polysaccharide pneumococcal vaccine contains 23 serotypes, defining the serotype antibody responses to be measured is a major concern, and no reliable data are available on cutoff values. Most studies use the same cutoff concentration for all serotypes despite differences in immunogenicity. Finally, guidelines for interpreting the global antibody response to caps-PS have not been validated.

The present study was undertaken to determine reference (cutoff) values for the serotype-specific antibody response to caps-PS and to validate criteria for defining patients with a defect in the antibody response to caps-PS.

Materials and Methods

MATERIALS

Pneumovax[R] was obtained from Aventis Pasteur MSD. Pneumococcal caps-PS of serotypes 3, 4, 9N, 14, 18C, 19F, and 22F were obtained from ATCC. C-PS was from Statens Serum Institute. NaCl 9 g/L was from Braun Medical NV./SA. Peroxidase-conjugated goat-antihuman IgG was from Nordic hninunological Laboratories. Goat serum and phosphate-buffered saline (PBS; 2.67 mmol/L KCl, 1.47 mmol/L KH2P04, 137.93 mmol/L NaCl, 8.06 mmol/L Na2HP04-7H20) were from Gibco BRL, Life Technologies Ltd. Polyoxyethylenesorbitanmonolaurate (Tween 20) and TMB (3,3'-5,5'-tetramethylbenzidine) were from Sigma-Aldrich NV/SA. [H.sub.2]S[O.sub.4] solution was from Merck KgaA. Covalink NH and Maxisorp 96-well ELISA plates were obtained from NUNC Brand Products, Nalge Nunc International.

DETERMINATION OF TYPE-SPECIFIC ANTIPOLYSACCHARIDE ANTIBODIES

Serum antibody concentrations to 5 pneumococcal serotypes (3, 4, 9N, 18C, and 19F) were measured by ELISA 3 weeks after immunization with Pneumovax. Covalink NH ELISA (for serotype 3) and Maxisorp 96-well ELISA plates (Nunc) (for serotypes 4, 9N, 18C, and 19F) were coated overnight at 37 [degrees]C with 100 [micro]L pneumococcal polysaccharide (final concentration 3.3 mg/L in NaCl, 9 g/L). After coating, the plates were washed 4 times with 200 [micro]L of 0.5 mL/L Tween 20 in PBS. Thereafter, the plates were blocked for 1 h at 37 [degrees]C with 100 [micro]L 100 mL/L goat serum in PBS per well. Serum was diluted 200-fold in absorption solution containing PBS with 20 mL/L goat serum, C-PS (5 mg/L), and caps-PS 22-F (5 mg/L). Thereafter, five 2-fold serial dilutions were prepared in absorption solution. The diluted samples were incubated for 30 min at room temperature. We added 50 [micro]L of the diluted serum samples to each well and incubated for 2 h at 37 [degrees]C. After 4 washes with 200[micro]L of 0.5 mL/L Tween 20 in PBS, peroxidase-conjugated goat antihuman IgG in a 1/5000 dilution was added to the wells. The plates were incubated for 1.5 h at 37 [degrees]C. After 4 washes with 200[micro]L of 0.5 mL/L Tween 20 in PBS, 100 [micro]L TMB was added for color development. After 30-min, the reaction was stopped by adding 100 [micro]L of 0.5 mol/L [H.sub.2]S[O.sub.4] to each well. Thereafter, plates were read at 450 mn. Absorbance data were converted to antibody concentrations with the computer program CDC ELISA (Plikaytis BD, Holder PF, Carlone GM, Program ELISA for Windows, version 09/27/2004, US Department for Health & Human Services), which uses a 4-parameter logistic-log method to perform a curve-fitting procedure. As a reference (standard), the US Pneumococcal Reference Serum Lot 89-SF was used.

The ELISA described above is based on the 3rd-generation assay format (22) (absorption with C-PS and caps-PS 22-F) with some modifications. We initially performed the assay described by Wernette et al. (22) using conventional polystyrene microtiter plates (Maxisorp) for all serotypes. This method displayed a bad linearity for measurement of antibodies to serotype 3, as shown in Fig. 1 in the Data Supplement that accompanies the online version of this manuscript at http://www.clinchem.org/content/vol53/issue3. Linearity for serotype 3 was appreciably improved by using the assay described above, in which the method of Sanders et al. (16) was applied using Covalink NH 96-well plates. A linearity analysis for this assay is shown in Fig. 2 in the online Data Supplement. In the assay described by Sanders et al. (16), coating was performed overnight in NaCl 9 g/L at 37 [degrees]C, whereas in the protocol of the 3rd generation assay format (22) (described in http://www.vaccine.uab.edu), coating was performed in PBS for 5 h at 37 [degrees]C, and the plates were stored at 4 [degrees]C. The assay of Sanders et al. (16) used a peroxidase-labeled antihuman antibody, whereas the method of Wernette (22) used an alkaline-phosphatase-conjugated antibody. Fig. 3 in the online Data Supplement shows the correlation between the assay of Wernette and the assay described in this protocol.

The ELISA was performed in 2005-2006 by 2 experienced (>10 years) technologists who were not blinded to the results of the other test. Outliers were not excluded. The sera were frozen before analysis.

CONTROL AND PATIENT POPULATIONS

We examined the antibody response of 37 child and 39 adult controls (recruited between 2000 and 2005) and 82 consecutive patients (recruited between 2000 and 2003) who were referred to our institution because of recurrent infections of the upper and lower airways. Recurrent infections of the upper respiratory tract were defined as at least 5 episodes (in a 1-year period) of upper respiratory tract infections complicated by otitis media or chronic (longer than 3 weeks duration) draining ears. Recurrent infection of the lower respiratory tract was defined as at least S lower respiratory tract infections in a 1-year period with radiographic evidence of pneumonia in at least 2 of these periods. The age distribution of the child controls was 1, 8, 12, and 16 for the age groups, 3-4, 5-6, 7-9, and 10-15 years. No children aged [less than or equal to]2 years were included in the control group. The adults were healthy students 19-30 years of age (median 21 years of age). The age distribution of the patient group was 46, 17, 11, and 8 for the age groups 3-4, 5-6, 7-9, and 10-23 years. No individuals were excluded from the tests.

Approval of the study was granted by the local ethical committee of the Catholic University Leuven.

STATISTICAL METHODS

Differences in antibody responses between the adult and the child controls and between the different age groups were evaluated by use of Kruskall-Wallis ANOVA with posthoc Mann-Whitney tests (and Bonferoni adjustment). A [chi square] test was performed when indicated. Passing and Bablok analysis was performed to evaluate linearity. Statistical analysis was performed with Analyze It for Excel (version Windows 2003).

Results

IMPRECISION OF THE ASSAY FOR QUANTIFICATION OF SEROTYPE-SPECIFIC ANTIBODIES

Estimates of interrun imprecision (as CV, n = 17-20 runs per serotype) were 9%, 6%, 18%, 8%, and 8%, for, respectively, serotypes 3, 4, 9N, 18C, and 19F at concentrations of 1.6, 9.9, 1.1, 1.2, and 7.7 mg/L.

POSTVACCINATION ANTIBODY TITERS IN CONTROLS. CUTOFF CONCENTRATIONS FOR ANTIBODIES TO SEROTYPES 3, 4, 9N, 18C, AND 19F

Table 1 shows anti-caps-PS antibody concentrations to serotypes 3, 4, 9N, 18C, and 19F in 39 adult controls and 37 healthy children immunized with Pneumovax[R]. Table 1 of the on-line Data Supplement shows reference values (n = 72) for serotype 14. For serotype 3 and serotype 4, antibody concentrations in children were higher than in adults (P = 0.008 Mann-Whitney U-test).

We evaluated age-dependent differences in the ability to develop antibodies against different polysaccharides (14). In children <16 years of age, no statistically significant difference was observed among age groups (Table 2). A serotype-specific response below the 5th percentile (Table 1) was defined as an inadequate response.

With a cutoff concentration for the total group (adults and children), 67 (88%) of the 76 controls responded adequately to a115 serotypes. One (1%) adult did respond adequately to only 1 serotype. Two (3%) individuals (1 child and 1 adult) did not respond adequately to 3 of the 5 serotypes, 4 (5%) (all adults) did not respond adequately to 2 of the 5 serotypes, and 2 (3%) (1 child and 1 adult) did not respond adequately to 1 serotype. The results of the controls who did not respond to 1 or more of the 5 tested serotypes are shown in Table 3. In the total control group, all individuals responded adequately to 5 serotypes.

EVALUATION OF THE ANTI-CAPS-PS ANTIBODY RESPONSE IN INDIVIDUALS REFERRED TO OUR INSTITUTION FOR MEASUREMENT OF CAPS-PS ANTIBODIES

The antipolysaccharide antibody concentrations after vaccination with Pneumovax in 82 patients referred to our institution for measurement of anti-caps-PS antibodies in the context of evaluation of the humoral immune function are shown in Table 3. The response to all 5 tested serotypes tested was adequate in 48 (58%) of the 82 patients, whereas 34 (41%)had an inadequate response to at least 1 serotype. Inadequate responses were seen in 16, 5, 4, 5, and 4 patients to 1, 2, 3, 4, and 5 serotypes, respectively. This distribution was statistically different from the distribution found in the control population (P = 0.0007, [chi square]). Nine patients (11%; 95% CI: 17.7%-4.2%) had a defective response to 4 or 5 serotypes, a condition observed in only 1 (1.3%; 95% CI: 0%-3.8%) control individual (P = 0.03, [chi square]). The ages of these children were 3 years (n = 3), 4 years (n = 2), 5 years (n = 3), and 14 years (n = 1).

Table 2 in the on-line Data Supplement shows detailed results for the patients and controls who did not react to at least 1 of the 5 serotypes tested. For each serotype, 4 (5%) of 76 control individuals had an inadequate response, a rate that corresponded with the cutoff, which was set at the 5th percentile. In the patient group, 8 (10%), 11 (13%), 17 (21%), 13 (16%), and 29 (35%) of individuals did not react to serotype 3, 4, 9N, 18C, and 19F, respectively. These data suggest that the immune response to serotype 19F is more sensitive than the immune response to serotype 3 to detect an impaired antipolysaccharide response. On the other hand, a defective immune response to serotype 3 is more predictive for a general failure to react to polysaccharide antigens. In many patients, an impaired immune response to serotype 3 is accompanied by an impaired immune response to other serotypes.

The patient group comprised a substantial number of 3-year-old children, which was not the case in the control group. Therefore, we confirmed that the antibody concentrations found in the patient group of 3 and 4 years of age were comparable to the antibody concentrations in the patient group of >4 years of age. The evidence is given in the on-line Data Supplement, (Text 1).

Discussion

The main problem in the evaluation of the antibody response to caps-PS is the lack of standardized criteria for interpretation of the functional test. There is no consensus about which serotypes of the 23-valent pneumococcal vaccine should be tested. Different studies select different serotypes and often it is not mentioned why particular serotypes are tested (Table 4). We selected serotypes 3, 4, 9N, 18C, and 19F because they are among the most frequently isolated serotypes in Belgium (23), and thus analysis of the immune response to these serotypes is relevant in Belgium.

Furthermore, there is no consensus about the cutoff that should be used for each serotype and about the number of serotypes to which a patient has to respond to be classified as a responder or a nonresponder. Javier et al. defined an abnormal response as a postimmunization antibody concentration not reaching either 1.3 mg/L or a 4-fold increase above the preimmunization concentration for at least 5 of the 9 serotypes (7). Epstein et al. considered a normal response a 2-fold or more increase in antibody titer, or an absolute increase to >0.3 mg/L in 8 or more of the 12 serotypes tested (5). Zora et al. used the same criteria per serotype as Epstein et al., but considered acceptable an antibody response to at least 6 of 12 measured serotypes (19). Sanders et al. defined a normal response to caps-PS as a 2-fold or greater increase in serum antibody titer and a postimmunization antibody concentration of at least 20% (20 U/L) of a hyperimmune plasma pool of healthy volunteers immunized with Pneumovax[R] (16). Nonresponsiveness was defined as nonresponsiveness to 5 or more of the 7 serotypes tested (16). Gigliotti et al. and Ambrosino et al. described a patient with a selective absence of IgG antibody response to all the serotypes tested (4, 20). Ekhdal et al. expressed the anti-caps-PS concentrations as geometric means. Patients with geometric mean postvaccination antibody concentrations <10% of the reference standard of the Statens Seruminstitut (Copenhagen, Denmark) were designated nonresponders (21). All patients tested did not respond to any of the 4 serotypes tested (21). Finally, Wasserman et al. stated that an adequate response to an individual serotype should be defined as a 4-fold or higher increase in serum antibody titer or a postimmunization antibody concentration of 1.3 mg/L or greater (6). Failure to respond normally to >50% of the serotypes tested should be considered abnormal (6). Taken together, several criteria are being used to evaluate the anti-caps-PS antibody response. A standardized evidence-based protocol is obviously lacking.

In our study, postimmunization antibody concentrations were compared with a hyperimmune plasma pool standard (FDA US Pneumococcal Reference Serum Lot 89-SF), as was done in several previous studies (14,16) and recommended by the guidance protocol for the 3rd-generation pneumococcal antibody ELISA (22).

The cutoff concentrations (5th percentile) determined in our study varied between 0.31 and 1 mg/L. Esposito et al. (18) suggested that IgG anticapsular antibody concentrations > 1 mg/L are required for protection for the majority of the serotypes (particularly serotype 1 and 19), whereas for serotype 4, an IgG antibody concentration of [greater than or equal to]0.15 mg/L seems to be protective. Sorensen et al. (13,14) and Wasserman et al. (6) estimated a protective concentration to be 1.3 mg/L, whereas Epstein (5) and Zora et al. (19) suggested a protective concentration of 0.3 mg/L. Protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. An antibody concentration of 0.35-0.4 mg/L is generally considered to be sufficient for protection (24).

All individuals of our control population responded well to at least 2 of the 5 tested serotypes except 1 adult who responded to only 1 serotype. More of the patients with a defect in the anti-caps-PS antibody response did not respond to 4 or 5 of the 5 tested serotypes (n = 9). An inadequate response to 4 or 5 of the 5 tested serotypes indicates a specific antipolysaccharide antibody deficiency. Our results also indicate that an inadequate response to serotype 3 predicts unresponsiveness to other serotypes. This finding has also been reported by Sorensen et al. (14), who found that patients who failed to respond to serotype 3 at any age also failed to develop protective concentrations of antibodies against the other serotypes.

Our reference values were obtained in a relatively small number of control persons. Moreover, our reference values in adults have been obtained with young adults, and cannot be applied to old adults.

Bacterial capsular polysaccharides induce antibodies primarily by T cell-independent mechanisms. Therefore, antibody response to most pneumococcal capsular types is generally poor or inconsistent in children aged <2 years whose immune systems are immature. This is the reason why no children aged 2 years or less were included in the control group. The cutoff concentrations proposed in this manuscript are valid for children >4 years of age. To overcome the problem of nonresponsiveness in children younger than 2 years, a conjugated vaccine (Prevnar[R]) was developed. This contains 2 [micro]g of serotype 4, 9V, 14, 18C, 19F, and 23F and 4 [micro]g of serotype 6B. Use of the conjugated vaccine will have an influence on the choice of serotypes to diagnose specific anti-caps-PS antibody deficiency. Once a patient has been vaccinated with Prevnar, the serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F can no longer be used for antibody measurement, because the evaluation would assess the response to protein antigens rather than to polysaccharide antigens. For patients who have previously been vaccinated with the conjugated vaccine, other serotypes must be chosen. Uddin et al. compared the total pneumococcal IgG antibody, IgG subclass antibody titers, and serotype-specific antibody concentrations (4, 6B, 9V, 14, 18C, 19F, 23F, 1, and 5) in children who had received 2 doses Prevnar followed by a dose of Pneumovax to evaluate the most useful assays in clinical practice (25). They determined that children after Prevnar vaccination produced mostly IgG1 antibodies in contrast to the IgG2 antibody response to natural infection with S. pneumoniae or pneumococcal polysaccharide vaccines. Uddin et al. concluded that serotype-specific IgGs were useful in determining protection against specific pneumococcal strains, and showed that Pneumovax did not broaden protection against non-Prevnar serotypes (25). The increased clinical use of Prevnar and possible use of the next generation conjugated vaccines, such as 9-valent (26-28) and 11-valent (29), will make it necessary to determine the response to additional serotypes in the future. On the other hand, potential new antigens for vaccine formulations, such as the surface proteins PspA, PspC, PsaA, pneumolysin, BVH-3, and BVH-11, will also need the establishment of reference values.

In conclusion, we established reference intervals for the specific antipolysaccharide immune response to 5 serotypes. Among 76 healthy controls (3-30 years of age), 75 adequately responded to at least 2 of these 5 serotypes, and 73 (96%), of the controls responded to at least 3 of these 5 serotypes. Patients who did not respond to at least 4 of the 5 serotypes were diagnosed as nonresponders.

X. Bossuyt is a senior clinical investigator of the Fund for Scientific Research--Flanders (FWO-Vlaanderen).

Received September 15, 2006; accepted December 28, 2006. Previously published online at DOI: 10.1373/clinchem.2006.080051

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AXEL JEURISSEN, [1] [dagger] LEEN MOENS, [1] [dagger] MARC RAES, [2] GREET WUYTS, [1] LUC WILLEBRORDS, [1] KATE SAUER, [2] MARIJKE PROESMANS, [2] JAN L. CEUPPENS, [3] KRIS DE BOECK, [2] and XAVIER BOSSUYT [1] *

[1] Laboratory Medicine, Immunology, University Hospital Leuven, Belgium.

[2] Department of Pediatrics, University Hospital Leuven, Belgium.

[3] Department of Internal Medicine, Allergy, University Hospital Leuven, Belgium.

[[dagger]] These authors contributed equally to this study.

* Address correspondence to this author at: Laboratory Medicine, Immunology, GHB-Herestraat 49, B-3000 Leuven, Belgium. Fax 32-16-34-79-31; e-mail Xavier.bossuyt@uz.kuleuven.ac.be.
Table 1. Median values, 5th and 25th to 75th percentile for the
antibody responses to caps-PS in control individuals

 5th percentile, Median, 25th-75th
 mg/L mg/L percentile,
 mg/L

Caps-PS3 All 0.67 4.5 2.1-8.5
 Children (a) 0.99 5.4 3.6-9.5
 Adults 0.58 2.9 1.6-6.0
Caps-PS4 All 0.45 3.8 2.1-8.4
 Children (a) 0.73 5.7 2.9-10
 Adults 0.37 2.9 1.2-5.0
Caps-PS9N All 0.46 4.8 2.5-10
 Children 0.78 5.4 2.7-9.6
 Adults 0.23 4.4 1.9-11
Caps-PS18C All 0.31 4.3 1.6-8.6
 Children 0.55 3.8 1.3-5.8
 Adults 0.31 4.9 1.9-9.3
Caps-PS19F All 1.04 5.7 2.6-15.6
 Children 1.66 6.4 3.8-15.9
 adults 0.91 4.4 2.0-15.4

(a) Statistically significantly different from adults
(Mann-Whitney U-test).

Thirty-seven child and 39 adult controls were immunized
with Pneumovax, and antibody titers were measured against
serotypes 3, 4, 9N, 18C, and 19F.

Table 2. Age-dependent anti-caps-PS antibody concentrations

 Antibody concentration: median
 (25-75th percentile), mg/ L

 Age (years)

Serotype 5-6 (n = 8) 7-9 (n = 12)
Caps-PS3 8.9 (4.3-18.2) 5.1 (4.4-8.7)
Caps-PS4 4.6 (3-8.8) 7.6 (4.4-10.1)
Caps-PS9N 4.3 (1.6-8.4) 7.7 (4.1-10)
Caps-PS18C 1.6 (0.8-4.3) 4.7 (2.2-7.5)
Caps-PS19F 4.5 (2.4-7.8) 6.2 (4.0-18.2)

Serotype 10-15 (n = 16) 18 (n = 39)
Caps-PS3 4.1 (1.8-9.6) 2.9 (1.6-6)
Caps-PS4 3.9 (2.2-10.2) 2.9 (1.2-5.1)
Caps-PS9N 4.8 (2.3-7.0) 4.4 (1.9-10.9)
Caps-PS18C 3.5 (1.3-8.6) 4.7 (1.9-9.3)
Caps-PS19F 9.7 (5.2-31) 4.4 (2.0-15.4)

Thirtyseven child and 39 adult controls were immunized
with Pneumovax, and antibody titers were measured
against serotypes 3, 4, 9N, 18C, and 19F. The
anti-caps-PS antibody concentrations were classified
according to age. The values found in 1 child 3 years
of age were 10.4 mg/L, 45.1 mg/L , 19.1 mg/L, 12 mg/L,
and 6.4 mg/L for serotype and 3, 4, 9N, 18C, and 19F,
respectively.

Table 3. Controls and patients were vaccinated with
Pneumovax.

Number of serotypes Controls (n = 76) Patients (n = 82)
to which an inadequate
response was generated

0 67 48
1 2 16
2 4 5
3 2 4
4 1 5
5 0 4

Antipolysaccharide antibodies to 5 serotypes were measured
3 weeks after vaccination. The table shows the number of
patients and controls with an inadequate response to
0, 1, 2, 3, 4, and 5 serotypes. The differences between
controls and patients were significant (P = 0.0007, [chi square]).

Table 4. Serotypes tested in different studies.

Reference Serotypes Measured

Ambrosino et al. (4) 3, 6, 14, 23
Epstein et al. (5) 1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C
 19F, 23F
Javier et al. (4) 1, 3, 4, 6B, 9V, 14, 18C, 19F, 23F
Sorensen et al. (12) 3, 7F, 9N, 14
Sorensen et al. (13) 1, 3, 4, 6B, 9V, 14, 18C, 19F, 23F
Sanders et al. (15) 3, 4, 6A, 9N, 14, 19F, 23F
Rijkers et al. (16) 3, 4, 6A, 9N, 14, 19F, 23F
Esposito et al. (17) 3, 4, 5, 7F, 9V, 14, 18C, 6B, 19F, 23F,
Zora et al. (18) 1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C
 19F, 23F
Gigliotti et al. (19) 1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C
 19F, 23F
Ekhdal et al. (20) 3, 6A, 19F, 23F
Uddin et al. (25) 1, 4, 5,6B, 9V, 14, 18C, 19F, 23F
This study 3, 4, 9N, 18C, 19F
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Title Annotation:Clinical Immunology
Author:Jeurissen, Axel; Moens, Leen; Raes, Marc; Wuyts, Greet; Willebrords, Luc; Sauer, Kate; Proesmans, Ma
Publication:Clinical Chemistry
Date:Mar 1, 2007
Words:5012
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