Serum samples. A total of 19 serum samples and one immune globulin preparation were used in the different assays. Sera were obtained after informed consent and protocol exemption by the CDC. Seven sera from normal, healthy, adult donors (age range, 24 to 60 years) were obtained through Emory Donor Services, Atlanta, GA; 12 unlinked cord blood sera were provided by Alberto Villaseñor Sierra (CIBO, IMSS, Jalisco, México) and contained only IgG antibodies. These 12 sera were selected from a larger bank of 69 cord blood sera collected from healthy mothers. Sera were selected randomly if enough volume was available and if ELISA was positive (cutoff level of 0.09). An immune globulin preparation was also used (Gamunex 10%; Talecris Biotherapeutics, Inc., Clayton, NC). Sera were stored frozen at −70°C in 500-μl aliquots until use.
Complement source. The complement source was sterile serum from 3- to 4-week-old baby rabbits (Pel-Freez, Brown Deer, WI) previously used in the Hib SBA (
15). Although the concentration of cross-reactive antibodies was not measured in the complement lots, three different complement lots were qualified for use on both assays by testing active and heat-inactivated complements. The lot chosen (8028) had 14% killing (level of acceptability is less than 25% killing) compared to time zero inoculum, where the inactivated complement had no killing.
Bacterial strains. Twelve bacterial strains were used in the SBA assays (Table ). Six strains were obtained from the CDC Active Bacterial Core surveillance (ABCs) from Georgia (1989 to 2007) (GA41512, GA41513, GA18491, GA11151, GA44497) (
12) and Oregon (M8881). Five strains were obtained from the CDC Arctic Investigations Program (AIP) (2000 to 2007) (AK1435C3, AK137339, AK137341, AK137342, AK137865). The last strain (ATCC 9006) was obtained from the American Type Culture Collection (ATCC). Seven isolates were obtained from sterile sites and caused invasive disease, and five were obtained from the colonized nasopharynx. Three isolates (GA41512, GA41513 [
12], and M8881 [Satola personal communication] had the partial IS
1016-bexA deletion, a mutation possibly associated with increased virulence in Hia (
1).
| TABLE 1.Haemophilus influenzae type a strains used for SBA evaluation |
Each Hia strain was plated on chocolate II solid medium overnight. Few colonies were selected from the medium and added to brain heart infusion (BHI) broth, supplemented with 2% Fildes enrichment (BBL, Becton Dickinson and Co., Sparks, MD), and then incubated at 37°C and 5% CO2 until optical density at 600 nm reached an average of 0.4. Aliquots were flash frozen and stored at −70°C for future use. A frozen aliquot of each strain was diluted to yield 1,000 bacteria in a 20-μl volume (amount added to each well).
Standard slide agglutination capsule serological testing was performed as described by the manufacturer of the antiserum (Bacto-Difco Diagnostic Systems), and PCR molecular capsule typing was performed as described by Falla et al. (
5).
SBA assays. SBA assays (with a viability endpoint [CFU counts] and with a fluorometric endpoint) were performed as described by Romero-Steiner et al. (
15) for Hib, which is a modification of the method described by Schlesinger and Granoff (
17).
SBA assays were performed on four separate days for each strain by two independent researchers blinded to the assay results using either method.
Ten negative-control sera with negative SBA titers using the reference strain AK1435C3 were used but were not included in the comparison of strains or the final analysis.
SBA assays. (i) SBA assay with a viability count endpoint. Serum samples were tested with a starting dilution of 1:8. Two-fold serum serial dilutions were made in 10 μl of Hanks buffer with Ca2+ and Mg2+ (Life Technologies, Grand Island, NY) supplemented with 2% Fildes enrichment. Each bacterial strain was diluted to yield 1,000 bacteria in 20 μl inoculum per well. After 15 min of incubation of the serum and the bacterial strain at 37°C and in 5% CO2, 20 μl of baby rabbit complement was added to each well. An additional 30 μl of the dilution buffer was added to each well to bring the total volume of the reaction to 80 μl. After an incubation of 60 min at 37°C in 5% CO2, 5 μl from each well was plated onto chocolate agar plates using a tilt method for better definition of the CFU (ChocII; BBL, Becton Dickinson and Co., Sparks, MD). After 16 h of incubation at 37°C in 5% CO2, viability counts were performed and SBA titers were determined. SBA titers were defined as the reciprocal of the serum dilution that resulted in more than 50% killing compared to the growth in the complement controls.
(ii) SBA assay with a fluorometric endpoint. alamarBlue (Trek Diagnostics, Westlake, OH) is a commercially available metabolic indicator. In the presence of viable bacteria, alamarBlue is reduced and a color change from blue to pink occurs. The reduced compound is also fluorescent with an emission wavelength of 590 nm if excited by a UV light source at a wavelength of 530 nm. Therefore, the SBA assay using alamarBlue could be used as an indirect method for the measurement of metabolism of the surviving bacteria after SBA assay, and the fluorescent units collected represent a viability endpoint.
The same protocol for the SBA assay using viability counts as endpoints applies except that 30 μl of alamarBlue buffer was used once 20 μl of the complement was added to each well. The alamarBlue buffer consisted of 16% alamarBlue, 64% Hanks buffer (containing Ca
2+, Mg
2+, and 2% Fildes enrichment), and 20% BHI broth (BBL). alamarBlue was found to be stable under SBA assay conditions and did not result in bacterial death in the absence of antibodies (
15). After a 6-h incubation period at 37°C in 5% CO
2, assay plates were read in a fluorometer (model FL 600 synergy; BIO-TEK Instruments Inc., Winooski, VT). Reagent blanks (without bacteria) were used in each plate. SBA titers were defined as the reciprocal of the serum dilution with 50% of the fluorescent units (FU) detected in the complement controls.
(iii) SBA assay reproducibility. The reproducibility of the SBA assays is evaluated using the immune globulin preparation with the 12 bacterial strains in 46 different independent assay runs.
(iv) SBA assay specificity for the Hia polysaccharide capsule. The specificity of SBA assay for the Hia polysaccharide capsule was determined by performing a competitive SBA assay in the presence of 100 μg/ml of purified reagent-grade (<1.2 μg/ml protein) Hia-specific capsular polysaccharide (
11) (and comparing the results with SBA titers obtained in the absence of added polysaccharide). For this evaluation, we used five selected bacterial strains (GA41512, GA41513, GA18491, GA11151, AK1435C3) and five selected healthy adult donor sera. We also evaluated the effect of heterologous Hib polysaccharide (provided by Moon Nahm, University of Alabama at Birmingham) in the Hia SBA assay. For this evaluation, we used the reference strain AK1435C3 and the same adult sera. Hib polysaccharide was added also at 100 μg/ml.
ELISA. Anti-Hia polysaccharide IgG concentrations (μg/ml) were determined on cord blood sera using an Hia ELISA. The Hia ELISA is based on an anti-Hib polysaccharide IgG ELISA method previously described (
6,
16). This optimized ELISA used a standard reference material which was assigned an anti-Hia polysaccharide IgG concentration (4.13 μg/ml). This anti-Hia IgG concentration was determined through a heterologous ELISA using the Hib standard reference material (FDA1983) as a calibrator serum (Schmidt, unpublished) based on the cross-standardization method described by Concepcion and Frasch (
4).
Sera with negative ELISA (levels less than 0.01) were used as negative controls but were not included in the analysis.
Statistical analysis. Correlations between SBA assays were determined by Pearson's product moment correlation coefficient by use of Sigma Plot and Sigma Stat software, version 2.0 (SPSS, Inc., Chicago, IL). Significant differences among assays were determined by Student's t test or the Mann-Whitney rank sum test for data not normally distributed. The significance level was set at a P value of less than 0.05. The concordance correlation coefficients (CCC) were calculated to estimate the degree of agreement between pairs of log2-transformed SBA titers. Paired comparisons were made for each strain and AK1435C3 (CDC reference strain).
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