Serum samples
Four quality control (QC) sera with very high (QCVH), high (QCH), medium (QCM), or low (QCL) titer sera prepared by mixing sera from 2 to 3 individuals (age range = 26 to 42 years) and were previously described [11, 12]. Their reference ranges of anti-Hib antibody titer were assigned after performing anti-Hib-antibody ELISA assay for more than 50 times [11]. Their reference ranges (mean ± standard deviation [SD]) were 43.00 ± 6.54 μg/mL, 4.38 ± 0.50 μg/mL, 1.52 ± 0.18 μg/mL, and 0.27 ± 0.07 μg/mL for QCVH, QCH, QCM, and QCL, respectively [11]. These sera were stored in 200-μL aliquots at −70 °C.
Ten pre-immune sera and 80 post-immune sera were selected based on their serum availability from a cohort of infants participating in an immunogenicity study of the Hib vaccine in Korean infants [12]. Anti-Hib IgG levels were previously determined for these residual sera [12] and 0.15 μg/mL was used as the lower limit of assay [11]. They were vaccinated with a single Hib vaccine (PRP-T or PRP-OMP).
A high throughput SBA assay
SBA was performed as described [13] with modifications described below. All serum samples were heated at 56 °C for 30 min before testing was performed in duplicate. The heat inactivated sera were serially (three fold) diluted in a dilution buffer (Hanks’ buffer with Ca2+ and Mg2+ [Life Technologies, Grand Island, NY, USA] and 0.1 % gelatin). A frozen aliquot of Hib Eagan strain [14] was diluted in the dilution buffer to yield 750 colony forming unit (CFU) in 10-μL. Twenty μl of diluted serum was mixed with 10 μL of Hib solution and 10 μL of baby rabbit complement (Pel-Freez Biologicals, Brown Deer, WI, USA) in a microwell. The mixture was incubated for 2 min at 25 °C with shaking at 700 rpm and incubated for 30 min at 37 °C in a CO2 incubator without shaking. After stopping the reaction by placing the plates on ice for 15 min, 10 μL of the reaction mixture was plated on an approximately 1 cm by 3 cm area of a brain-heart-infusion (BHI) agar plate with 2 % Fildes enrichment (Becton Dickinson and Company, Sparks, MD, USA). After the fluid was absorbed into the agar, molten BHI agar (0.75 %) with Fildes enrichment (2 %) and 25 mg/L 2, 3, 5-triphenyl tetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) was poured on top of the bottom agar layer.
The plates were incubated at 37 °C in a 5 % CO2 incubator for 16 h. Surviving bacterial colonies on the plates were counted with NICE (NIST [National Institute of Standards and Technology]’s Integrated Colony Enumerator); a free software [15] available from Dr. J. Whang in the US NIST) (http://www.nist.gov/pml/electromagnetics/grp05/nice.cfm). The colony counts were used to determine the serum bactericidal index (SBI). The SBI of a serum was defined as the dilution of the serum that results in half as many colonies as are seen with complement controls. If an undiluted serum sample killed 50 %, then the SBI is 4 in our system. To reduce the effect of variable activation of the alternative pathway on SBI result, each assay run included a complement control containing bacteria and baby rabbit complement with no serum. This complement control was used as 0 % killing in the SBI calculation. A control serum was included in each assay to monitor assay reproducibility. A detailed SBA method is posted on the following website: http://www.vaccine.uab.edu/.
Reproducibility of SBA assays
The reproducibility of the Hib SBA was evaluated using the four QC sera, QCVH, QCH, QCM, and QCL. To determine intra-assay variability, sera were tested 5 times in one assay run. To determine short-term inter-assay variability, 11 independent assays using the same lot of reagents were performed but on different days.
To determine the effect of varying assay components, the assay was performed once with two different batches of bacterial stock and once with two different batches of complement. The overall mean ± SD and the coefficient of variation (CV) of SBIs were calculated.
To assess long term assay performance, SBA was performed with three QC sera (QCVH, QCH, and QCL) over a 6 year period and their bactericidal indices were plotted on a Levey-Jennings chart. During this period, the assay involved four different lots of baby rabbit complement, two different lots of Hib bacteria, and three assay operators.
Statistical analysis
Correlations between SBAs were determined by Pearson’s correlation. Significant differences among assays were determined by Student’s t test. Comparisons between paired data were done by chi-square or Fisher’s two-tailed exact test. The significance level was set at a P value of <0.05.
Ethics statement
The study protocol was approved by the Institutional Review Board of Ewha Womans University Hospital. The study was conducted in accordance with good clinical practices (national regulations and ICH E6) and the principles of the Helsinki Declaration. Informed written consent was obtained from all participants or their parents or legal guardians following a detailed explanation of the study.