Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides.

Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.

Development of A Standardized Opsonophagocytosis Killing Assay for Group B Streptococcus and Assessment in an Interlaboratory Study.

The placental transfer of antibodies that mediate bacterial clearance via phagocytes is likely important for protection against invasive group B Streptococcus (GBS) disease. A robust functional assay is essential to determine the immune correlates of protection and assist vaccine development. Using standard reagents, we developed and optimized an opsonophagocytic killing assay (OPKA) where dilutions of test sera were incubated with bacteria, baby rabbit complement (BRC) and differentiated HL60 cells (dHL60) for 30 min. Following overnight incubation, the surviving bacteria were enumerated and the % bacterial survival was calculated relative to serum-negative controls. A reciprocal 50% killing titer was then assigned. The minimal concentrations of anti-capsular polysaccharide (CPS) IgG required for 50% killing were 1.65-3.70 ng/mL (depending on serotype). Inhibition of killing was observed using sera absorbed with homologous CPS but not heterologous CPS, indicating specificity for anti-CPS IgG. The assay performance was examined in an interlaboratory study using residual sera from CPS-conjugate vaccine trials with international partners in the Group B Streptococcus Assay STandardisatiON (GASTON) Consortium. Strong correlations of reported titers between laboratories were observed: ST-Ia r = 0.88, ST-Ib r = 0.91, ST-II r = 0.91, ST-III r = 0.90 and ST-V r = 0.94. The OPKA is an easily transferable assay with accessible standard reagents and will be a valuable tool to assess GBS-specific antibodies in natural immunity and vaccine studies.

Quantifying Circulating IgY Antibody Responses against Select Opportunistic Bacterial Pathogens and Correlations with Body Condition Factors in Wild American Alligators, Alligator mississippiensis.

Little is known about the disease ecology of American alligators (Alligator mississippiensis), and especially how they respond immunologically to emerging infectious diseases and zoonotic pathogens. In this study, we examined serum samples collected from wild alligators in Florida (2010-2011) and South Carolina (2011-2012, 2014-2017) for antibody responses to multiple bacteria. Immunoglobulin Y (IgY) was purified from serum to generate a mouse monoclonal antibody (mAb AMY-9) specific to the IgY heavy chain. An indirect ELISA was then developed for quantifying antibody responses against whole cell Escherichia coli,Vibrio parahaemolyticus, Vibrio vulnificus, Mycobacterium fortuitum, Erysipelothrix rhusiopthiae, and Streptococcus agalactiae. In Florida samples the primary differences in antibody levels were between January-March and late spring through summer and early fall (May-October), most likely reflecting seasonal influences in immune responses. Of note, differences over the months in antibody responses were confined to M. fortuitum, E. rhusiopthiae, V. vulnificus, and E. coli. Robust antibody responses in SC samples were observed in 2011, 2014, and 2015 against each bacterium except E. coli. All antibody responses were low in 2016 and 2017. Some of the highest antibody responses were against V. parahaemolyticus, M. fortuitum, and E. rhusiopthiae. One SC alligator estimated to be 70+ years old exhibited the highest measured antibody response against V. parahaemolyticus and M. fortuitum. By combining data from both sites, we show a clear correlation between body-mass-indices (BMI) and antibody titers in all six of the bacteria examined. Our study provides a critical antibody reagent and a proof-of-concept approach for studying the disease ecology of alligators in both the wild and in captivity.

Serologic Testing of US Blood Donations to Identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Reactive Antibodies: December 2019-January 2020.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019, with subsequent worldwide spread. The first US cases were identified in January 2020. METHODS: To determine if SARS-CoV-2-reactive antibodies were present in sera prior to the first identified case in the United States on 19 January 2020, residual archived samples from 7389 routine blood donations collected by the American Red Cross from 13 December 2019 to 17 January 2020 from donors resident in 9 states (California, Connecticut, Iowa, Massachusetts, Michigan, Oregon, Rhode Island, Washington, and Wisconsin) were tested at the Centers for Disease Control and Prevention for anti-SARS-CoV-2 antibodies. Specimens reactive by pan-immunoglobulin (pan-Ig) enzyme-linked immunosorbent assay (ELISA) against the full spike protein were tested by IgG and IgM ELISAs, microneutralization test, Ortho total Ig S1 ELISA, and receptor-binding domain/ACE2 blocking activity assay. RESULTS: Of the 7389 samples, 106 were reactive by pan-Ig. Of these 106 specimens, 90 were available for further testing. Eighty-four of 90 had neutralizing activity, 1 had S1 binding activity, and 1 had receptor-binding domain/ACE2 blocking activity >50%, suggesting the presence of anti-SARS-CoV-2-reactive antibodies. Donations with reactivity occurred in all 9 states. CONCLUSIONS: These findings suggest that SARS-CoV-2 may have been introduced into the United States prior to 19 January 2020.