Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis

Abstract

Serological diagnosis of pertussis is mainly based on anti-pertussis toxin (PT) IgG antibodies. Since PT is included in all acellular vaccines (ACV), serological assays do not differentiate antibodies induced by ACVs and infection. Adenylate cyclase toxin (ACT) is not included in the ACVs, which makes it a promising candidate for pertussis serology with the specific aim of separating infection- and ACV-induced antibodies. A multiplex lateral flow test with PT and ACT antigens was developed to measure serum antibodies from pertussis-seropositive patients (n = 46), healthy controls (n = 102), and subjects who received a booster dose of ACV containing PT, filamentous hemagglutinin, and pertactin (n = 67) with paired sera collected before and one month after the vaccination. If the diagnosis was solely based on anti-PT antibodies, 98.5–44.8% specificity (before and after vaccination, respectively) and 78.2% sensitivity were achieved, whereas if ACT was used in combination with PT, the sensitivity of the assay increased to 91.3% without compromising specificity. No increase in the level of anti-ACT antibodies was found after vaccination. This exploratory study indicates that the use of ACT for serology would be beneficial in combination with a lower quantitative cutoff for anti-PT antibodies, and particularly in children and adolescents who frequently receive booster vaccinations.

Oral fluid-based lateral flow point-of-care assays for pertussis serology

“…Apparently, the LF assay was not able to quantify ACT antibodies from oral fluid samples with sufficient specificity: only 20 samples had a detectable signal response of over 0.12 t/c-ratio, and no differences in ACT antibody concentration were noted between samples of low (<50 aU) and high (>50 aU/ml) anti-PT IgG concentrations. This may reflect our previous finding with sera, both LF and ELISA [16], ACT antibodies were roughly only one-tenth the quantity compared to those of PT following pertussis infection. Although oral fluid anti-PT antibodies correlate strongly with serum titres [15], the overall concentration of IgG in oral fluid is approximately 1000 times lower than in serum [39].…”

“…Anti-ACT antibodies were studied as an additional antigen to potentially improve diagnostics instead of measuring solely PT-specific antibodies [16,17]. Apparently, the LF assay was not able to quantify ACT antibodies from oral fluid samples with sufficient specificity: only 20 samples had a detectable signal response of over 0.12 t/c-ratio, and no differences in ACT antibody concentration were noted between samples of low (<50 aU) and high (>50 aU/ml) anti-PT IgG concentrations.…”

“…The LF test strips were assembled as previously described [16]. In brief, 100 ng per test of rabbit anti-goat IgG (G4018; Sigma-Aldrich) as the control line, 100 ng per test of purified native PT (GlaxoSmithKline Biologicals) as the first test line and 250 ng per test of CyaA-adenylate cyclase toxoid [26] as the second test line were dispensed in 10 mM Tris-HCl (pH 8.0) buffer evenly on a nitrocellulose membrane (Hi-Flow Plus HF90; Millipore).…”

“…Therefore, quantitative anti-PT immunoassays and population-specific cut-offs are needed for diagnostics with known personal vaccination history and possible exposure information. An additional approach to potentially improve diagnosis was to include B. pertussis antigens that are not included in current acellular vaccines, such as adenylate cyclase toxin (ACT) [16,17].…”

“…An additional approach to potentially improve diagnosis was to include B. pertussis antigens that are not included in current acellular vaccines, such as adenylate cyclase toxin (ACT) [16, 17].…”