Development and validation of magnetic bead pentaplex immunoassay for simultaneous quantification of murine serum IgG antibodies to acellular pertussis, diphtheria and tetanus antigens used in combination vaccines.

We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Assay validation experiments were done using a panel of serum samples. The results are expressed in IU/ml using WHO reference mice serum. The standard curve was linear with 4PL logistic fit over an eight 2-fold dilution range with LOQ of 0.003, 0.022, 0.005 IU/ml for PT, FHA and PRN and 0.016 U/ml for T and D antigens indicating sensitivity. No interference was observed in monoplex versus multiplex measurements. Specificity was demonstrated by ≥90% homologous and ≤15% heterologous inhibition for all the antigens. The assay was reproducible, with a mean coefficient of variation (CV) of ≤10% for intra-assay duplicates and ≤25% for interassays using different lots of beads and analyst. Accuracy was demonstrated wherein the ratio of observed vs. assigned unitages were within 80-120%. The study suggests that the Pentaplex (MIA) platform meets all the criteria for the serological assay combination vaccines with additional advantages of high throughput, reduced sample volumes, faster analysis with reduced manpower in contrast to conventional monoplex ELISA.

Immunoadjuvant potential of Asparagus racemosus aqueous extract in experimental system.

The immunoadjuvant potential of Asparagus racemosus (Willd.) Family (Liliaceae) aqueous root extract was evaluated in experimental animals immunized with diphtheria, tetanus, pertussis (DTP) vaccine. Immunostimulation was evaluated using serological and hematological parameters. Oral administration of test material at 100 mg/kg per day dose for 15 days resulted significant increase (P = 0.0052) in antibody titers to Bordtella pertussis as compared to untreated (control) animals. Immunized animals (treated and untreated) were challenged with B. pertussis 18323 strain and the animals were observed for 14 days. Results indicate that the treated animals did show significant increase in antibody titers as compared to untreated animals after challenge (P = 0.002). Immunoprotection against intra-cerebral challenge of live B. pertussis cells was evaluated based on degree of sickness, paralysis and subsequent death. Reduced mortality accompanied with overall improved health status was observed in treated animals after intra-cerebral challenge of B. pertussis indicating development of protective immune response. Present study indicates applications of test material as potential immunoadjuvant that also offers direct therapeutic benefits resulting in less morbidity and mortality.