Background/Case Studies: Plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools as part of tetanus hyperimmune programs. This testing serves as a quality control test and helps estimate the antibody potency of the product. There are currently no anti-tetanus antibody assays for this quality control testing. The aim was to validate a quantitative Anti-Tetanus Toxoid IgG enzyme immunoassay kit using an automated enzyme-linked immunosorbent assay (ELISA) processor.
Study
Design/Methods: Specificity of the VaccZyme Anti-Tetanus Toxoid Ig G Immunoassay Kit was determined by testing 20 replicates of sample diluent using the kit on an automated ELISA processor. Analytical sensitivity of the kit was performed by performing a probit analysis on samples containing various concentrations of anti-tetanus immunoglobulin (0, 0.375, 0.75, 1.5, and 6 IU/mL) using the 2nd international Standard for Anti-Tetanus Immunoglobulin Human; NIBSC code: 13/240. Each concentration was tested a minimum of 10 replicates for 3 days. The mean, standard deviation (SD), and %CV were calculated for each concentration of all samples in the probit analysis. Linearity of the immunoassay kit was performed by testing samples at various concentrations of anti-tetanus immunoglobulin (0, 8, 16, 24, 32, and 40 IU/mL) by using the 2nd International Standard for Anti-Tetanus Immunoglobulin Human; NIBSC code: 13/240. Each concentration was tested a minimum of 5 replicates. The mean, SD, %CV, and % recovery was calculated for each concentration in the linearity study and the limit of quantitation (LOQ) was also calculated.
Results/Findings: The specificity of the assay was acceptable, all 20 replicates of the sample diluent were nonreactive when screened. The probit analysis of the assay determined the 95% LOD of the assay was 1.56 IU/mL. The qualitative accuracy of the assay passed since all anti-tetanus toxoid samples at 3 and 6 IU/mL were detected. All anti-tetanus concentrations tested had a %CV ≤ 15% for intermediate precision and a %CV ≤ 15% for precision within a run. Linear regression analysis demonstrated that r2 was >0.95, % recovery was between 85 – 115%, and %CV was ≤ 15%. The linear range was determined to be 8 – 40 IU/mL and Limit of Quantitation (LOQ) of the assay was calculated to be 8 IU/mL. Conclusions: The data presented shows the successful validation of the Human Anti-Tetanus Toxoid IgG Immunoassay Kit for use on an automated ELISA processor. Validation studies of this assay demonstrate excellent accuracy, precision and linearity.
Importance of research: For specialty products produced from human plasma, such as antibody collected and concentrated from a hyperimmunization program, new and innovative ways for measuring analytes is needed whenever a new program or immunization protocol is established. This abstract highlights how an existing technology can be modified and validated to be used for this purpose.
