Development of a serological test, ELISA in curiel, to evaluate
the immunogenic activity of diphtheria vaccines

Diphtheria is an infectious disease prevented by vaccination. To ensure the

safety and immunogenicity of these vaccines, the regulatory entities demand in

vitro and in vivo techniques. In vitro techniques, such as ELISA, reduce the

number of animals, are more sensitive, faster and cheaper, then, currently work

is focused on its implementation. Therefore, the purpose of this work was to

develop a serological assay, ELISA in curiel, that allow the determination of the

immunogenic activity of this type of vaccines. The batches of reference

materials (RM) were prepared: Diphtheria coating toxoid, curiel serum,

calibration curve and positive control. The optimal conditions of the assay were

established, the interval and linearity of the curve were defined, as well as the

intra- and inter-assay precision and the specificity. The results of the serological

test, ELISA in curiel, and the in vivo seroneutralization test dose were

compared. The calibration curve was generated with a standard serum and a

good linear adjustment was achieved with a R2 ≥ 0,98; stablishing 10Lf/mL as

the optimal coating concentration, 1/12000 as the dilution of conjugate and an

incubation temperature of 37ºC. The variation coefficients in the intra- and inter

assay precision tests in the intervals established for each one were ≤ 10% and

≤ 20% respectively, showing an adequate correlation between both tests. The

results obtained endorse the employment of this quantitative ELISA for the

evaluation of the immunogenic activity of antidiphtheric vaccines.