Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines

Abstract

Enzyme linked immunosorbent assay (ELISA) has been widely used to measure antibody titers for evaluating the immunogenicity of a vaccine. However, there is as yet no generally accepted way of expressing the ELISA results in the case of experimental vaccines, since there is usually no uniform standard. Both end point and single dilution methods have significant disadvantages. In this paper, we obtained reproducible data with fewer dilutions of samples by addition of serially diluted standard serum to each ELISA plate. Since this ELISA method gives reliable antibody titer with less labor than other methods, it can strongly support vaccine development.

Fig. 1

Comparison of ELISA antibody units derived from a single versus multiple serial dilutions. Antibody units were calculated from O.D.₄₀₅ of a single dilution referring to a standard curve and are plotted on the X-axis. The same sample was serially diluted to generate a complete dilution curve. From the dilution curve, the reciprocal of the dilution (Reci. Dilution) giving an O.D.₄₀₅ = 1 was calculated (on the Y-axis). (a) Eight anti-Pfs25 rabbit sera were tested with Pfs25 coated plates. Spearman Rank Correlation (SRC) of two data sets is 1.0 (p<0.0001). (b) Ten anti-Pvs25 rhesus monkey sera were tested with Pvs25 coated plates. SRC of two data sets is 1.0 (p<0.0001).

Fig. 2

Effect of varying substrate incubation time on O.D. ₄₀₅ and on ELISA antibody units. Anti-AMA1 rhesus monkey sera were tested. After adding substrate to the ELISA plates, O.D. ₄₀₅ of a standard and test samples were read at 20, 35 and 60 minutes without adding stopping buffer. (a) O.D. ₄₀₅ of the serially diluted standard sera at the three time points; (b) % increase above background of O.D. ₄₀₅ of each sample compared with the O.D. at 20 min; (c) % increase of antibody units of each samples compared with the antibody units at 20 min.

Fig. 3

Effect of exposure time of coating antigen on ELISA plates. ELISA plates were coated with specific antigens overnight or for two days before washing. Aliquoted and frozen sera were freshly thawed just before applying to the coated ELISA plates. (a) Anti-Pvs25 rhesus monkey serum was tested with Pvs25 coated plates (2 plates for each condition). (b) Anti-AMA1 mouse serum was tested with AMA1 coated plates (3 plates for each).

Fig. 4

Quality control parameters for an ELISA standard curve. The R² of a standard curve (fitted to a 4- parameter hyperbolic curve) is plotted on the X-axis and the arithmetic mean value of O.D.₄₀₅ of 4 blank wells on the same plate is plotted on the Y-axis. The data (114 plates) from mice immunized with Pvs25, Pfs25 or AMA1, rabbits immunized with Pfs25, and monkeys immunized with Pvs25 or AMA1 are included in this figure.

Fig. 5

Impact of O.D. ₄₀₅ on ELISA antibody units. Animal sera were serially diluted and tested by ELISA (78 samples, 261 data points). A relative unit for each dilution was calculated by comparison with the units that came from the dilution which gave an O.D. ₄₀₅ closest to 1. The relative units of 1, as a definition, are not shown in this figure. Sera were collected from mice immunized with Pfs25 or Pvs25, rabbits immunized with Pfs25, and monkeys immunized with Pvs25.

Fig. 6

Relationship of ELISA O.D. ₄₀₅ to percent coefficient of variation (%CV) of triplicate wells. The arithmetic mean O.D.₄₀₅ of triplicate wells tested by ELISA is plotted on the X-axis and the %CV of antibody units obtained from triplicate wells is plotted on the Y-axis. The data (1241 points) include mice immunized with Pvs25, Pfs25 or AMA1, rabbits immunized with Pfs25, and monkeys immunized with Pvs25 or AMA1.

Fig. 7

Reproducibility of ELISA antibody units. Anti-AMA1 rhesus monkey sera (112 samples) were tested by ELISA twice at an interval of 15 months. The sera were aliquoted and frozen at -80°C before use. The concordance between the two sets of the data is highly significant (coefficient was 1.01; 95%CI, 0.97-1.06; p<0.001).