Molerular Immunology. Vol. 26, No. 3. pp. 269-214. 19X9 Printed m Great Britain. 0161.5890/89 S3.00f0.00 Pergamon Press plc zyxwvu ESTIMATION OF THE AVIDITY OF ANTIBODIES IN POLYCLONAL ANTISERA AGAINST STREPTOCOCCUS zyxwvutsrqponml PNEUMONIAE TYPE 3 BY INHIBITION ELISA COVERT J. VAN DAM, ANDRE F. M. VERHEUL, GUY J. W. J. ZIGTERMAN,* MARINUS J. DE REUVER and HARM SNIPPET Department of Immunology, Laboratory of Microbiology, State University of Utrecht, Catharijnesingel 59. 3511 GG Utrecht. The Netherlands and *Intervet International, P.O. Box 31. 5830 AA Boxmeer, The (First received 15 April 1988; accepted in reviscdfbrn~ 1 September 1988) zyxwvutsrqponmlkjihgfedcb Abstract-The reliability of the determination of antibody avidity in polyclonal sera by indirect sandwich ELISA was studied. Binding of IgM and IgG (sub)classes in unpurified serum to Streptococcuspneumoniae type 3 capsular polysaccharide, which was coated onto ELISA plates, was inhibited with different inhibitors. The inhibitor concn at which 50% inhibition of antibody binding to the ELISA coat was achieved, was used as a measure for antibody avidity. As this 50% inhibition value is dependent upon the dilution of the serum and thus upon the initial amount of free antibody, it is necessary to define (a narrow range of) final ELISA absorbance values to which the dilutions of non-inhibited sera have to be adjusted. The shapes of the serum dilution curves have a good correlation with the numerical 50% inhibition values of the antibody avidity. The inhibition ELISA is suitable to compare the avidity values of the different antibody isotypes, but two remarks should be made: (1) antibody heterogeneity should be considered to influence the results and prevent the accurate measurement of absolute numerical avidity values. Because in the ELISA system merely antibody “activity” is measured, comparison of the efficacy of vaccines by means of the 50% inhibition (avidity) value of various antibody (sub)classes can still be performed in a reliable way; (2) results of the determination of the 50% inhibition values of the different antibody (sub)classes showed them to be dependent on the molecular ratio between antibody (sub)class levels. More aspects of the determination should be taken into account, like shapes of simple dilution curves, influences of various inhibitor concns in the diluent and whole (extended) inhibition curves. INTRODUCTION In developing new vaccines several parameters are used to evaluate the efficacy of the vaccine. One of these parameters concerns the specificity of the vac- cine. This specificity can be measured as the avidity of the antibodies (or antiserum) for the antigen (Ag) against which the immune response is directed. The avidity is a parameter which is difficult to quantify and only a few techniques are available (Nieto et al., 1984; Devey et al., 1988). Zigterman et al. (1988) developed an ELISA for measuring antibody (Ab) levels in sera from mice immunized with vaccines against Streptococcus pneu- moniue type 3 (S3). In an ELISA system only relative amounts of antibodies can be determined and some authors use for ELISA data the expression “antibody activity” to designate a resultant of Ab concn and affinity (de Savigny and Voller, 1980). In this paper, inhibition of the anti-S3 sera by inhibitors derived from S3 polysaccharide (S3PS) was performed to estimate the avidity of these anti-S3 sera. In this “inhibition ELISA” avidities of IgM or IgG sub- classes could be measured in native sera by the use of tAuthor to whom correspondence should be addressed different (sub)class specific goat anti-mouse immuno- globulin-horseradish peroxidase (HRPO) conjugates. Several aspects of the inhibition process in the in- hibition ELISA, which influence the measurement of the antibody (Ab) avidity, were studied. MATERIALS AND METHODS Materials Non-ionic block polymers (NBP; for chemical properties see Zigterman et al., 1987) were obtained from BASF (Parsippany, NJ). Before use they were dispersed in phosphate buffered saline by intensive vortexing followed by sonification (MSE sonicator, 2 min, 40% of full scale energy). Keyhole limpet haemocyanin (KLH) was purchased from Calbio- them (La Jolla, CA) and bovine gamma globulin (BGG) from Sigma Chemicals (St Louis, MO). Mice and immunizution Female (CBA/N x BALB/c)F, mice were raised and maintained at the Laboratory of Microbiology, State University of Utrecht. CBA/N breeding pairs were a generous gift from Dr W. T. Watson (Food and Drug Administration, Bethesda, MD). All mice were used at an age of about 10 weeks and were 269