Influence of hapten density on immunogenicity for anti-ciprofloxacin antibody production in mice

To generate antibodies against small molecules, it is necessary to couple them as haptens to large carriers such as proteins. However, the immunogenicity of the conjugates usually has no linear correlation with the hapten-protein ratio, which may lead to large variations in the character of the desired antibodies. In the present study, ciprofloxacin (CPFX) was coupled to bovine serum albumin (BSA) in five different proportions using a modified carbodiimide method. The conjugates were characterized qualitatively by spectrophotometric absorption and electrophoresis methods. Mass spectrometry and the trinitrobenzene sulfonic acid method were adopted to assay the density of conjugates quantitatively. As a result, CPFX-BSA conjugates with various hapten densities (21-30 molecules per carrier protein) were obtained. After immunization in mice, ELISA tests showed that the antisera titer increased gradually with the increase of hapten density. The antibody obtained from the mice showed high sensitivity toward CPFX. These results revealed the relationship between hapten density and immunogenicity as well as an optimized conjugation approach for immunization purposes.