The Specificity and Affinity of Polyclonal Antibodies Raised in Rabbits Against a Hapten Conjugated to Rabbit Serum Albumin

Abstract

In this study, rabbit antisera to hapten-rabbit serum albumin conjugates were investigated regarding antibody titer, affinity, specificity, and affinity distribution. Methyl phosphonic acid p-amino-phenyl 1,2,2-trimethylpropyldiester (MATP) served as model hapten. Four MATP-rabbit serum albumin conjugates with various hapten densities (with and without spacer) were synthesized and used for immunization of rabbits. Antisera were collected over a 130 day-period and characterized with different ELISA methods. We found that immunogens with rabbit serum albumin gave antisera with lower titers, but similar affinity as compared to polyclonal or monoclonal antibodies obtained with non-rabbit protein as carrier protein. Immunogens with a low hapten density led to higher final titers without affecting antibody affinity or specificity. Immunogens containing a bridging group resulted in higher antibody affinity with a changed specificity. The pattern of antibody affinity distribution differed considerably among individual rabbits and showed a non-Gaussian subpopulation distribution.

The differential dynamics of antibody subpopulation expression during affinity maturation in a teleost

“…The anti-TNP antibody affinities were determined by a partitioning ELISA-based analysis originally described by Nieto et al for murine sera [23] and adapted for use in salmonids [28]. The characteristic affinity of each of affinity subpopulation (aK) is determined by the equation:…”

“…Since its original development this method has been widely employed with humans [24,25], and a variety of animals including mice [26,27] rabbits [28], ducks [29], and trout [21,22]. Further, both by mathematical derivation and empirical analysis the estimate of the weighted average affinity (aK) was found comparable to the average affinity (K 0 ) as determined by classical methodology (Farr technique).…”

Optimization and Immunological Characterization of a Photochemically Coupled Lysergic Acid Diethylamide (LSD) Immunogen

“…Depending on the size of the protein and the distance between it and the hapten, areas surrounding the point of attachment may be obscured from the immune system. This is of particular importance for small molecules such as drugs of abuse, for which immunogen design plays a fundamental role in determining the overall antibody specificity ( − ). As a result, cross-reactivity studies can provide valuable insights into possible structural attachments which have taken place in the immunogen.…”