Catalytic Antibodies Induced by a Zwitterionic Hapten

Abstract

A zwitterionic hapten 4 featuring both positively and negatively charged functional groups was designed and synthesized with the goal of generating catalytic antibodies for the hydrolysis of ester 6 and amide 7. Of the 36 monoclonal antibodies specific to BSA-4 (bovine serum albumin) that were isolated, six accelerated the hydrolysis of 6. Two catalytic antibodies with distinctively different and representative kinetic behaviors were selected for detailed kinetic studies. Whereas H8-2-6F11 showed burst kinetic behavior, which can be attributed to the formation of an acyl intermediate, H8-1-2D5 did not, but it did exhibit high multiple turnover activity. The rate of hydrolysis of 6 catalyzed by H8-1-2D5 followed Michaelis-Menten kinetics; the apparent values of the Michaelis-Menten constant Km and the catalytic constant kcat were 488 microM and 3.5 min(-1), respectively. The catalytic rate enhancement (kcat/kun) observed for H8-1-2D5 was 1.3 x 10(5), which is approximately two orders of magnitude greater than those for monofunctional haptens. Thus H8-1-2D5 compares well in catalytic activity with antibodies isolated by a related approach called heterologous immunization.

Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

“…Since the maximum buried surface areas of small haptens in antibody-hapten complexes are estimated to be approximately 400 Å 2 [50,51,52,53,54,55,56], it was predicted that hapten 1 , consisting of a pentacyclic skeleton (ABCDE ring, calculated water accessible surface area was 398 Å 2 ) and a cyclic acetal connected to a linker, would be sufficiently large to occupy the antibody-combining site, whereas leaving the acetal and linker moiety free from interactions with the antibody-combining site [44]. In addition, hapten 1 was designed to induce mAbs that would bind to the left wing of CTX3C or 51-hydroxyCTX3C in an orientation suitable for sandwich ELISA.…”

“…After three injections of KLH-conjugate 4 , the titer of serum immunoglobulin G (IgG) was determined by ELISA using BSA-conjugate 5 . The mouse showing the highest titer was boosted; after 3 days, the spleen was taken from the mouse, and the splenocytes were fused with X63-Ag8.653 myeloma cells using an electro cell fusion apparatus as reported previously [53,54]. All hybridomas producing IgGs that bound to BSA-conjugate 5 were subcloned twice or three times to provide six hybridomas producing BSA-conjugate 5 -binding mAbs.…”

Catalytic Antibodies as Designer Proteases and Esterases

“…Zwitterionic hapten 73 was used to induce catalytic antibodies for the hydrolysis of ester 74 (Scheme ) . The phosphonate is expected to induce an acidic residue in the catalytic site that would stabilize the oxyanion of the transition state, whereas the protonated amine should induce a basic residue that deprotonates a water molecule to transform it into hydroxide for the nucleophilic attack.…”

Development of an Enzyme-Linked Immunosorbent Assay for the Determination of the Linear Alkylbenzene Sulfonates and Long-Chain Sulfophenyl Carboxylates Using Antibodies Generated by Pseudoheterologous Immunization

“…In the field of catalytic antibodies, several research groups have made use of heterologous immunization strategies to elicit antibody immunoresponse against two different epitopes of the molecules, simplifying hapten synthesis. − Thus, although an ideal hapten preserving both groups would have a spacer arm at the ortho or meta positions, the synthetic pathway leading to these types of chemical structures could be troublesome. The heterologous immunization strategy devised by the group of Prof. Masamune − consists of successfully immunizing the animal with two different but structurally related haptens. It has mainly been used for zwirterionic transition-state analogues with positive and negative charges, for which chemical preparation of the corresponding hapten could be problematic.…”