Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Abstract

Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a b-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG 1 ), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy -neither as a biotechnological strategy nor in the biological context of an immune response against infection -as raising inhibitory rVHHs.

Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays

“…The absence of reactivity against IgG 1 , IgG 2 , and IgG 3 was checked by the same ELISA. Briefly, screening was performed, and cross-reactivity was studied by coating microplates with 1 µg of the corresponding antigens (IgM, IgG 1 , IgG 2 , or IgG 3 , purified following the procedure described elsewhere 4 ) for 1 hr at 37°C, blocking overnight with 3% bovine serum albumin (BSA) in PBS and then incubating 50 µl of culture supernatants (1 hr at 37°C). Bound antibodies were detected using a commercial antibody (goat anti-mouse horseradish peroxidase [HRP]-labeled, i diluted 1:10,000), developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate j and stopped with the addition of 50 µl of 4N H 2 SO 4 .…”