Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids

Abstract

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

FIG. 1.

(A) Structure of camelid IgGs (data from reference 12). (B, top) Coomassie blue-stained SDS-PAGE gel of purified llama IgG isotypes resolved under nonreducing and reducing conditions. (a and b) IgG1 and IgG3 (eluted from protein G); (c) IgG2 (eluted from protein A). (Bottom) Western blot of gels identical to Coomassie blue-stained gels developed with polyclonal anti-llama IgG (H+L). Molecular masses (in kilodaltons) of protein standards run in parallel are indicated.

FIG. 2.

Reactivities of mouse MAbs with affinity-purified llama IgGs in ELISA. (A) IgG1; (B) IgG3; (C) IgG2. Twofold dilutions of supernatants were tested in triplicate wells against purified IgGs. Bound MAbs were detected with HRP-goat anti-mouse IgG. Columns represent mean optical densities for three replicate wells ± standard deviations of the means (I bars). Asterisks denote MAbs selected for further evaluation.

FIG. 3.

Binding of anti-llama IgG1 MAb (27E10) to fragments of llama IgG1. (A) Coomassie blue-stained gel of llama IgG1 digested for 0 or 6 h with pepsin in acetate buffer, pH 4.5. Estimated molecular masses (in kilodaltons) are indicated. (B) Western blot showing the reactivity of 27E10 (α-1) with llama IgG1, which was digested for 0 or 6 h with pepsin and developed with HRP-goat anti-mouse antibodies. poly, HRP-goat anti-llama IgG (H+L) against pepsin-cleaved IgG1.

FIG. 4.

Specificities of MAbs for native camelid IgGs. The anti-llama IgG1, IgG3, and IgG2 MAbs, 27E10, 8E1, and 19D8, respectively, were coupled with CN-Br activated Sepharose 4B and used to purify IgGs from camelid sera. (A) Llama serum; (B) alpaca serum; (C) guanaco serum; (D) camel serum. Eluted IgG fractions were resolved by SDS-PAGE under nonreducing and reducing conditions and stained with Coomassie blue. Estimated molecular masses (in kilodaltons) are indicated.

FIG. 5.

Reactivities of HCAb-specific MAbs with eluates from the anti-IgG3 and anti-IgG2 affinity columns in Western blots. Affinity-purified HCAbs from llama (A) and alpaca (B) sera were resolved by SDS-PAGE, blotted onto nitrocellulose, and developed with anti-IgG2 and anti-IgG3 MAbs (20 μg/ml) or normal mouse serum. α-3, anti-IgG3 (8E1) MAbs; α-2, anti-IgG2 (19D8) MAbs; -ve, negative (normal mouse serum). Estimated molecular masses (in kilodaltons) are indicated.

FIG. 6.

Detection of rPtAPI-specific IgG isotypes in llama sera by ELISA. (A) Llamas from a P. tenuis-endemic area (New York State). Sera from diseased llamas, confirmed to be P. tenuis infected, contained predominantly IgG1 specific for rPtAPI. No antigen-specific IgG isotypes were detected in the uninfected llama serum. Polyclonal goat anti-llama IgG (H+L) did not distinguish infected from uninfected llamas. (B) Llamas from P. tenuis-free area (Washington State). Cross-reactive IgG1 as well as IgG3 and/or IgG2 were detected in sera from uninfected llamas. The polyclonal goat antibodies yielded consistently high readings for all seven sera. Columns represent mean optical densities of sera diluted 1:100 (tested in triplicate) ± standard deviations (T bars).