Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG1, IgG2 and IgG3); IgG2 and IgG3 are HCAbs whereas the IgG1 has the conventional structure. Different subtypes of IgG1 (IgG1a and IgG1b) and IgG2 (IgG2a, IgG2b and IgG2c) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG2a, IgG2b, IgG2c and IgG3 by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG1. The anti-IgG1 serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH1 domain of the IgG1. The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n = 20) analyzed by ELISA gave the following values of immunoglobulins: IgG1 = 6.168 ± 1.628 mg/ml; IgG2 = 0.684 ± 0.310 mg/ml; IgG3 = 1.232 ± 0.410 mg/ml; total IgG = 8.933 ± 1.815 mg/ml and IgM = 1.027 ± 0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG3 subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA–DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG1. No anti-dextran response was observed in any of the isotypes analyzed.