ELISA试验研制的IgM和从美洲驼.docVIP

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 Abstract Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chainantibodies, HCAbs). Three IgG isotypes have been identified (IgG1, IgG2 and IgG3); IgG2 and IgG3 are HCAbs whereas the IgG1has the conventional structure. Different subtypes of IgG1 (IgG1a and IgG1b) and IgG2 (IgG2a, IgG2b and IgG2c) have been classifiedaccording to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred asVHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbshas 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 weproduced specific antisera against llama IgM and IgG1. The anti-IgG1 serum was obtained by immunizing rabbits with arecombinant fusion protein formed by GST fused to the CH1 domain of the IgG1. The anti-IgM serum was obtained by immunizingrabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgGand 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 andIgM = 1.027 _ 0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG3 subtype is thepredominant 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 e