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. 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 system (10 34 have prompted researchers to explore their use as immunological tools. There are three published reports of the involvement of HCAbs in camelid immunity specifically in the response to trypanosome infection and to immunization with bacterial proteins (12 30 33 The production of IgG isotypes in response to infection with parasitic nematodes has not yet been investigated. A limiting factor for investigations into camelid immunity has been a shortage of well-characterized isotype-specific reagents. Currently polyclonal antibodies that react broadly with camelid IgGs are available from commercial sources. Monoclonal antibodies (MAbs) specific for dromedary (was confirmed in diseased llamas by CGP 3466B maleate observation of nematodes within the central nervous system during necropsy. Sera from llamas in Pullman Washington an area in which is not endemic were kindly provided by William Foreyt of Washington State University. Antibodies. CGP 3466B maleate Polyclonal goat CGP 3466B maleate anti-llama IgG (H+L) conjugated to horseradish peroxidase (HRP; Bethyl Laboratories Inc. Montgomery Tex.) was used in ELISA and Western blotting. Monoclonal mouse antibodies were detected with HRP-conjugated goat anti-mouse antibodies (ICN/Cappel Aurora Ohio). Three MAbs 270000000000 (anti-IgG1) 1900000000 (anti-IgG2) and 8E1 (anti-IgG3) were selected for use in the serologic assays. An ELISA to determine the isotype of the MAbs employed CGP 3466B maleate Rabbit Polyclonal to MMP-11. rat MAbs to mouse isotypes and HRP-conjugated mouse anti-rat κ-chain antibodies (BD PharMingen San Diego Calif.). Purification of llama IgGs. Llama IgG isotypes were purified using affinity chromatography as described elsewhere (12 33 Briefly serum was first loaded onto a protein G-Sepharose 4B column (Sigma Chemical Co. St. Louis Mo.) and the unbound fraction was collected and loaded on a protein A-Sepharose 4B column (Sigma Chemical Co.). IgG3 was eluted from protein G with 0.15 M NaCl-0.58% acetic acid (pH 3.5) and IgG1 was eluted with 0.1 M glycine-HCl (pH 2.7). IgG2 was eluted from protein A with 0.15 M NaCl-0.58% acetic acid pH 4.5. Fractions were neutralized immediately with 0.1 M Tris-HCl pH 9.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots. Serum and chromatographically separated IgGs were resolved in discontinuous polyacrylamide gels (16). Minigels were used except for the determination of apparent molecular masses; instead proteins were resolved in standard 15-cm-long gels. Samples and molecular mass markers (Bio-Rad Laboratories Hercules Calif.) were boiled for 7 min in sample preparation buffer with or without 2-mercaptoethanol before being loaded in the gels. Gels were stained with Coomassie blue or were blotted onto nitrocellulose membranes. Western blotting procedures CGP 3466B maleate were performed at room temperature. Incubation periods were 1 h unless specified otherwise. Membranes were blocked with 5% skim milk in Tris-buffered saline and washed with Tris-buffered saline containing 0.05% Tween 20 and 0.1% bovine serum albumin (Sigma). Primary antibodies were diluted in blocking solution and conjugates were diluted in blocking solution containing 10% normal goat serum. Antibody binding was detected with a chemiluminescent substrate (ECL reagent; Amersham Pharmacia Biotech Piscataway N.J.) and autoradiography. Films were scanned and images were prepared using Adobe Photoshop and Microsoft Powerpoint. Production of monoclonal antibodies. Mice were injected intraperitoneally with 100 to 300 μg of purified llama IgGs mixed in complete Freund’s adjuvant (Sigma). One mouse per isotype was injected intravenously with purified IgG in Dulbecco’s phosphate-buffered saline (DPBS) 30 days postimmunization and.