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      The complement system of the goat: Haemolytic assays and isolation of major proteins

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          Abstract

          Background

          The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats ( Capra aegagrus hircus) and to characterize the major goat complement system proteins.

          Results

          The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H.

          Conclusions

          Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

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          Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

          A. Sjohölm, Henk A. M. Seelen, M Loos … (2004)
          Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.
          Article 0 comments Cited 81 times – based on 0 reviews     Review now
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            Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.

            T Steck, G Fairbanks, Arian D Wallach (1971)
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              Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin.

              James N Arnold, C H Radcliffe, P Rudd … (2005)
              The glycoprotein IgM is the major antibody produced in the primary immune response to antigens, circulating in the serum both as a pentamer and a hexamer. Pentameric IgM has a single J chain, which is absent in the hexamer. The mu (heavy) chain of IgM has five N-linked glycosylation sites. Asn-171, Asn-332, and Asn-395 are occupied by complex glycans, whereas Asn-402 and Asn-563 are occupied by oligomannose glycans. The glycosylation of human polyclonal IgM from serum has been analyzed. IgM was found to contain 23.4% oligomannose glycans GlcNAc2Man5-9, consistent with 100% occupancy of Asn-402 and 17% occupancy of the variably occupied site at Asn-563. Mannan-binding lectin (MBL) is a member of the collectin family of proteins, which bind to oligomannose and GlcNAc-terminating structures. A commercial affinity chromatography resin containing immobilized MBL has been reported to be useful for partial purification of mouse and also human IgM. Human IgM glycoforms that bind to immobilized MBL were isolated; these accounted for only 20% of total serum IgM. Compared with total serum IgM, the MBL-binding glycoforms contained 97% more GlcNAc-terminating structures and 8% more oligomannose structures. A glycosylated model of pentameric IgM was constructed, and from this model, it became evident that IgM has two distinct faces, only one of which can bind to antigen, as the J chain projects from the non-antigen-binding face. Antigen-bound IgM does not bind to MBL, as the target glycans appear to become inaccessible once IgM has bound antigen. Antigen-bound IgM pentamers therefore do not activate complement via the lectin pathway, but MBL might have a role in the clearance of aggregated IgM.
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                Author and article information

                Journal
                Journal ID (nlm-ta): BMC Vet Res
                Journal ID (iso-abbrev): BMC Vet. Res
                Title: BMC Veterinary Research
                Publisher: BioMed Central
                ISSN (Electronic): 1746-6148
                Publication date Collection: 2012
                Publication date (Electronic): 26 June 2012
                Volume: 8
                Page: 91
                Affiliations
                [1 ]Animal Science Department, Universidad de las Palmas de Gran Canaria, Arucas, Las Palmas, 35413, Spain
                [2 ]MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
                Article
                Publisher ID: 1746-6148-8-91
                DOI: 10.1186/1746-6148-8-91
                PMC ID: 3413586
                PubMed ID: 22734447
                SO-VID: fdac6a98-1cab-406a-996f-4b5e4db9b28b
                Copyright © Copyright ©2012 Moreno-Indias et al.; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 30 August 2011
                Date accepted : 26 June 2012
                Categories
                Subject: Methodology Article

                ScienceOpen disciplines: Veterinary medicine
                Keywords: c3,goat,complement system,factor h,c1q
                Data availability:
                ScienceOpen disciplines: Veterinary medicine
                Keywords: c3, goat, complement system, factor h, c1q

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