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      Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein

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          Abstract

          Background

          Mycoplasma bovis ( M. bovis) is a major, but often overlooked, pathogen documented to cause respiratory disease, mastitis, and arthritis in cattle throughout China since 2008. Here, we report the development of a direct competitive enzyme-linked immunosorbent assay (Dc-ELISA) to detect M. bovis antibody.

          Results

          We used a recombinant P48 protein and monoclonal antibody (mAb) 10E. MAb 10E, prepared against the recombinant P48 protein of M. bovis, identified all M. bovis strains with no cross-reactivity with other related pathogens. Coating micro plates with P48 protein instead of whole M. bovis cells as well as the use of mAb 10E produced a specific and sensitive Dc-ELISA for M. bovis antibody detection with a cut-off percent inhibition (PI) value of 32%. Compared with two commercial indirect ELISA (i-ELISA) kits, our Dc-ELISA offered a higher positive detection rate in 165 clinical bovine serum samples.

          Conclusions

          A rapid, sensitive, and reliable serological diagnosis method was developed for M. bovis, which can facilitate M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology of M. bovis.

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          Most cited references24

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          Mycoplasma bovis: disease, diagnosis, and control.

          R.A.J Nicholas, R.D. Ayling (2003)
          Mycoplasma bovis is a major, but often overlooked, pathogen causing respiratory disease, mastitis, and arthritis in cattle. It is found worldwide and has spread into new areas, including Ireland and parts of South America, in the last decade. In Europe, it is responsible for at least a quarter to a third of all calf pneumonia although this may be an underestimate as few laboratories regularly monitor for mycoplasmas. Like all mollicutes, M. bovis is inherently refractory to certain groups of antibiotics because it does not possess a cell wall; furthermore evidence is accumulating that strains of M. bovis are becoming resistant to antibiotics, including tetracycline, tilmicosin and spectinomycin, traditionally used for their control. No vaccines are presently available for the control of M. bovis infections.
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            Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

            I Lysnyansky, Karla J Helbig, D Yogev … (2000)
            The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.
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              Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

              Yoshie Yamamoto, Akio Yamada, Osamu Fujita … (2012)
              A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.
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                Author and article information

                Contributors
                Ping Fu
                Zhenhong Sun
                Yuewei Zhang
                Ziqiang Yu
                Haiyan Zhang
                Dan Su
                Fei Jiang
                Wenxue Wu
                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: 2014
                Publication date (Electronic): 18 February 2014
                Volume: 10
                Page: 42
                Affiliations
                [1 ]Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
                [2 ]Shandong Vocational Animal Science and Veterinary College, Weifang, China
                Article
                Publisher ID: 1746-6148-10-42
                DOI: 10.1186/1746-6148-10-42
                PMC ID: 3942108
                PubMed ID: 24533468
                SO-VID: 00957573-d570-491a-8de7-615134c5a98c
                Copyright © Copyright © 2014 Fu 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 credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 3 November 2013
                Date accepted : 6 February 2014
                Categories
                Subject: Methodology Article

                ScienceOpen disciplines: Veterinary medicine
                Keywords: direct competitive elisa,mycoplasma bovis,monoclonal antibody
                Data availability:
                ScienceOpen disciplines: Veterinary medicine
                Keywords: direct competitive elisa, mycoplasma bovis, monoclonal antibody

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