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Clinical and Vaccine Immunology, November 2009, p. 1665-1674, Vol. 16, No. 11
1071-412X/09/$08.00+0     doi:10.1128/CVI.00223-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay{triangledown} ,{dagger}

Maja Neiman,1 Carl Hamsten,1 Jochen M. Schwenk,1 Göran Bölske,2 and Anja Persson1*

Department of Proteomics, School of Biotechnology, KTH-Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden,1 Department of Bacteriology, National Veterinary Institute, SE-75183 Uppsala, Sweden2

Received 25 May 2009/ Returned for modification 9 July 2009/ Accepted 24 August 2009

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10–6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

* Corresponding author. Mailing address: Department of Proteomics, School of Biotechnology, KTH-Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden. Phone: 46-8-5537 8017. Fax: 46-8-5537 8481. E-mail: anja{at}biotech.kth.se

{triangledown} Published ahead of print on 2 September 2009.

{dagger} Supplemental material for this article may be found at http://cvi.asm.org/.

Clinical and Vaccine Immunology, November 2009, p. 1665-1674, Vol. 16, No. 11
1071-412X/09/$08.00+0     doi:10.1128/CVI.00223-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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