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. 2022 Sep;133(3):1496-1505.
doi: 10.1111/jam.15655. Epub 2022 Jun 22.

A novel phage-displayed MilA ELISA for detection of antibodies against Myc. bovis in bovine milk

Mina Farzaneh  1 , Abdollah Derakhshandeh  1 , Abd Al-Bar Ahmed Al-Farha  2 , Kiro Petrovski  3   4 , Farhid Hemmatzadeh  4   5
Affiliations

A novel phage-displayed MilA ELISA for detection of antibodies against Myc. bovis in bovine milk

Mina Farzaneh et al. J Appl Microbiol. 2022 Sep.

Abstract

Aims: The aim of this study was to assess a phage-displayed MilA protein of Myc. bovis in an indirect ELISA for the detection of Myc. bovis antibodies in milk samples.

Methods and results: The desired sequence of milA gene was synthesized and cloned into pCANTAB-F12 phagemid vector. The expression of the MilA on the phage surface was confirmed by Western blotting. The recombinant phage was used in the development of an indirect ELISA to detect Myc. bovis antibodies in milk samples. There was a significant agreement between the results of phage-based ELISA and recombinant GST-MilA ELISA for the detection of Myc. bovis antibodies in milk samples.

Conclusions: The inexpensive and convenient phage-based ELISA can be used instead of recombinant protein/peptide ELISA as an initial screening of Myc. bovis-associated mastitis.

Significance and impact of study: Mastitis associated with Myc. bovis is a continuous and serious problem in the dairy industry. Sero-monitoring of Myc. bovis infection cases are one of the key factors for surveillance of the infections in dairy farms. Despite the existence of some commercially serological assays for Myc. bovis antibodies, they have some limitations regarding their sensitivity and availability. The development of accurate diagnosis tools could contribute to control programmes of Myc. bovis-associated mastitis in the dairy herds.

Keywords: Myc. bovis; MilA antigen; Milk; indirect ELISA; phage display.

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Conflict of interest statement

No conflict of interest declared.

Figures

FIGURE 1
FIGURE 1
PCR amplification of milA fragment. Lane M: DNA marker (GelPilot 1 kb plus ladder [100], QIAGEN), lane 1: Negative control containing nuclease‐free water, lane 2: Amplified milA fragment (519 bp).
FIGURE 2
FIGURE 2
Colony PCR by vector‐specific primer. Lane M: DNA marker (GelPilot 1 kb plus ladder [100], QIAGEN), lane 1: Negative control containing nuclease‐free water, lane 2: Colony containing non‐recombinant pCANTABF12 showed 364 bp band on agarose gel, lane 3: Colony containing milA‐ pCANTABF12 construct showed 778 bp band on agarose gel.
FIGURE 3
FIGURE 3
Silver staining SDS‐PAGE. Lane 1: Protein marker (BenchMark pre‐stained protein ladder (life technologies, Inc., Carlsbad, CA, USA)), lane 2: Recombinant phage displaying the MilA peptide (the presence of a band around 65 kDa indicated the expression of MilA on the phage surface), lane 3: Wild‐type phage.
FIGURE 4
FIGURE 4
Western blot analysis of the recombinant MilA phage and wild‐type phages. Lane 1: Protein marker (BenchMark pre‐stained protein ladder (life technologies, Inc., Carlsbad, CA, USA)), lane 2: MilA recombinant phage displaying a band around 65 kDa: Probed with anti‐E tag antibody (Abcam, USA), lane 3: Wild‐type phage: Probed with anti‐E tag antibody (Lane 3).
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