J Vet Sci.  2016 Dec;17(4):479-487. 10.4142/jvs.2016.17.4.479.

Development and evaluation of an immunochromatographic assay using a gp51 monoclonal antibody for the detection of antibodies against the bovine leukemia virus

• Kim EJ
• Cheong KM
• Joung HK
• Kim BH
• Song JY
• Cho IS
• Lee KK
• Shin YK

• KMID: 2412603
• DOI: http://doi.org/10.4142/jvs.2016.17.4.479

Abstract

Infection of cattle with bovine leukemia virus (BLV) has been observed and reported worldwide, including in Korea. The onsite identification of infected cattle would help decreasing and eradicating BLV infections on farms. Here, we present a new immunochromatographic assay that employs monoclonal antibodies (MAbs) for the detection of antibodies against BLV in the field. BLV envelope glycoprotein (gp)51 was expressed in E. coli, and MAbs against recombinant BLV gp51 were generated for the development of an immunochromatographic assay to detect BLV antibodies in cattle. The sensitivity and specificity of the assay were determined by comparing these results with those obtained from a standard enzyme linked immunosorbent assay (ELISA). A total of 160 bovine sera were used to evaluate the new immunochromatographic assay. Using ELISA as a reference standard, the relative specificity and sensitivity of this assay were determined to be 94.7% and 98%, respectively. Because of its high sensitivity and specificity, this BLV antibody detection assay would be suitable for the onsite identification of BLV infection in the field.

Keyword

antibody detection; bovine leukemia virus; immunochromatography

MeSH Terms

Animals
Antibodies, Monoclonal/*blood
Antibodies, Viral/*blood
Cattle
Enzootic Bovine Leukosis/*diagnosis/virology
Immunochromatography/*veterinary
Leukemia Virus, Bovine/*immunology
Mass Screening/*veterinary
Pilot Projects
Republic of Korea
Antibodies, Monoclonal
Antibodies, Viral

Figure

• 

  Fig. 1 Interpretation of the immunochromatographic assay results. (A) A serum sample that only produced the control line was considered positive. (B) A serum sample that produced both the test and control lines was considered negative.

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  Fig. 2 SDS-PAGE analysis of the recombinant bovine leukemia virus (BLV) gp51 protein. (A) Before purification. Lane 1, protein size marker; Lane 2, BL21(DE3) total cell lysate; Lane 3, BL21(DE3) cell lysate supernatant; Lane 4, recombinant gp51 protein in BL21(DE3) total cell lysate; Lane 5, recombinant gp51 protein in BL21(DE3) cell lysate supernatant. (B) After purification. M, marker; T, BL21(DE3) total cell lysate; S, BL21(DE3) cell lysate supernatant; F, flow through; 40, flow through after wash step; 60, flow through after wash step; E, eluted gp51 protein.

• 

  Fig. 3 Blocking ELISA using BLV and BLV antiserum for the selection of monoclonal antibodies (MAbs) with blocking ability. Of the 18 hybridoma clones that were tested, seven (hybridoma clones 8A8, 10B43, 9D72, 2E42, 8F76, 12F12, and 10H37) exhibited superior blocking ability.

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  Fig. 4 Reactivity of the selected MAbs against BLV gp51 in the BLV-gold conjugate immobilized on nitrocellulose membranes. Of the seven hybridoma clones tested, hybridoma clone 12F12 was selected, as it reacted strongly with BLV in the immunochromatography strip assay.

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  Fig. 5 Specificity of the immunochromatographic assay strip. The specificity of the immunochromatographic assay strip was evaluated with antisera against a range of bovine viral agents, including BVDV, BRSV, BHV-1-1, PI-3 and BLV. *Value read in the reader.

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  Fig. 6 Evaluation of the immunochromatographic assay strip using the BLV reference serum E05 diluted 1 : 10 in serum negative for BLV antibody. Lane 1, E05 serum diluted 1 : 10; Lane 2, BLV-antibody-negative serum 1; Lane 3, serum that was positive for the BLV antibody (NVSL, USA); Lane 4, BLV-antibody-negative serum 2.

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  Fig. 7 Reproducibility of the immunochromatographic assay strip. Sera that were positive and negative for the BLV antibody were tested in triplicate. Lane 1, serum that was positive for the BLV antibody (NVSL); Lane 2, field-collected bovine serum that was positive for the BLV antibody (as determined by ELISA); Lane 3, serum that was negative for the BLV antibody.

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