J Vet Sci.  2012 Mar;13(1):73-79. 10.4142/jvs.2012.13.1.73.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

• Lien YY
• Huang CH
• Sun FC
• Sheu SC
• Lu TC
• Lee MS
• Hsueh SC
• Chen HJ
• Lee MS

• KMID: 1365006
• DOI: http://doi.org/10.4142/jvs.2012.13.1.73

Abstract

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Keyword

chicken anemia virus; immunoaffinity column; immunohistochemistry; monoclonal antibody; VP1

MeSH Terms

Animals
Antibodies, Monoclonal/biosynthesis/genetics/*immunology
Antigens, Viral/analysis
Capsid Proteins/genetics/*immunology
Chicken anemia virus/genetics/*immunology
*Chickens
Circoviridae Infections/blood/immunology/*veterinary/virology
Escherichia coli/genetics
Immunohistochemistry/veterinary
Liver/virology
Mice
Mice, Inbred BALB C
Microscopy, Fluorescence/veterinary
Poultry Diseases/blood/immunology/*virology
Specific Pathogen-Free Organisms
Thymus Gland/virology

Figure

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  Fig. 1 Analysis of the VP1Nd129 protein expressed in recombinant Escherichia (E.) coli by SDS-PAGE (A) and Western blotting (B). The symbols '-' and '+' represent pre- and post-induction with 1 mM of isopropyl-β-D-thiogalactopyronoside in E. coli, respectively. Anti-His tag monoclonal antibody was used for recognizing the VP1Nd129 protein. The arrowheads are the symbol for expressed VP1Nd129 protein.

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  Fig. 2 Analysis of the specificity and reactivity of E3 monoclonal antibody (mAb) by Western blotting (A) and ELISA (B). Different amounts of purified VP1Nd129 or E. coli cell lysate were reacted with E3 mAb in both (A) and (B).

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  Fig. 3 Immunohistochemical labeling of chicken anemia virus (CAV)-infected chicken liver tissues. E3 mAb was used as the primary antibody for recognizing CAV antigen in liver tissue. (A) Negative control. (B) CAV-infected chicken liver tissue. Scale bars = 100 nm.

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  Fig. 4 Immunohistochemical labeling of CAV-infected chicken thymus tissues. E3 mAb was used as the primary antibody for recognizing CAV antigen in the thymus tissue. (A) Negative control. (B) CAV-infected chicken thymus tissue. Scale bars = 100 nm.

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  Fig. 5 Immunofluorescence assay of CAV-infected chicken MSB-1 cells. E3 mAb was used as the primary antibody for recognizing CAV VP1 antigen in the MSB-1 cells. The fluorescent agent 4',6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) were used to counterstain the cell nuclei of cultures and react with E3 mAb, respectively. CPE: CAV-infected MSB-1 cells showing a cytopathic effect.

• 

  Fig. 6 Western blotting of immunocolumn-purified CAV VP1 by E3 mAb (A). CAV-infected liver lysate reacted with E3 mAb and immunoaffinity resin was then used to precipitate the mAb-bound or unbounded antigen. Lane M: pre-stained protein marker (the molecular weights of respective bands are 116, 66, 45, 35, 25, and 18 kDa), Lane 1: uninfected liver lysate, Lane 2: CAV-infected liver lysate, Lane 3: VP1 protein co-precipitated with E3 mAb from tissue lysate of Lane 2, Lane 4: E3 mAb only, Lane 5: protein precipitated by E3 mab from tissue lysate of Lane 1. "H" and "L" indicate the IgG heavy and light chains, respectively. Identification of the CAV genome by PCR (B). CAV VP1 capsid was co-precipitated with E3 mAb and then used to as a DNA template for PCR amplification using primers specific for the VP1 and VP2 genes. Lane M: 1-kb DNA ladder marker, Lane 1: positive PCR control, Lane 2: uninfected liver lysate, Lane 3: CAV-infected liver lysate, Lane 4: sample co-precipitated with E3 mAb from CAV-infected liver lysate.

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