Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies

Abstract

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.

FIG. 1

Isolation of the APV M gene and production of recombinant protein. The M gene was isolated from pCR-XL-TOPO by PCR and subcloned into the pQE-30 expression plasmid. A two-step process was used to purify M proteins, a nickel-nitriolotriacetic acid column that binds the His₆, tag located at the NH₂ terminus of the protein and gel purification. (A) PCR product of APV/CO (lane 1) and APV/MN2A (lane 2) in an agarose gel. (B) Recombinant M protein from APV/CO (lane 1) and APV/MN2A (lane 2) in an SDS-polyacrylamide gel stained with Coomassie blue.

FIG. 2

Western blot analysis of APV-positive and -negative turkey sera with APV proteins and recombinant M proteins. The partially purified APV proteins and the recombinant M protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Each lane of the membrane was incubated with a 1:40 dilution of turkey serum followed by horseradish peroxidase-conjugated anti-turkey IgG (1:20,000 dilution) and detected by chemiluminescence. (A) Reactivity of the sera with APV proteins; (B) reactivity with recombinant M protein. Lanes 1 to 8 show the reactivity with positive turkey sera, and lanes 9 and 10 show the reactivity with negative sera.

FIG. 3

Presence of APV-specific IgG in sera of APV-infected turkeys detected by M protein ELISA. Twofold serial dilutions of known positive turkey sera (n = 10) were tested in plates coated with recombinant M protein. Pooled sera from known APV-negative flocks were used as negative control. The results are expressed as mean and standard error.

FIG. 4

Western blot analysis of turkey sera that were negative by M protein ELISA but positive by routine APV ELISA. Partially purified APV or recombinant M protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Each lane of the membrane was incubated with a sample of turkey serum (1:40) followed by horseradish peroxidase-conjugated anti-turkey IgG (1:20,000) before being subjected to chemiluminescence. (A) Reactivity with purified APV proteins; (B) reactivity with recombinant M protein. All 12 samples negative by M protein ELISA were not reactive with M protein or APV proteins, as shown for 10 representative samples (lanes 1 to 10). A sample positive by M protein ELISA (lane 11) was used as a positive control and reacted with the 30-kDa M protein in both antigen preparations.

FIG. 5

Comparison of the sensitivity of M protein ELISA with routine APV ELISA for detection of APV-specific IgGs from experimentally infected turkeys. Turkeys (n = 34) were experimentally inoculated with live APV (APV/MN1A), and serum was collected 4 weeks later. A 1:40 dilution of each sample was applied to plates coated with recombinant M protein or APV-infected cell lysate and detected using horseradish peroxidase-conjugated goat anti-turkey IgGs. The A₄₉₀ values from M protein-coated plates were consistently higher than those from cell lysate-coated plates (P < 0.001).