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A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Avian leukosis virus subgroup J poses a great threat to the poultry industry in China. To reduce the economic losses, a quick method for detection of ALV-J antigen is required for diagnosis and identification of the congenitally transmitting hens. In this study, we report the production and evaluation of one monoclonal antibody (MAb) suitable for achieving these goals. The gp85 gene of avian leukosis virus subgroup J CAUHM01 China isolates was subcloned into the expression vectors pGEX-6p-1 and pET28a and successfully expressed in E. coli. After immunizing BALB/c mice with recombinant His-Jgp85 protein, splenic cells from immunized mice were fused with SP2/0 myeloma cells to produce hybridomas. We isolated and characterized one ALV-J gp85-specific MAb by determining its titer, affinity and IgG subclass. In addition, we performed epitope mapping and determined the epitope for the MAb 1E3 to be 81-92 aa of ALV-J gp85 protein (LPWDPQELDILG). Bioinformatics analysis and IFA studies revealed that this epitope is conserved among all ALV-J isolates and that this antibody could serve as a useful reagent for ALV-J detection and diagnosis.

We describe the construction of a recombinant baculovirus containing the cloned DNA encoding the gp85 envelope glycoprotein of HPRS-103 (subgroup J) avian leukosis virus fused to the carboxy-terminus of the affinity tag glutathione-S-transferase. The fusion protein was efficiently secreted into the supernatant medium of the infected insect cell culture and could be purified in a single step using immobilized glutathione. An enzyme-linked immunosorbent assay using the recombinant protein was found to be specific and sensitive for detection of HPRS-103 virus-specific antibodies in the sera of infected birds.

A total of 72 White Leghorn grandparent hens was examined by ELISA for avian leukosis virus (ALV), ALV antigens and anti-ALV antibodies to identify and characterize the hens transmitting ALV to their embryos (transmitters) by using fertilized eggs. These hens were divided into 3 groups as no antibody and non-viremic (NANV) (49 hens), antibody-positive and non-viremic (APNV) (21 hens) and no antibody and viremic (NAV) (2 hens) by testing the sera for the presence of ALV and anti-ALV antibody. Egg albumen and embryos were tested for the presence of ALV and ALV antigens. As a result, no ALV was detected in both albumen and embryos in the NANV group. On the other hand, all albumen samples collected repeatedly from 3 hens of the APNV group and 2 hens of the NAV group contained infectious ALV, although the infectivity differed with the individual. Also, these 5 hens produced infected embryos at varying frequencies. However, on AP hen which shed neither ALV nor ALV antigens into the albumen produced an infected embryo at a lower rate. These results indicate that testing for infectious ALV in albumen from a newly laid egg per hen is effective to identify the transmitters to some extent. When virus titers in each of 8 tissue samples from the 6 transmitting hens were determined, the highest virus titers were found in washing from the ampulla of the oviducts in most of the shedders, suggesting that embryo infection is closely correlated with ALV produced at the oviduct, but not with ALV transferred from the other parts of the body.

An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.

An ELISA procedure for the detection of avian sarcoma-leukosis gs antigen in feather pulp of adult birds is described. The test can be used for identifying gs+ and gs- chickens in leukosis-free populations. The titres of exogenous and endogenous virus-coded gs antigens overlap and the ELISA can be used only for a preliminary screening of unknown chicken populations.

An enzyme-linked immunosorbent assay (ELISA) for avian leukosis virus group-specific antigen was used to study infections with and shedding of avian leukosis virus in a commercial flock of chickens with a known high incidence of infection. Avian leukosis virus group-specific antigen was detected in serum or cloacal washings from 76% of a group of 100 61-week-old hens. With eggs collected during the next 3 weeks, antigen was detected in the albumen of 88% of the eggs from ELISA-positive hens and 2% of the eggs from ELISA-negative hens. Results of assays for infectivity correlated closely with the ELISA results. Serum and cloacal specimens were almost equally sensitive in detecting infection; however, a higher proportion of cloaca-positive hens (97%) than serum-positive hens (91%) shed virus in their eggs. Similar results were obtained from a second sampling of eggs taken when the hens were 84 weeks old, after an intervening molt. Avian leukosis virus infection was correlated with reduced egg production and increased mortality. Eggs were produced by 100% of the ELISA-negative birds during both sampling periods, whereas only 69% of the ELISA-positive birds produced eggs at 61 weeks and 76% produced eggs at 84 weeks. All birds that were ELISA negative at 61 weeks survived the experiment. Of 14 ELISA-positive birds that died and were examined postmortem between 61 and 84 weeks, 8 had malignancies, and the remainder died of a variety of nonmalignant diseases.