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Development of an Enzyme-Linked Immunosorbent Assay to Determine the Seroprevalence of Bovine Leukemia Virus Antibodies in Humans


Breast cancer is a leading cause of morbidity and mortality worldwide. About 5-10% of breast cancer cases are associated with hereditary factors (such as mutations of the BRCA-1 or BRCA-2 gene), but the exact causes for most breast cancers are unknown. The remaining 90% of breast cancer cases may potentially be caused by external initiators such as radiation, chemical carcinogens, or infectious agents. Infectious agents cause about 23% of all malignancies in developing countries, and approximately 8% of malignancies in developed countries. Currently, six viruses are causally associated with human cancers: Epstein-Barr virus (EBV), human T-lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), and human herpes virus 8 (HHV-8). No infectious agent has yet been causally associated with human breast cancer. One candidate virus is bovine leukemia virus (BLV), a retrovirus closely related to HTLV and the causative agent of enzootic bovine lymphosarcoma. In cattle, BLV is transmitted by transfer of infected lymphocytes via blood or milk. Humans are potentially exposed to BLV by consumption of cow's milk and meat.

One part of the process of proving that an infectious agent causes a particular disease is proving that the organism infects humans. Production of immunoglobulins specific to the organism is often evidence of infection. Utilizing an immunoblot assay with a chemiluminescent endpoint, Buehring et al. demonstrated the presence of anti-BLVp24gag IgG, IgM, and IgA in human sera. Competition studies with pre-immune and immune goat sera further verified the human anti-BLV specificity. Since immunoblotting is a labor-intensive and inefficient method compared to ELISA, an ELISA assay was developed in this study to detect anti-BLVp24gag IgG and IgM in human serum and plasma samples. Receiver operator curve analysis was used to compare the ELISA to the immunoblot. The ELISA method developed here has poor ability (IgG AUC=0.51, IgM AUC= 0.56) to discriminate between people with and without antibodies to BLV p24gag, compared to immunoblot. ROC analysis is concordant with the unimodal IgG and IgM frequency distributions.

The cut-off value derived from ROC analysis for IgG is 77800 RLUs, which gives a sensitivity of 76.47% and a specificity of 26.1%. The cut-off value for IgM is 26244 RLUs, giving a sensitivity of 80.9% and a specificity of 28.05%. Initial competition studies with a monoclonal anti-human secondary antibody indicates that the Assay may show specificity for the BLV p24 protein. However, an appropriate antigen control was not available to rule out non-specific reactions with the antigen matrix. Therefore, this ELISA needs further development to assure specificity of the assay for the recombinant BLV p2gag protein before solid conclusions can be made regarding the seroprevalence of BLV antibodies in humans.

However, this developmental stage ELISA was used to estimate the frequency of anti-BLVp24gag IgG and IgM in our study population, using the ROC derived cut-off points. In the 0-3 month age category, the frequency of IgG is 14.2%, declining to 0% by the age of 6 months. The frequency of IgM in the 0-3 month age group is 23.9%, increasing to 70% by 6 months of age. The presence of IgG in the 0-3 month age group is most likely maternal IgG that wanes by age 6 months, while IgM is indicative of the child's own immune response to a new antigen. The level of IgG peaks at 30-39 years of age, plateaus, and then begins to decrease at 60 years of age. IgM peaks in adolescence and then begins a slow decline after 30 years of age, but never falls below 60% prevalence. Continuing high IgM titers may be indicative of constant reexposure to BLV via dairy and meat consumption, or to episodes of viral reactivation. The relatively high overall percentage of IgM seropositivity (73.3%) compared to IgG seropositivity (49.3%) may be due to the low median age of the study population.

Using the preliminary ELISA method being developed here, this study may indicate that both vertical and sexual transmission of BLV potentially occur. However, presence of antibodies to the BLV p24gag protein do not necessarily indicate infection, but may result from exposure to the antigen from consumption of dairy products. In addition, confirmation of the specificity of this ELISA is necessary to make firm conclusions. Further prospective studies are warranted in order to determine precisely at what point(s) in the perinatal period vertical transmission could occur, and to elucidate whether sexual transmission is truly occurring. Prospective studies may also permit a study sample more representative of the target population to be developed.

In conclusion, widespread consumption of BLV-contaminated dairy and beef may potentially be responsible for a significant proportion of breast cancer cases worldwide. If the pathogenicity of BLV for humans is established, the implications are far-reaching and may indicate the need for primary preventative measures to avert continuing infection of humans.

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