Validation of an automated ELISA system for detection of antibodies to Aleutian mink disease virus using blood samples collected in filter paper strips.

BACKGROUND: Aleutian mink disease virus (AMDV) is the cause of a chronic immune complex disease, Aleutian disease (AD), which is common in mink-producing countries. In 2005, implementation of an AMDV eradication programme in Finland created a need for an automated high-throughput assay. The aim of this study was to validate an AMDV-VP2 -recombinant antigen ELISA, which we developed earlier, in an automated assay format for the detection of anti-AMDV antibodies in mink blood and to determine the accuracy of this test compared with the reference standard (counter-current immunoelectrophoresis, CIEP). METHODS: A blood sampling method based on filter paper 12-strips (blood combs) and a device to introduce these strips to an ELISA plate for elution of the samples were developed. Blood and serum samples were collected from 761 mink from two farms with low (2%) and high (81%) seroprevalences of AMDV infection in 2008. ELISA sensitivity and specificity were estimated with a Bayesian 2-test 2-population model that allowed for conditional dependence between CIEP and ELISA. Agreement between the two tests was assessed with kappa statistic and proportion agreement. RESULTS: The sensitivity and specificity of the automated ELISA system were estimated to be 96.2% and 98.4%, respectively. Agreement between CIEP and ELISA was high, with a kappa value of 0.976 and overall proportion agreement of 98.8%. CONCLUSIONS: The automated ELISA system combined with blood comb sampling is an accurate test format for the detection of anti-AMDV antibodies in mink blood and offers several advantages, including improved blood sampling and data handling, fast sample throughput time, and reductions in costs and labour inputs.

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant VP2 capsids for the detection of antibodies to Aleutian mink disease virus.

Aleutian disease (AD), a common infectious disease in farmed minks worldwide, is caused by Aleutian mink disease virus (AMDV). Serodiagnosis of AD in minks has been based on detection of AMDV antibodies by counterimmunoelectrophoresis (CIE) since the 1980s. The aim of this study was to develop and evaluate an enzyme-linked immunosorbent assay (ELISA) based on recombinant virus-like particles (VLPs) for identifying AMDV antibodies from mink sera. AMDV capsid protein (VP2) of a Finnish wild-type strain was expressed by the baculovirus system in Spodoptera frugiperda 9 insect cells and was shown to self-assemble to VLPs (with an ultrastructure similar to that of the actual virion). A direct immunoglobulin G ELISA was established using purified recombinant AMDV VP2 VLPs as an antigen. Sera from farmed minks were collected to evaluate the AMDV VP2 ELISA (n = 316) and CIE (n = 209) based on AMDV VP2 recombinant antigen in parallel with CIE performed using a commercially available traditional antigen. CIE performed with the recombinant antigen had a sensitivity and specificity of 100% and ELISA a sensitivity of 99% and a specificity of 97%, with reference to CIE performed with the commercial antigen. The results show that the recombinant AMDV VP2 VLPs are antigenic and that AMDV VP2 ELISA is sensitive and specific and encourage further development of the method for high-throughput diagnostics, involving hundreds of thousands of samples in Finland annually.