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

Abstract

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.

Figure 1

Flow diagram of the sampling and number of mink undergoing CIEP and automated AMDV-VP2 ELISA tests.

Figure 2

Histograms of automated AMDV-VP2 ELISA results of CIEP-positive and -negative samples from mink. A. Farm 1 (high prevalence), 361 samples. B. Farm 2 (low prevalence), 400 samples. C. All 761 samples. X-axis shows the categories (at 0.05 intervals) of net ELISA optical density (OD) values. Y-axis depicts the number of samples within each category.

Figure 3

Box-and-whisker plot graph of AMDV-VP2 ELISA results for CIEP-positive and -negative samples from mink. The bottom of the box shows the 25^th percentile, the line within the box indicates the median, and the top of the box shows the 75^th percentile. Ends of the whiskers are at 25^th percentile – (1.5 × interquartile range) and 75^th percentile + (1.5 × interquartile range). Outliers are indicated as small circles. OD = optical density.

Figure 4

End-point titration curve of automated AMDV-VP2 ELISA using a high-positive mink serum. The limit of detection of the positive serum was 1:10,000 in ELISA and 1:100 in CIEP. OD = optical density.

Figure 5

Photograph of the filter paper blood comb. The blood comb is used for the blood sampling of mink in the automated AMDV-VP2 ELISA system. Twelve mink can be sampled with one comb. Except for the tips, the combs are coated with a thin film to prevent the comb absorbing dilution buffer. On the upper part of the comb, there is room for farm identification, date, and serial number.

Figure 6

Photograph of the device utilized in the automated AMDV-VP2 ELISA. The device introduces eight blood combs simultaneously to an ELISA plate for the elution of antibodies.