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The IDEXX HerdChek* Bovine Spongiform Encephalopathy (BSE) Antigen EIA Test Kit has been aApproved by the European Commission (EC)

Announcement

IDEXX Laboratories is pleased to announce that the IDEXX HerdChek* Bovine Spongiform Encephalopathy (BSE) Antigen EIA Test Kit has been approved by the European Commission (EC) for postmortem testing in cattle. The test is also approved by the United States Department of Agriculture (USDA). The novel capture technology used in this kit—the Seprion ligand—is licensed from Microsens Biotechnologies, UK.

The IDEXX HerdChek Bovine Spongiform Encephalopathy (BSE) Antigen EIA Test Kit is an antigen-capture enzyme immunoassay (EIA) for detection of the abnormal conformer of the prion protein (PrP^Sc). The kit detects abnormal prions from postmortem brain tissues (obex) from cows with BSE. This kit is intended for veterinary use only.

Prion nomenclature

The endogenous cellular form of the prion protein is identified as PrP^c, where "c" represents cellular. Abnormal prions are composed of PrP^Sc, where "sc" represents a generic term for the infectious prion.

IDEXX BSE assay technology

Current postmortem TSE tests for the detection of prions in bovine, ovine and cervids must destroy the PrP^c and leave any PK-resistant PrP^Sc behind. The anti-prion protein antibodies used in these assays cannot distinguish the normal, globular, proteinase K-sensitive (α-helix-rich) form of the protein (PrP^c) from the abnormal, aggregated and relatively proteinase K-resistant form (PrP^Sc), in which the secondary structure is dominated by ß-sheet.¹ Thus, a complex sample preparation procedure is required to eliminate the normal prion protein in the sample homogenate before immunoassay, usually involving a proteinase K digestion step carried out under closely controlled conditions. Under- or overdigestion problems with this protease step can compromise the specificity and sensitivity of the subsequent immunoassay. As an alternative to seeking PrP^Sc-specific antibodies, the Seprion ligand is used to selectively capture the native conformation of the rogue prion protein.²

IDEXX BSE test value proposition

Our trusted ELISA platform revolutionizes BSE testing by combining a simple homogenization step with a straightforward EIA. This technology eliminates the need for complicated proteinase K sample preparation, resulting in unmatched ease-of use and efficiency for BSE laboratories.

Truly a second-generation kit, the IDEXX HerdChek BSE EIA Test makes it very easy to process a large number of samples in either automated or manual formats. For those who prefer automation, the kit can process nine plates (828 samples) from sample incubation to result in less than five hours. When used manually, the test still provides high throughput, allowing one person to process four plates (368 samples) from sample encubation to result in less than five hours.

IDEXX BSE EIA protocol

Test samples are prepared by homogenizing brain tissue, diluting an aliquot homogenate with plate diluent, and applying directly to the plate. The Seprion ligand is immobilized on the surface of the microtiter plate. Due to the specific nature of the PrP^Sc capture, there is no need for proteinase K treatment of samples or PrP^Sc concentration prior to the capture. Once applied to the plate, the disease-associated conformer binds to the immobilized ligand with high affinity, while normal prion protein is not captured on the plate. Unbound material is removed by washing. After incubation with conditioning buffer, the captured antigen is detected using PrP-specific antibody-HRPO conjugate. After washing and addition of substrate, positive samples are identified by the development of color in the microwells. The test cutoff=negative control OD+0.12. The principles of the assay are illustrated in Figure 1. The EIA portion of the IDEXX BSE test has been validated for use in manual or automated platforms.

Refer to the product insert for complete instructions.

IDEXX BSE assay performance: European Union data (phases I and II)

The IDEXX BSE EIA test has been evaluated on bovine brain populations from Europe. The BSE status of all positive samples used in these studies were confirmed to be PrP^Sc-positive by immunohistochemistry (IHC) or an EU-approved BSE rapid test. Assays were performed using the IDEXX BSE EIA standard protocol described in the package insert.

Analytical sensitivity—dilution series
The EU lab evaluation (phase I) required a dilution series of a BSE-positive homogenate. Positive brain tissue was serially diluted to 1:200 in negative bovine brain homogenate, and then tested in replicate across three different IDEXX BSE EIA plates. Additional 1:400 and 1:800 sample dilutions were added at IDEXX's request. Mean OD values relative to cutoff and standard deviations are graphically represented for each dilution in Figure 2. The number of replicates was variable for each dilution because of limited homogenate volume. All dilutions tested positive for all replicates up to the 1:400 dilution; four of five 1:800 dilutions were detected as positive.

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Diagnostic sensitivity and poor-quality negatives
Diagnostic sensitivity was tested using two hundred-fifty (n=250) samples from BSE-positive animals provided as part of the lab evaluation (phase I) and field evaluation (phase II). Two hundred (n=200) poor-quality samples were used to test the effect of autolyzed samples on the specificity of the test. Figure 3 shows the OD relative to cutoff values for the 200 confirmed-positive samples tested on the IDEXX BSE EIA test during phase II of the EU field trials. The BSE EIA test identified all 200 positive samples correctly. The fifty BSE-positive samples tested during the lab evaluation also tested positive on the IDEXX BSE EIA, demonstrating an assay sensitivity of 100% (50/50=100%).

Figure 3 also shows results for poor-quality negative samples. The test demonstrated 100% specificity for this population. The positive (red) and negative (gray) populations are clearly separated, with most of the BSE-positive samples testing greater than 20-fold higher than the cutoff for each plate.

Figure 3.

Diagnostic specificity
A negative population of 10,715 animals, including one hundred and fifty (n=150) samples from phase I and two hundred (n=200) poor-quality negatives from phase II (field trials), were tested using the BSE EIA test according to the standard assay protocol. The samples were confirmed negative by either IHC or an EU-approved test method. The IDEXX BSE EIA test performed with 99.99% specificity, with a mean sample OD relative to cutoff value of 0.325 and a standard deviation of 0.082. The mean of the population was 8.2 standard deviations from the test cutoff, and demonstrated a normal distribution (Figure 4).

Table 1 shows an overall summary of diagnostic sensitivity and diagnostic specificity of the IDEXX HerdChek BSE Antigen Test Kit during the EU evaluation. The IDEXX BSE test showed an excellent performance during this evaluation and successfully gained the EC approval.

Table 1. Summary of IDEXX BSE assay performance during the EU evaluation (phase I and phase II)

*Four samples were initially reactive; three initially reactive samples were retested as negative.

Summary
The IDEXX BSE EIA test is an antigen-capture enzyme immunoassay (EIA) for detection of the abnormal conformer of the prion protein (PrP^Sc) in cattle. The IDEXX BSE EIA test is USDA- and EC-approved. The test performed with 100% sensitivity and 99.99% specificity during the EU validation studies. The test is designed to rapidly identify samples containing disease-associated PrP^Sc with minimal sample handling, and provides high throughput with or without automation. The simplified sample preparation eliminates the need for extra laboratory equipment, and allows customers to perform high-volume screening with ease and precision.

References

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