October 2007 Issue
Validation of a 90-minute TSE Assay: An Ultra-Short Microtiter-Based EIA
Introduction
The IDEXX HerdChek* Bovine Spongiform Encephalopathy Antigen Test Kit (BSE EIA) and IDEXX HerdChek* Bovine Spongiform Encephalopathy-Scrapie Antigen Test Kit (BSE-Scrapie EIA) are designed to rapidly detect the abnormal conformer of the prion protein (PrPS) in postmortem brain samples, preferably from the medulla oblongata (obex), with minimal sample handling and high throughput. IDEXX has developed the new Ultra-Short assay protocol option that decreases EIA assay time to 90-minutes while maintaining the same high level of sensitivity and specificity attained with the approved Standard (4-hour) and Short (2.2-hour) protocols. In the 90-minute protocol, a 32°–37°C (89.6°–98.6°F) incubator is used during all assay incubation steps and a slow-speed plate shaker is used during the sample incubation step to accelerate PrPS capture from the viscous brain homogenate sample. The enhanced binding kinetics provided by the elevated temperature and plate shaker allow for a significant reduction in the sample incubation and conjugate incubation time as compared to the Standard and Short protocols.
In the new 90-minute Ultra-Short protocol, a 32°–37°C (89.6°–98.6°F) incubator is used during all assay incubation steps and a slow-speed plate shaker is used during the sample incubation step to accelerate PrPS capture.
Objective
The study described below was conducted to compare the performance of the Ultra-Short 90-minute transmissible spongiform encephalopathy (TSE) assay protocol to the Standard 4-hour assay protocol for bovine and ovine populations. Diagnostic sensitivity and specificity were evaluated as well as analytical sensitivity.
Methods and Materials
In the IDEXX HerdChek* BSE and BSE-Scrapie Antigen test kits, bovine or small ruminant brain samples are prepared by homogenizing 0.3 ± 0.05g of tissue and diluting the homogenized samples in a plate diluent. The diluted samples are then applied directly to the ligand-coated antigen capture plate. In the 4-hour protocol, which was used as the approved method for these studies, the samples are incubated on the plate for a 2–3 hour static incubation period, while in the 90-minute protocol the samples are incubated for 20–25 minutes on a slow-speed plate shaker (200 ± 100 rpm). In the 4-hour protocol, all incubations are at room temperature (18°–25°C or 64.4°–77.0°F ), while in the 90-minute protocol, all assay incubations are performed at 32°–37°C (89.6°–98.6°F). Following the sample incubation, plates are washed to remove unbound materials. After a short conditioning buffer step, the captured antigen is detected using a PrP-specific antibody that has been conjugated to horseradish peroxidase (HRPO). The conjugate incubation time for the 4-hour protocol is 60–75 minutes versus 25–30 minutes for the 90-minute protocol. After the conjugate incubation, plates are washed to remove unbound conjugate and a peroxidase substrate is added. Color development is related to the relative amounts of PrPS captured by the ligand immobilized in the microtiter plate well. The Ultra-Short 90-minute assay protocol option decreases assay time by approximately 100 minutes at the sample incubation step and 35 minutes at the conjugate incubation step, compared to the 4-hour protocol. The 4-hour, 2.2-hour, and 90-minute assay protocols are compared in Figure 1.
Results
Diagnostic Specificity—Bovine
A total of 2,100 normal bovine brain samples were tested on the Standard 4-hour and Ultra-Short 90-minute IDEXX HerdChek* BSE assay protocols for evaluation of diagnostic specificity. Of the 2,100 normal bovine samples tested, all were correctly identified as negative on both assay protocols, resulting in a specificity of 100% for both methods. The population histograms and bin/frequency ranges are shown in Figures 2 and 3 for the Standard 4 hour and Ultra-Short 90-minute protocols, respectively. Data is depicted as OD values relative to test cutoff (OD/Cutoff).
The negative population distributions for the two protocols are roughly equivalent and demonstrate the new Ultra-Short protocol to have excellent specificity.
Diagnostic Specificity—Small Ruminant
A total of 2,008 normal small ruminant brain samples were tested on the 4-hour and 90-minute IDEXX HerdChek* BSE-Scrapie assay protocols for evaluation of diagnostic specificity. Of the 2,008 normal small ruminant samples tested, all were correctly identified as negative on both assay protocols, resulting in a specificity of 100% for both methods. The population histograms and bin/frequency ranges are shown in Figures 4 and 5 for the 4-hour and 90-minute protocols, respectively. Data is depicted as OD values relative to test cutoff (OD/Cutoff).
The negative population distributions for the two protocols are roughly equivalent and demonstrate the new Ultra-Short protocol to have excellent specificity.
Diagnostic Sensitivity—BSE
A total of 20 BSE-positive bovine brain tissue samples were tested using the IDEXX HerdChek* BSE Standard 4-hour and Ultra-Short 90-minute assay protocols for evaluation of diagnostic sensitivity. Each sample was tested in quadruplicate on both the approved Standard protocol and the new Ultra-Short protocol. Mean OD values are listed in Table 1 as well as the ratio of 90-minute OD value to 4-hour OD value (90-minute versus 4-hour). Of the 20 samples tested, all were correctly identified as positive on both protocols, for a diagnostic sensitivity of 100% for each method. The OD relative to cutoff values are summarized as an XY scatter plot (Figure 6). The R2 correlation for the two sets of data is 0.977, demonstrating excellent correlation between the two protocols.
Table 1: BSE
Positive Population (mean OD
values)
4231:170
Sample # |
Protocol |
90-minute vs. 4-hour |
90-minute
Mean ± Std Dev |
4-hour
Mean ± Std Dev |
| 1 |
3.00 ± 0.008 |
3.09 ± 0.056 |
0.97 |
| 2 |
4.00 ± 0.000 |
3.96 ± 0.043 |
1.01 |
| 3 |
4.00 ± 0.000 |
3.99 ± 0.024 |
1.00 |
| 4 |
3.85 ± 0.121 |
3.70 ± 0.094 |
1.04 |
| 5 |
2.97 ± 0.080 |
3.06 ± 0.149 |
0.97 |
| 6 |
4.00 ± 0.000 |
3.92 ± 0.096 |
1.02 |
| 7 |
2.08 ± 0.115 |
2.34 ± 0.113 |
0.89 |
| 8 |
2.49 ± 0.063 |
2.40 ± 0.092 |
1.04 |
| 9 |
4.00 ± 0.000 |
3.94 ± 0.077 |
1.02 |
| 10 |
3.97 ± 0.055 |
3.89 ± 0.081 |
1.02 |
| 11 |
4.00 ± 0.000 |
4.00 ± 0.000 |
1.00 |
| 12 |
4.00 ± 0.000 |
4.00 ± 0.000 |
1.00 |
| 13 |
4.00 ± 0.000 |
4.00 ± 0.000 |
1.00 |
| 14 |
3.96 ± 0.046 |
3.68 ± 0.026 |
1.08 |
| 15 |
3.98 ± 0.034 |
3.90 ± 0.079 |
1.02 |
| 16 |
3.97 ± 0.039 |
3.98 ± 0.040 |
1.00 |
| 17 |
4.00 ± 0.000 |
3.94 ± 0.077 |
1.01 |
| 18 |
2.71 ± 0.087 |
2.80 ± 0.119 |
0.97 |
| 19 |
3.99 ± 0.028 |
3.97 ± 0.051 |
1.00 |
| 20 |
4.00 ± 0.000 |
3.97 ± 0.051 |
1.01 |
Diagnostic Sensitivity—Scrapie
Twenty scrapie-positive ovine brain tissue samples were tested using the HerdChek* BSE-Scrapie Standard 4-hour and Ultra-Short 90-minute assay protocols for evaluation of diagnostic sensitivity. Each sample was tested in quadruplicate on both the approved Standard protocol and the new Ultra-Short protocol. Mean OD values are listed in Table 2 as well as the ratio of 90-minute OD value to 4-hour OD value (90-minute versus 4-hour). Of the 20 samples tested, all were correctly identified as positive on both protocols, for a diagnostic sensitivity of 100% for each method. The OD relative to cutoff values for the negative and positive populations are summarized as an XY scatter plot (Figure 7). The R2 correlation of for the two sets of data is 0.999, demonstrating good correlation between the two protocols.
Table 2: Scrapie Positive Population (mean OD values)
| Sample # |
Protocol |
90-minute vs. 4-hour |
90-minute
Mean ± Std Dev |
4-hour
Mean ± Std Dev |
| 1 |
3.97 ± 0.0568 |
3.95 ± 0.034 |
1.01 |
| 2 |
3.96 ± 0.049 |
3.94 ± 0.070 |
1.00 |
| 3 |
4.00 ± 0.000 |
4.00 ± 0.000 |
1.00 |
| 4 |
4.00 ± 0.000 |
3.97 ± 0.033 |
1.01 |
| 5 |
3.96 ± 0.050 |
3.97 ± 0.054 |
1.00 |
| 6 |
3.90 ± 0.074 |
3.89 ± 0.101 |
1.00 |
| 7 |
3.97 ± 0.037 |
3.89 ± 0.079 |
1.02 |
| 8 |
3.98 ± 0.036 |
3.94 ± 0.068 |
1.01 |
| 9 |
4.00 ± 0.000 |
3.96 ± 0.088 |
1.01 |
| 10 |
3.97 ± 0.031 |
3.96 ± 0.079 |
1.00 |
| 11 |
3.96 ± 0.054 |
3.96 ± 0.078 |
1.00 |
| 12 |
3.93 ± 0.058 |
3.92 ± 0.098 |
1.00 |
| 13 |
3.89 ± 0.147 |
3.94 ± 0.092 |
0.99 |
| 14 |
3.92 ± 0.058 |
3.97 ± 0.066 |
0.99 |
| 15 |
3.96 ± 0.045 |
3.96 ± 0.050 |
1.00 |
| 16 |
3.96 ± 0.057 |
4.00 ± 0.000 |
0.99 |
| 17 |
4.00 ± 0.000 |
4.00 ± 0.000 |
1.01 |
| 18 |
3.91 ± 0.079 |
3.97 ± 0.036 |
0.99 |
| 19 |
3.93 ± 0.052 |
3.88 ± 0.097 |
1.01 |
| 20 |
3.91 ± 0.111 |
3.91 ± 0.073 |
1.00 |
Analytical Sensitivity—BSE Conjugate
For evaluation of analytical sensitivity, the BSE dilution panels used for batch release testing of the IDEXX HerdChek* BSE EIA Test Kit were evaluated using the Standard 4-hour and Ultra-Short 90-minute protocols. The BSE panel is made up of a BSE-positive brain homogenate that has been diluted into normal bovine brain homogenate to final dilutions of 1:55, 1:100 and 1:218 (representing BSE panel members 1, 2 and 3 respectively). Each panel member was tested in triplicate on two kit lots using the 4-hour and 90-minute protocols.
Analytical Sensitivity—BSE-Scrapie Conjugate
For evaluation of analytical sensitivity, the scrapie dilution panel used for batch release testing of the IDEXX HerdChek* BSE-Scrapie EIA Test Kit was evaluated using both the 4-hour and new 90-minute protocols. The details for composition of the scrapie panel are described in the preceding section. Each panel member was tested in triplicate using the 4-hour and 90-minute protocols.
Mean OD/cutoff values for the scrapie dilution series (SRB-CC Conjugate) are shown in Figure 9 (error bars indicate standard deviation). Scrapie panel results for the 90-minute protocol closely align with the Standard 4-hour protocol test results.
Results
The sensitivity and specificity performance characteristics of the new Ultra-Short 90-minute protocol for the IDEXX BSE EIA and the IDEXX BSE-Scrapie EIA are equivalent to the Standard 4-hour protocol.
IDEXX Ultra-Short protocol saves up to 1.5 hours on the EIA portion of the assay as compared to other TSE microtiter plate tests, and increases overall laboratory productivity.
Two IDEXX TSE Assays Sport Faster Protocols and Complete Automation
IDEXX is pleased to announce that the IDEXX HerdChek* Bovine Spongiform Encephalopathy Antigen Test Kit (BSE EIA) and the HerdChek* Bovine Spongiform Encephalopathy-Scrapie Antigen Test Kit (BSE-Scrapie EIA) have received Community Reference Laboratory and USDA approval for two new time-saving and productivity-enhancing features:
- Ultra-Short protocol
- Full automation (automation from Step 1)
The new Ultra-Short protocol EIA takes only 90 minutes, making the IDEXX BSE and BSE-Scrapie Ag protocols the fastest tests available. IDEXX Ultra-Short protocol saves up to 1.5 hours on the EIA portion of the assay as compared to other TSE microtiter plate tests and increases your laboratory’s overall productivity.
Additionally, IDEXX HerdChek* BSE and BSE-Scrapie Antigen test kits offer the only fully automated and traceable protocol from the tissue homogenate to the final test result. A single robotic instrument can perform the entire assay.
IDEXX offers three kits in the HerdChek* transmissible spongiform encephalopathy (TSE) product line, two of which are available with the Short and Ultra-Short protocols.
Proper Maintenance of BSE EIA Ultra-Short Protocol Incubators
An additional piece of equipment for users of the new HerdChek* BSE and BSE Scrapie Ultra-Short protocol is a plate incubator. This piece of equipment shortens reagent incubation times (please refer to Figure 1 in the Field Notes article in this newsletter). The incubator must have minimal airflow and be able to maintain a temperature of 32°–37°C (89.6°–98.6°F). Since the plate incubator is critical for reliable Ultra-Short protocol results, it must be maintained and checked for temperature accuracy on a routine basis.
Maintenance and calibration of all laboratory equipment is essential for accurate and reproducible results. Maintenance routines depend upon the amount of daily testing performed in your laboratory. Always refer to your equipment manufacturer’s guide for their recommendations.
Events Around the World
- Florianopólis, Brazil—October 16–19, 2007
Congresso da ABRAVES 2007
- Angers, France—October 17–19, 2007
ADILVA 2007
www.adilva.com
- Reno, Nevada, USA—October 17–24, 2007
50th Annual AAVLD/USAHA Meeting—Come see us at Booth 203 and 205!
www.aavld.org
- Tsukuba, Japan—October 29–November 2, 2007
9th International Colloquium on Paratuberculosis
www.soc.nii.ac.jp/jsp3/9ICP
- São Paulo, Brazil—November 5–9, 2007
XX RAIB; Il Simpósio sobre Manejo Sanitário de Reprodução de Bovinos
www.infobibos.com/raib2007
- Melbourne, Australia—November 11–14, 2007
WAVLD 2007—Come see us at Booth #1!
www.wavld2007.com
- Rome, Italy—November 14–16, 2007
SIDiLV—IX Congresso Nazionale
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