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Prepared by
Claude E. Barton, DVM; Robert F. Devlin, PhD; Robert M. Studholme
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System Overview
A semi-automated test system for brucellosis antibody presumptive testing in cattle and swine has been developed. The system utilizes brucella antigen that has been in use for many years, uses automated reading of the agglutination pattern to minimize subjectivity and enhance lab-to-lab uniformity and is designed for high volume applications.
Background
The Cooperative National Brucellosis Program currently utilizes three different presumptive tests in the serologic diagnosis of brucellosis in cattle and swine. They are the Buffered Acidified Plate Antigen (BAPA) test, The Rapid Screening Test (RST), and the Particle Concentration Fluorescence Immunossay (PCFIA). The BAPA and RST are very economical tests but are prone to subjectivity and variation between individual technicians and laboratories. The PCFIA, while less subjective, is comparatively much more expensive.
The RAP test was developed as a procedure that could standardize presumptive testing of cattle and swine serums for brucellosis, reduce the subjectivity and variation of the manual tests, and reduce overall costs of presumptive testing in the brucellosis eradication program. While incorporating sophisticated instrumentation, a major objective throughout its development has been to maintain low per-test cost within this high volume environment. The technology of the RAP test is adapted from the Autolex® Latex Agglutination test for pseudorabies that was developed by Viral Antigens, Inc., Memphis, TN.
Three versions of this test have been evaluated over the past 18 months. The first utilized diluted BAPA antigen. The second utilized diluted Card test antigen. While the instrumentation performed consistently well, in both cases the antigen-antibody ratio was such that there was a tendency toward prozoning in high titered serums. The third, the RAP test reported here, utilizes undiluted Card test antigen in a ratio to serum that optimizes the agglutination process while minimizing the prozone phenomena. During development, a total of approximately 60,000 serum samples have been tested with this system.
Materials And Methods
1. BIO-TEK® Ceres 900C Autoreader with 690 nm filter. The system includes: Color monitor, Bar-code scanner, Printer, Rotator (45 degree), Brucellosis RAP test software. (BRU RAP V3.01), Operator's Manual, ELISA plates, 96 well, round bottom (Falcon 3910), Pipet tips, 1 - 200 ul. (Costar), Card test antigen (USDA, APHIS, VS, Reagents Section, NVSL), Bar-code labels, Eppendorf type, 30 ul. pipettors, Multichannel pipettor (8-channel).
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Test Procedure:
1. Add 30 ul. of serum to each well.
2. Negative control serum is added to wells
__A12, B12, and C12.
3. Positive control serum is added to wells
__D12, E12, and F12.
4. Apply bar-code label to end of the ELISA plate.
5. Add 20 ul. of card test antigen to each well -
__use 8 channel pipettor.
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6. Read bar-code with bar-code reader.
7. Place plate on BIO-TEK® reader. Reader shakes for 15 seconds and does the initial scan.
8. Place plate on 45 degree rotator for 10 minutes.
9. Remove plate and allow to settle for 20 minutes.
10. At the prompt signal from the autoreader, read bar-code.
11. Place plate on BIO-TEK® reader. Final scan is done.
12. Reader stores and prints test results.
13. Remove plate and discard.
Card antigen is mixed with the sample in an ELISA well and read in a scanning ELISA plate reader. The materials are then mixed for 10 minutes and allowed to settle for 20 minutes before reading a second time. Bar codes are used to prevent mix up of plates. Up to 12 plates can be in process at any time.
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The Instrumentation
The BIO-TEK® Ceres 900c Scanning Autoreader uses a tungsten lamp filtered to 690 nm and fiber optics to illuminate 12 wells across the ELISA plate as it is drawn into the reader. Twelve detectors are used to read the total light transmitted at 26 locations within each well.
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Each of the 26 initial reads are subtracted from the final reads generating transmission change profiles representing the degree of agglutination of the Card test antigen. With no antibody present in the sample there is no change in the scan profile from the initial scan to the final scan. When antibody is present the Card antigen agglutinates and forms a pellet in the bottom of the well reducing the transmission in the center and increasing the transmission at the edge. The degree of agglutination is represented as %AGG.
Percent agglutination (%AGG) is a parameter defined by subtracting the change in the transmission in the center of the well from the change in transmission at the edge.
The final scan is subtracted from the initial scan at each position resulting in a display of the percent agglutination within each well position.
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%AGG Calculation
1) Determine the maximum reading from position 1 to 6,
2) Determine the maximum reading from position 21 to 26,
3) Take the mean of 1) & 2),
4) Determine the minimum reading from position 8 to 18,
5) Subtract the minimum from the mean
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A hard copy report is printed indicating the positives, negatives, and potential problem samples. The plate ID, read timing and other information are provided at the top of each printout. Samples can be identified, using a plate set-up feature of the software program, to provide the case and tube numbers.
A positive and negative control are run in column 12 of each ELISA plate. These results are summarized at the top of the results printout and stored on the reader for comparisons from plate-to-plate.
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Readings are controlled by parameters stored in the reader. These parameters setup assay timing, scan wavelength and error checking. Each reading is compared with these parameters to alert the operator if the reading is out of these preset limits. Also, the reference value to designate a sample as positive or negative is stored in this file.
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