T. pallidum (Syphilis)
PREPARATION FOR ASSAY
1. Bring all reagents and samples to room temperature (20-25oC) and mix gently before beginning the test.
2. Have all reagents and samples ready before the start of the assay. Once the test has begun it must be performed without any interruptions to get the most reliable and consistent results.
3. Use new disposable tips for each specimen.
ASSAY PROCEDURE (30/15)
1. Secure the desired number of coated wells in the holder. Mark data sheet with sample identification.
2. Dispense 50 ul of Sample diluent into well #1 as a blank, The negative Control into well #2, The positive Control into well #3, and Weakly Reactive Control into well #4 and the Patient Samples into remaining well.
3. Dispense 50 ul TP Enzyme Conjugate into each well except blank well.
4. Incubate for 30 minutes at room temperature.
5. Wash Five times with the Washing buffer.
6. Dispense 100 ul of Solution A and 100 ul of Solution B.
7. Incubate for 15 minutes at room temperature.
8. Stop reaction by adding 50 ul of Stop Solution in each well.
9. Zero a micro reader on the blank and measures absorbance of each well at 450 nm.
1. Dispense 50 ul of the sample diluent into well 2, well 3 to well 8 etc
2. Dispense 50 ul of patient serum or plasma onto well 1 and 50 ul onto well 2.
3. Mix well 2 thoroughly, then transfer 50 ul of the mixture from well 2 to well 3 . Mix thoroughly. Repeat this dilution procedure to well 8. Discard 50 ul from the last well.
4. Well 1 through 8 represent a dilution series as follows:
Well 1 2 3 4 5 6 7 8
Dil undil 1:1 1:2 1:4 1:8 1:16 1:32 1:64
5. Proceed with the test as described in the Assay Procedure from Step 6 to Step 12
INTERPRETATION OF RESULTS
Specimens yielding absorbance reading greater than Weakly Reactive at 450 nm should be reported as positive for antibodies against Teponema pallidum. Absorbance less than Weakly Reactive within 10% limit, a new sample after two weeks should be retested with the old sample. If O.D. is less than Weakly Reactive, the sample is considered negative.
VALIDATION OF TEST
1. Negative Control: mean absorbance value should be <0.2 units.
2. Positive Control mean absorbance value should be greater than Weakly Reactive, 2X of value.
3. A test may be validated if the above criteria are met.
1. Portony, J., W. Garson, and C.A. Smith. 1957. Rapid plasma regain test for Syphilis. Pub. Health Rep. 72:761-766.
2. Manual Tests for Syphilis, Public Health Service Publication, No. 411, 1969.
3. Hunter, E.F. Deacon, W.E. and Meyer, P.E. An improved FTA Test for Syphilis, The Absorption Procedure(FTA-ABS). Public Health Report 79,410-412, 1964.
4. Ijsselmuiden, O.E. and L.M. Schouls et al. 1989. Sensitivity and Specificity of an Enzyme-Linked Immunosorbent assay Using the recombinant DNA-Derived Treponema pallidum Protein TmA for serodiagnosis of assessing the Effect of Antibiotic Therapy. J. Clin. Microbiol.. 27:152-157.
5. Lesinsky, J., J. Krach, and E. Kadziewicz. 1974.Specificity, Sensitivity, and Diagnostic value of the TPHA test. Br. J. Vener. Dis. 50:334-340.