Syphilis is a venereal disease caused by the spirochaete organismTreponema palladium. This disease develops 3 stages over several years, separated by asymptomatic stages during which diagnosis can only be made by serological tests. As this organism cannot be cultured on artificial media, the diagnosis of Syphilis depends on the correlation between the clinical data and the serological test results.
Antibodies become detectable at about 3-4 weeks following exposure, and may remain at detectable levels for long periods after treatment. Two groups of antibodies are formed: non-treponemal antibodies which react with nonspecific antigens (VDRL or RPR test); treponemal antibodies which react with the specific antigens to T.palladium (like TPHA test). Antibodies specific to non-treponemal antigens are found in active disease and the levels decrease after successful treatment. Specific antibodies persist long after the infection has been treated. It is necessary to test both groups of antibodies since non-treponemal antibodies may arise for reasons other than Syphilitic infection.
Principle
The TPHA tests detect human T-palladium antibodies based upon an indirect hemagglutination method (HAI). Fowl erythrocytes are sensitized with T. palladium fragments. In the presence of specific anti-T.palladiumantibodies, sensitized erythrocytes (Test Cells) agglutinate and induce a characteristic structure on the microtiter plate (appearance of even layer) in the bottom of the well.
Unspecific reactions are detected using control cells which are unsensitized fowl erythrocytes.
Simple protocol
Qualitative test (An alternative method and a semi-quantitative approach are also available, please refer to the insert):
Dispense 190 µL of diluent into well 1.
Dispense 10 µL of serum into well 1 and mix.
Discard 150 µL from well 1.
Transfer 25 µL of the mixture from well 1 to well 2.
Add 75 µL of Test Cells resuspended (CT) to well 1 and 75 µL of Control Cells resuspended (CC) to well 2.
Tap plate gently to mix the contents thoroughly.
Cover and let stand 45 to 60 minutes at room temperature. Do note move the plates. (The plates can be left overnight).
Final dilutions in wells 1 and 2 are 1/80.
Easy-to-read and easy-to-interpret results
The reading is made at the end incubation. The images below correspond to reactions carried out in U-well microtiter-plates TPHA-0004. Other plates can provide different images.
Results are expressed given the agglutination intensity (- to 4+).
All samples showing a positive or weakly positive result (+/- to 4) must be tested with a quantitative procedure.
Examples of positive results:
Examples of negative results:
Reagents and material
Test Cells:
T. palladium treponemal sensitized formolised tanned fowl erythrocytes in buffer
Quantity: 1×8.5 ml
Control Cells:
Unsensitized formolised tanned fowl erythrocytes in buffer
Quantity: 1×8.5 ml
Diluent:
Rabbit serum in buffer
Quantity: 1×20 ml
Positive control:
Serum prediluted (1/20) in buffer containing antibodies to T. palladium
Quantity: 1×1 ml
Negative control:
Serum prediluted (1/20) in buffer free of antibodies to T. palladium
Quantity: 1×1 ml
Stability and storage
When stored at 2-8°C and protected from light, the reagents are stable until the expiry date stated on the label. Do not freeze any of the reagents. Reagents must be stored upright. Performance
Prozone effect
Titre of 1/164000 has been detected by this test with no prozone (Hook) effect.
Correlation
TPHA test has been evaluated by a European reference center. These samples originated from antenatal clinics, genito-urinary clinicals and Public Health Laboratories. 1084 serum have been tested.
Results with TPHA test:
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