IMMUTREP TPHA Ref OD221/OD071/OD081

Treponema pallidum haemagglutination test for the

Serodiagnosis of Syphilis.
o o
Store at 2 C to 8 C. DO NOT FREEZE.
For in-vitro diagnostic use only.
INTRODUCTION AND INTENDED USE PRECAUTIONS
Syphilis is a complex disease which is normally IMMUTREP TPHA reagents contain material of human
sexually transmitted. The causative organism, origin and have been tested and confirmed negative for
Treponema pallidum cannot be grown on conventional HCV, HIV I and HIV II antibodies, and HBsAg by
laboratory culture media or in the tissue culture. approved procedures at single donor level. Because no
Infection is normally diagnosed by detecting antibodies test can offer complete assurance that products derived
specific for T. pallidum in the patient’s serum or CSF. from human source will not transmit infectious agents it
Antibody becomes detectable at about 3-4 weeks is recommended that the reagents within this kit be
following exposure, and may remain at detectable handled with due care and attention during use and
levels for long periods after treatment. Two groups of disposal. All reagents should, however, be treated as
antibodies are formed: one reacting with the non- potential biohazards in use and for disposal. Do not
treponemal antigens used in the VDRL/Carbon Antigen ingest.
and RPR tests, and the other reacting with the specific
antigens of T.pallidum. Antibody to non-treponemal IMMUTREP TPHA reagents do not contain dangerous
antigens is found (normally) in active disease and the substances as defined by current UK Chemicals
levels subside after successful treatment. Specific (Hazardous Information and Packaging for Supply)
antibody persists long after the infection has been regulations. All reagents should, however, be treated
successfully treated. It is necessary to test for both as potential biohazards in use and disposal. Final
groups of antibody since the non-treponemal antibody disposal must be in accordance with local legislation.
may arise for reasons other than Syphilitic infection.
IMMUTREP TPHA is a specific, sensitive passive IMMUTREP TPHA reagents contain 0.095% sodium
haemagglutination test for the detection of antibodies to azide as a preservative which may be toxic if ingested.
Treponema pallidum in serum or CSF. Sodium azide may react with lead and copper plumbing
For professional use only. to form highly explosive salts. On disposal, flush with
large quantities of water.
PRINCIPLE OF THE TEST
IMMUTREP TPHA comprises T. pallidum sensitised STORAGE
formalised tanned fowl erythrocytes; unsensitised Reagents must be stored upright at temperatures
formalised tanned fowl erythrocytes, diluent and control between 2oC to 8oC.
sera. When diluted positive samples are mixed with
sensitised erythrocytes, antibody to the sensitising The kit will perform within specification until the stated
antigen causes agglutination of the cells. The cells expiry date as determined from date of product
form a characteristic pattern of cells in the bottom of a manufacture and stated on kit and components. Expiry
microtitration plate well. In the absence of antibody, date is the last day of the month on the bottle and the
they form a compact button in the well. kit label. Do not use reagents after the expiry date.

This test has been calibrated to WHO Reference Serum Exposure of reagents to excessive temperatures should
for Serodiagnostic tests for Treponemal Infections- Ref be avoided. Do not expose to direct sunlight.
3-1980 +/- one double dilution to ensure the correct
sensitivity. DO NOT FREEZE ANY OF THE REAGENTS as this
will cause irreversible damage.
CONTENTS
SPECIMEN COLLECTION AND PREPARATION
Ref Ref Ref Obtain a sample of venous blood from the patient and
OD081 OD071 OD221 allow a clot to form and retract. Centrifuge clotted
blood sample and collect clear serum. Fresh serum
samples are required.
850
Obtain a sample of CSF from the patient.

Do not use haemolysed, contaminated or lipaemic

Test Cells 8.5ml 2x8.5 ml 2x33 ml samples for testing as this will adversely affect the
T. Pallidum antigen coated preserved fowl erythrocytes results.
(approx 0.36% w/v) in buffer. Working Strength.
Samples may be stored at 2oC to 8oC for up to 48 hours
Control Cells 8.5ml 2x8.5ml 2x33 ml
prior to testing. If longer storage is required, store at –
Preserved fowl erythrocytes (approx 0.36% w/v) in 20oC for up to 1 year. Thawed samples must be mixed
buffer. Working Strength.
prior to testing.
DIL 20ml 2x20ml 3x57ml
Diluent. Selected rabbit serum (approximately 0.4%) in Do not repeatedly freeze-thaw the specimens as this
buffer. Working Strength. will cause false results.
Control + 1ml 1ml 9ml
Positive Control. Serum prediluted (1/20) in buffer REAGENT PREPARATION
containing antibodies to T. pallidum. Working Strength. All reagents should be brought to room temperature
Control – 1ml 1ml 9ml (20oC to 25oC) and fully resuspended prior to use. Do
Negative Control. Serum prediluted (1/20) in buffer free not induce foaming.
of antibodies to T. pallidum. Working Strength.
CELL 2 2 0 The Cell Droppers are provided for use with the Test
DROPPERS and Control Cell suspensions. These droppers
dispense 75l drops and these integral droppers should
INSTRUCTION 1 1 1 be placed on their corresponding suspensions as
LEAFLET follows:
Red Dropper – Test Cells
MATERIALS REQUIRED BUT NOT PROVIDED White Dropper – Control Cells
Dynex M24A U-well microtitration plates are
recommended. Microtitration droppers to deliver 25l or
Micropipettes to deliver 10, 25, 75, 100l and 190l
volumes.
Note: 75l droppers do not fit and are not supplied for
bottles in the 850 Test Kit.
 LIMITATIONS OF USE
RESULTS AND INTERPRETATION
The use of samples other than serum or CSF have not been validated in this test.
Kit controls or known level value samples should be tested with each test run. The
No serological haemagglutination test can discriminate between antibody due to kit negative control should give a negative result after 45 minutes. The kit positive
T.pallidum infection and antibody due to infection with other pathogenic treponemes, control should give a positive result after 45 minutes. If levels of controls or users
i.e. T.pertenue and T.carateum. known samples do not give expected results, test results must be considered invalid.
No other interfering factors have been specifically identified however positive results
should be confirmed, eg. by FTA-Abs, and complemented by clinical findings. Screening Procedure
Agglutinated cells form an even layer over the bottom of the well. Non-agglutinated
There is no reuse protocol for this product. cells form a compact button in the centre of the well. Weakly agglutinated cells form
a characteristic ring pattern. Agglutination of the Test Cells but not the Control Cells
A low or suspected positive result should be re-assessed. Diagnosis should not be indicates the presence of specific antibody to T.pallidum. Absence of agglutination
made solely on the findings of one clinical assay. When making an interpretation of indicates that antibody is below the limit of detection of the system. Do not use the
the test it is strongly advised to take all clinical data into consideration. Control Cell pattern as an indication of a negative result since they give a more
compact button of cells.
The test may also be negative in early active Syphilis or in late latent Syphilis. To Agglutination of the Control Cells as well as the Test Cells indicates the presence of
complete the profile of results to aid the physician, it is also recommended that a anti-cell antibody. In this event the test is not valid and should be repeated.
VDRL/Carbon Antigen or RPR test is performed on the patient’s sample since these
tests will detect an active case of Syphilis. OMEGA’s IMMUTREP VDRL, Should the test not be valid the test should be repeated after first performing an
IMMUTREP CARBON ANTIGEN and IMMUTREP RPR are available for this absorption of the test serum. To achieve this, dilute the test serum 1/4 with Control
purpose. Cells and allow to stand at room temperature for 45-60 minutes. After centrifuging
ASSAY PROCEDURE the sample (1000rpm/5 mins) dilute the supernatant 1/5 in Diluent. Test this dilution
directly, without any further dilution, using Test and Control Cell suspensions. A
Allow samples and reagents to reach room temperature and ensure that samples and confirmatory FTA ABS test is also recommended.
all reagents are fully resuspended before use. Samples do not require any
pretreatment. Quantitative Procedure
As screening procedure. The titre is the highest dilution showing agglutination. The
QUALITATIVE ( SCREENING ) PROCEDURE Reactive Control serum should produce a titre within one doubling dilution of 1/2560.
The starting dilution for the quantitative procedure is 1/80.
Each test requires 4 wells of a microtitre plate. Titres of 1/164000 have been detected with IMMUTREP TPHA with no prozone
1. Dispense Diluent into the microtitration plate as follows: ( Hook ) effect.
 25l in rows 1, 3 & 4 and 100l in row 2.
2. Dispense 25l of each sample into a well in row 1. TROUBLESHOOTING
 Mix well and transfer 25l from row 1 to row 2.
 Mix well and transfer 25l from row 2 to row 3. Hemagglutination tests are sensitive to the effects of heat, direct sunlight and
 Mix well and discard 25l from row 3. vibration. Keep away from such sources during test incubation periods.
 Transfer 25l from row 2 to row 4.
Do not allow saliva to contaminate the samples or reagents as this will cause
 Mix well and discard 25l from row 4
erroneous results.
3. Add 75l of well mixed Control Cells to row 3.
4. Add 75l of well mixed Test Cells to row 4. Use a separate disposable tip for each sample to prevent cross contamination.
Tap plate gently to mix.
The final dilutions in row 3 and 4 are 1/80. Replace caps on all reagents immediately after use.
5. Cover and let stand at room temperature for 45 to 60 minutes (alternatively
the plates can be left overnight). Do not allow reagent to run down the sides of the well. Prior to the start of the assay
6. Examine for agglutination patterns. bring all reagents to room temperature (20oC to 25oC). Gently mix all reagents by
Note: Kit controls are prediluted and should be added directly into individual wells in gentle inversion or swirling.
row 3 and 4 ( no diluent required ).
For use by operatives with at least a minimum of basic laboratory training.
ALTERNATIVE ONE WELL DILUTION PROTOCOL FOR SCREENING.
1. Dispense 190l of diluent into row 1 Do not use damaged or contaminated kit components.
2. Dispense 10l of sample to row 1 and mix
3. Discard 150l from row 1 Kit components are matched and should not be interchanged.
4. Add 25l from row 1 to row 2
5. Add 75l of well mixed Test Cells to row 1 EVALUATION DATA
Add 75l of well mixed Control Cells to row 2
Tap plate gently to mix. Samples were tested at a European reference centre. These samples originated
The final dilutions in wells in rows 1 and 2 are 1/80. from Antenatal Clinics, Genito – Urinary Medical Clinical and Public Health
6. Cover and let stand at room temperature for 45 to 60 minutes (alternatively Laboratories.
the plates can be left overnight). Positive Samples Negative Samples Total
7. Examine for agglutination patterns. Syphilis Positive 203 3 206
Syphilis Negative 3 669 672
QUANTITATIVE PROCEDURE 206 672 878
This study demonstrates:
If it is intended to routinely quantitate positive results the screening procedure may be
A sensitivity of 98.5%
modified by omitting the Control Cells and preparing only one final dilution. Most
A specificity of 99.6%
samples will be negative or genuinely positive, and the Control Cells may be used in
Reproducibility of IMMUTREP TPHA is 100% (+/- one doubling dilution).
the quantitative procedure below.
1. Prepare dilutions in a microtitration plate as follows: REFERENCES
 For each sample, dispense 25l of diluent into each well in one column of the
plate. For titrations of controls dispensing should commence from row 3. 1. Tomizawa, T.and Kasamatsu, S. Jap. J. Med. Sci. Biol. 19,305 (1966).
 Transfer 25l from row 2 of the original screening plate to row 1 of the 2. Rathlev, T., Brit. J. Vener. Dis., 43,181 (1967).
quantitative plate. 3. Tringali, G., Ann. Sc. Pav., 12,311 (1970).
 Mix and discard 25l. 4. Uete, T., Fukazawa., S., Ogi. K. and Takeuchi, Y., Brit., J. Vener. Dis., 47,73
 Transfer 25l from row 2 of the original screening plate to row 2 of the (1971)
quantitative plate. 5. Garner, M.F., Backhouse, J. L, Daskalopoulos, G. and Walsh, J.L., 48,474
 Prepare 25l doubling dilutions from row 2 to row 8 ( for controls doubling (1972)
dilutions should commence from row 3 ). 6. Johnston, N.A., Brit. J. Vener. Dis. 1972, 48,474 (1972)
2. Add 75l of well mixed Control Cells to row 1. 7. Sequeira, P.J.L. and Eldridge, A. E., Brit. J. Vener. Dis. 43,242 (1973 )
3. Add 75l of well mixed Test Cells to rows 2 to 8. 8. Lensinski, J., Krach, J. and Kadziewics, E., Brit. J. Vener. Dis. 50, 33 (1974)
4. Mix by gentle tapping. 9. O’Neill, P., Warner, R.W. and Nicol, C.S., Brit. J.Vener. Dis. 49,427 (1973)
The final dilutions in row 1 and row 2 are 1/80. 10. Young, H., Henrischen, C. and Robertson, D.H.H., Brit. J. Vener. Dis. 50,341
5. Cover and let stand for 45 to 60 minutes at room temperature (or overnight). (1975)

Note: Kit controls are prediluted and 25l should be added directly into 8010 Issue 5A Revised May 2015
individual wells in rows 1, 2 and 3 with doubling dilutions commencing from  Omega Diagnostics Ltd 2015.
row 3 (no diluent is required in row 1 or row 2).

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