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TPA (SYPHILIS) IgG; Page



Atlas Link

12720 Dogwood Hills Lane, Fairfax, VA 22033 USA

Phone: (703) 266-5667, FAX: (703) 266-5664

http://www.atlaslink-inc.com, info@atlaslink-inc.com



TREPONEMA PALLIDUM

(TPA) IgG ELISA

For in vitro diagnostic use.

Catalog No. 1463
INTENDED USE

The Atlas Link (AL) TREPONEMA PALLIDUM (TPA) IgG test is an Enzyme-Linked Immunosorbent Assay (ELISA) for the detection and semi-quantitative determination of IgG antibody toTreponema pallidum in human serum and as an aid in the diagnosis of syphilis.


SUMMARY

Syphilis is a contagious and infectious systemic disease of humans caused by the spirochete organism Treponema pallidum and characterized by periods of active florid manifestations and by years of symptomless latency. It can affect any tissue or vascular organ of the body and be passed from a mother to her unborn child. Syphilis may be fatal or a seriously disabling disease causing irreversible damage to the cardio-vascular, central nervous or musculoskeletal systems.

Syphilis is traditionally classified as acquired or congenital, each being further subdivided on the basis of the natural course of the disease. Treponema pallidum is a delicate spiral organism approximately 0.25 micron wide and from 5 to 20 microns long. It can be examined under darkfield microscope or identified by fluorescent techniques. In acquired syphilis, infection is usually transmitted by sexual intercourse. The incubation period of syphilis can vary from 1 to 13 weeks, but usually from 3 - 4 weeks (1). Untreated patients with primary or secondary syphilis having active lesions are the most infectious, and the risks of contagion are greatest during the first 2 years of infection. At the site of innoculation the primary chancre develops as a red papule which soon erodes forming a painless ulcer. Most primary chancres occur in the genital area. In secondary syphilis manifestations usually begin to appear about 6 weeks to 6 months after infection. Virtually every organ and tissue of the body is affected, including most body fluids (2). Over 80% of patients have mucocutaneous lesions, 50% have generalized enlargement of the lymph nodes, and about 10% have lesions of the eyes, bones and joints, meninges, liver, and spleen. Mild constitutional symptoms of malaise, headache, anorexia, nausea, aching pains in the bones, and fatigability are often present.

Congenital syphilis is the result of passage of T. pallidum across the placenta. Clinical manifestations may be present at birth but are more often seen at 3 weeks to 6 months of age. Signs of early congenital syphilis include skin rashes, mucous membrane lesions, anemia, and painful osteochondritis of the long bones. Clinical manifestations of late-onset congenital syphilis include blindness, deafness, deformed bones and teeth, and gummas (2).

For nearly two decades, the fluorescent treponemal antibody-absorption test (FTA-ABS) has been considered the method of choice in confirming the diagnosis of syphilis. A number of conditions have been reported to cause false-positive results, and they can be divided into two groups: the first embraces false reactions that are related to any biologic phenomena occurring in the patient; the other are biologic false-positives caused by the patient's clinical condition. Among the latter, diseases that produce abnormal antibodies, notably lupus erythematosus (LE), are by far the most common (3). Two types of antibodies are produced by T. pallidum: nontreponemal antibodies (reagin) and treponemal antibodies. The nontreponemal tests measure both IgG and IgM antilipid antibodies formed by the host in response both to lipoidal material released from damaged host cells early in infection and to lipid from the treponeme itself. Antigens for these tests are basically modifications of VDRL antigen (4) that contains cardiolipin, cholesterol, and lecithin and therefore lack specificity and are reactive in a variety of other conditions (5). The CDC recommended FTA-ABS test method uses a sorbent extract of Treponema reiter to reduce antibody interferences caused by reagin antibodies. Interpretation of results due to staining patterns and the presence of the whole organism's multiple binding site antigens can still result in biologic false-positive results in the FTA-ABS test (6).

The AL TPA IgG test uses a purified antigen and a sorbent extract to reduce biologic interferences and provide for accurate true -positive detection of treponemal antibodies.

The AL TPA IgG ELISA kit provides all the necessary reagents for the rapid semi-quantitative determination of IgG antibody to syphilis in human sera.

PRINCIPLE

The AL TPA diagnostic test kit uses the ELISA technique for the detection of IgG antibodies to purified T. pallidum antigen. The purified antigen is bound to the surface of the polystyrene wells. Patient serum samples to be assayed for antibody are first diluted in sorbent to bind non-specific antibodies and incubated at room temperature with the purified antigen bound to the solid surface of the microassay well. If antibody is present in the patient's serum, antigen-antibody complexes are formed. After washing the unbound serum from the well, horseradish peroxidase conjugated anti-human IgG is added to the wells and allowed to incubate. The conjugate will bind to human antibody which is present. After washing the unbound conjugate from the wells, substrate/chromogen is added and incubated. The enzyme conjugate present will react with the H2O2 substrate and tetramethylbenzidine (TMB) chromogen, resulting in blue color development. The addition of 1N H2SO4 stops the enzymatic reaction and turns the blue color to yellow. The absorbance of the solution, measured at 450 nm, is directly related to the concentration of antibody to T.pallidum.


MATERIALS SUPPLIED

Each kit contains the following components in sufficient quantities to perform the number of tests indicated on the package label.

1. T.pallidum antigen (inactivated), coated microassay plate: 96 wells, configured in twelve 1x8 strips. (96 T: one plate)

2. Sorbent Diluent: ready to use. Contains Treponema reiter extract in buffer, and proclin (0.1%) as preservative. (96 T: one bottle, 30 mL)

3. Calibrator: human serum. Sodium azide (0.1%) and pen/strep (0.01%) added as preservatives, with factor printed on vial label. (96 T: one vial, 0.250 mL)

4. Positive Control: human serum. Sodium azide (0.1%) and pen/strep (0.01%) added as preservatives, with range printed on vial label. (96 T: one vial, 0.250 mL)

5. Negative Control: human serum. Sodium azide (0.1%) and pen/strep (0.01%) added as preservatives, with range printed on vial label. (96 T: one vial, 0.250mL)

6. Horseradish-peroxidase (HRP) Conjugate: ready to use. Goat anti-human IgG, containing proclin (0.1%) as a preservative. (96 T: one bottle, 16 mL)

7. Wash Buffer (20X concentrate): dilute 1 part concentrate + 19 parts deionized or distilled water. Contains TBS, Tween-20 and proclin (0.1%) as a preservative, pH 7.2 + 0.2. (96 T: one bottle, 60 mL)

8. Chromogen/Substrate Solution: Tetramethylbenzidine (TMB), ready to use. (96 T: one bottle, 15 mL)


PRECAUTIONS

1. The human serum components used in the preparation of the controls and calibrators in this kit have been tested for the presence of antibody to human immunodeficiency virus (HIV), as well as Hepatitis B surface antigen and found negative. Because no test methods can offer complete assurance that HIV, Hepatitis B virus, or other infectious agents are absent, specimens and human-based reagents should be handled as if capable of transmitting infectious agents. Note: The Center for Disease Control and the Center for Devices and Radiological Health recommend that potentially infectious agents be handled at the Biosafety Level 2 (CDC/NIH manual Biosafety in Microbiology and Biomedical Laboratories, 1993).

2. The components in this kit have been quality control tested as a Master Lot unit.

3. Do not use reagents beyond the stated expiration date marked on the package label.

4. All reagents must be at room temperature (21-25° C) before running assay. Remove only the volume of reagents that are needed. Do not pour reagents back into vials as reagent contamination may occur.

5. Before opening Control and Calibrator vials, tap firmly on the benchtop to ensure that all liquid is at the bottom of the vial.

6. Use only distilled or deionized water and clean glassware.

7. Do not let wells dry during assay; add reagents immediately after completing wash steps.

8. If washing steps are performed manually, wells are to be washed three times. Five wash cycles are necessary if a washing manifold or automated equipment is used.

9. Sodium azide inhibits Conjugate activity. Clean pipette tips must be used for the Conjugate addition so that sodium azide is not carried over from other reagents.

10. It has been reported that sodium azide may react with lead and copper in plumbing to form explosive compounds. When disposing, flush drains with water to minimize build-up of metal azide compounds.

11. Never pipet by mouth or allow reagents or patient sample to come into contact with skin.

12. If a sodium hypochlorite (bleach) solution is being used as a disinfectant, do not expose to work area during actual test procedure because of potential interference with enzyme activity.

13. Avoid contact of sulfuric acid with skin or eyes. If contact occurs, immediately flush area with water.

14. Caution: Liquid waste at acid pH must be neutralized prior to adding to sodium hypochlorite solutions (bleach ) to avoid formation of poison gas.

15. For in vitro diagnostic use only.


MATERIALS REQUIRED BUT NOT SUPPLIED

1. Stop solution - 1N sulfuric acid (H2SO4) - (One part H2SO4 (18M) to 35 parts dH2O).

2. Graduated cylinder (100 mL).

3. Flask (1L).

4. Timer - 0 to 60 minutes.

5. Micropipettes capable of accurately delivering 10-200 L volumes (less than 3% cv).

6. Deionized or distilled water.

7. Paper towels.

8. Wash bottle, semi-automated or automated wash equipment.

9. Single or dual wavelength microplate reader with 450 nm filter (If dual wavelength is used, set the reference filter to 600-650 nm.) Read the operators' manual or contact the instrument manufacturer to establish linearity performance specifications of the reader.

10. Test tubes for serum dilution.

11. Disinfectant solution (e.g., dilute household bleach 1:10 with distilled water) or disposal basin. Do not use bleach while running test, it may interfere with conjugate activity.



Note: Use only clean, dry glassware.
STORAGE AND SHELF LIFE OF REAGENTS

1. Store unopened kit between 2° and 8° C. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.

2. Unopened microassay plates must be stored between 2° and 8° C. Unused strips must be immediately resealed in a sealable bag with desiccant and humidity indicator, and returned to storage at 2° and 8° C.

3. Store HRP Conjugate, Sorbent Diluent, and 20X Wash Buffer between 2° and 8° C.

4. Store the Calibrator, Positive Control and Negative Control between 2° and 8° C.

5. Store 1X (diluted) Wash Buffer at room temperature (21° to 25° C) for up to 5 days, or one week between 2° and 8° C.



6. Store the Chromogen/Substrate Solution between 2° and 8° C.

Note: If constant storage temperature is maintained, reagents and substrate will be stable for the dating period of the kit. Refer to package label for expiration date. Precautions were taken in the manufacture of this product to protect the reagents from contamination and bacteriostatic agents have been added to the liquid reagents. Care should be exercised to protect the reagents in this kit from contamination.


SPECIMEN COLLECTION

A blood sample should be collected by qualified personnel using approved aseptic venipuncture techniques. Clarify serum samples containing visible particulate matter by centrifugation. The samples may be stored at 2-8° C if testing is to be done in approximately one week. If stored longer, they should be frozen at -20° C or lower. Do not use hyperlipemic, hemolytic, heat treated or contaminated samples.


PREPARATION OF REAGENTS

1. All reagents must be removed from refrigeration and allowed to come to room temperature ( 21 - 25° C) before use. Return all reagents to refrigerator promptly after use.

2. All samples and controls should be vortexed before use.

3. Dilute 60 mL of the 20X Wash Buffer to 1200 mL with distilled and/or deionized H20. Mix well.


SERUM TREATMENT

Immunoassays for the detection of T. pallidum are known to be sensitive to non-specific interfering antibodies. This kit overcomes non-specificity by utilizing a sorbent solution which diminishes competing spirochete and reagin antibodies, which may be responsible for false positive reactions.


GENERAL PROCEDURE

1. Determine the number of patients to be assayed. For each assay the Calibrator should be run in duplicate. Also the Positive Control, Negative Control, and a reagent blank (RB) should be run on each assay. Check software and reader requirements for the correct Calibrator/Control configurations.


Example Configuration:


1A

RB

2A

Patient #4

1B

NC

2B

Patient #5

1C

Cal

2C

Patient #6

1D

Cal

2D

Patient #7

1E

PC

2E

Patient #8

1F

Patient #1

2F

Patient #9

1G

Patient #2

2G

Patient #10

1H

Patient #3

2H

Patient #11

2. For each test serum, Calibrator and Control to be assayed prepare a 1:21 serum dilution. Add 10 L of each serum sample to 200 L of Serum Diluent . Mix well.

3. Remove the number of wells needed from the plate bag and arrange in a strip holder. The remaining strips should be resealed in the plate bag with desiccant and humidity card. The bag should be reheat sealed or rolled over and the end taped. If the color of the humidity card changes from blue to pink the strips should not be used.

4. Transfer 100 L of the prediluted samples to the reaction wells, using a multichannel pipette. Withdraw and expel each sample at least three times to ensure proper mixing of the sample before transferring to the reaction plate. Use new fresh pipettes tips for each sample. Add 100 L of Serum Diluent to the reagent blank.

5. Incubate the plate at room temperature (21-25° C) for 20 minutes +1 minute.

6. Wash the reaction plate three times with 1x Wash Buffer. Shake all of the liquid out of the wells. With a wash bottle, automated or semi-automated wash system fill each well with 250-300 L Wash Buffer making sure no air bubbles are trapped in the wells. Shake all of the Wash Buffer out of the wells. Repeat the wash two more times. A total of up to 5 washes may be necessary with automated equipment. After the last wash shake out the Wash Buffer and remove residual Wash Buffer by tapping the plate firmly on a paper towel. The Wash Buffer can be collected in a basin and treated with 0.5% sodium hypochlorite (bleach) at the end of the days run.

7. Add 100 L of the Conjugate to each well of the reaction plate, including reagent blank.

8. Incubate each well of the reaction plate at room temperature (21-25° C) for 20 minutes + 1 minute.

9. Repeat wash as described in Step 6.

10. Add 100 L of the TMB Substrate to each well of the reaction plate, including reagent blank.

11. Incubate each well of the reaction plate at room temperature (21-25° C) for 10 minutes + 1 minute.

12. Add 100 L of the Stop Solution to each well, including reagent blank, at the same rate as the TMB Substrate was added. Positive samples will turn from blue to yellow. Tap plate to ensure mixing.

13. Read the plate using a spectrophotometer at a wavelength of 450 nm. If dual wavelength is used, set the reference filter to 600-650 nm. Measure each optical density (OD) against the reagent blank. The plate should be read within 30 minutes of assay completion.
CALCULATION OF RESULTS

Absorbance must be converted into ISR Values by using the following formula:


Sample Absorbance (450 nm)

ISR = __________________________________________

Calibrator Absorbance (450 nm) X Factor*
* The Factor is empirically derived by the manufacturer by analyzing cut-off data. Factors must not be interchanged between kit lot numbers. Factors for each kit lot are listed on the packing list and the Calibrator vial.
INTERPRETATION OF RESULTS


ISR

Results

Interpretation


<0.90

Negative

No detectable antibody to TPA by the ELISA test. Such individuals are presumed to be uninfected with syphilis and to be susceptible to primary infection.


0.91-1.09

Equivocal

Samples should be retested.


>1.10

Positive

Indicates presence of detectable antibody to TPA by the ELISA test. Indicative of current or previous infection. The individual may be at risk of transmitting syphilis infection, but is not necessarily currently contagious.


QUALITY CONTROL

For the assay to be considered valid the following conditions must be met:

1. Calibrator and Controls must be run with each test run.

2. Reagent blank (when read against air blank) must be < 0.150 Absorbance (A) at 450 nm.

3. Negative Control must be < 0.250 A at 450 nm (when read against reagent blank).

4. Each Calibrator must be > 0.250 A at 450 nm (when read against reagent blank).

5. Positive Control must be > 0.500 A at 450 nm (when read against reagent blank).

6. AL recommends that a Positive Control of known reactivity be included in each assay, run as part of the user's quality control program.

7. If above criteria are not met on repeat, contact AL Technical Service.
LIMITATIONS

1. The user of this kit is advised to carefully read and understand the package insert. Strict adherence to the protocol is necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of the incubation steps and control of incubation temperature are essential for accurate results.

2. Some antinuclear antibodies have been found to cause a false positive reaction on some ELISA tests.

3. A negative result does not rule out a current or recent infection. An equivocal result should be retested in duplicate, along with a second sample drawn several days later. Samples that remain equivocal after repeat testing should be retested by an alternate method, e.g., immunofluorescence assay (IFA).

4. Interpret results from an immunosuppressed patient with caution.

5. If comparisons with other methods are required, always perform both tests simultaneously.

6. Do not scratch coated wells during washing and aspiration. Wash and fill all reagents without interruption. While washing, check that all wells are filled evenly with washing solution, and that there are no residues in the wells.

7. Instructions for using appropriate photometers are to be observed; check adjustment of proper wave length (450 nm) and reference wave length (620 nm, optional) respectively.

8. The values obtained from this assay are intended to be an aid to diagnosis only. Each physician must interpret the results in light of the patient's history, physical findings and other diagnostic procedures.
EXPECTED VALUES

Pregnant women should be assessed by antibody analysis for antibody status to syphilis. If a patient develops clinical signs and symptoms, a serum specimen should be collected and paired with a second one collected within 5 days. The presence of IgM or a significant rise in IgG antibody, together with clinical symptoms, is diagnostic for recent infection. Neonates with positive results should be monitored over several months for a significant rise in titer due to possible congenital infection. Both the mother's and child's serum should be tested simultaneously.


PERFORMANCE CHARACTERISTICS
REPRODUCIBILITY

Three studies were performed to assess the precision of the test results. Five sera run in triplicate with values ranging from negative to high positive were used with three lots of kits for inter-lot assay results. Six sera were used for 5 days in two wells each for inter-day assay results. Six sera were used in ten wells each for intra-run assay.

The results are as follows:

INTER - LOT ASSAY








Serum 1


Serum 2

Serum 3

Serum 4

Serum 5

mean

3.04

0.31

0.91

1.82

4.43

S.D.

0.22

0.06

0.13

0.12

0.29

C.V.

7.2%

19.9%

14.7%

6.4%

6.5%

INTER - DAY ASSAY







Serum 1


Serum 2

Serum 3

Serum 4

Serum 5

Serum 6

mean

0.42

2.16

2.83

3.38

4.15

0.34

S.D.

0.06

0.25

0.17

0.14

0.26

0.06

C.V.

14.3%

11.7%

6.0%

4.1%

6.3%

17.6%

INTRA - RUN ASSAY







Serum 1

Serum 2

Serum 3

Serum 4

Serum 5

Serum 6

mean

0.25

0.44

1.32

2.19

3.37

4.79

S.D.

0.05

0.06

0.06

0.08

0.08

0.17

C.V.

21.2%

12.9%

4.5%

3.9%

2.4%

3.5%


CROSS-REACTIVITY

A series of 59 patient's sera confirmed positive for antinuclear antibodies (ANA), rheumatoid factor, cardiolipin, and Lyme were obtained from outside clinical laboratories. These positive sera were tested by commercial kits for ANA, RF, Lyme and cardiolipin antibody. These sera were then run in the AL TPA IgG ELISA test. No positive reactions were found on the AL kit, but eight samples were positive by the reference IFA method for reactivity to FTA-ABS. This study demonstrates that the AL TPA IgG test does not contain cross-reacting proteins to ANA, rheumatoid factor, Cardiolipin, or Lyme even though the FTA-ABS test showed positive reaction to eight Lyme samples. It is assumed this is due to the purified antigen and conjugate specificity used in the AL kit.



SENSITIVITY AND SPECIFICITY

A study was performed using 219 patient sera obtained from outside clinical laboratories. These samples were tested using both the AL TPA IgG ELISA test and a commercially available IFA test following the manufacturers' package inserts. Two samples were found to be false negative on the AL test as compared to the IFA and one sample was false positive as compared to the IFA kit. Using the above data criteria, the AL TPA IgG ELISA test has a 97.2% sensitivity and 99.3% specificity as compared to the results obtained on the IFA kit. The following data was obtained:

IFA Relative Relative

+ - Sensitivity Specificity



AL + 72 1

TPA IgG 97.2% 99.3%

- 2 144 (72/74) (144/145)



Agreement = 216/219 = 98.6%




REFERENCES

1. Fitzgerald, T. J. 1986. Treponema. In: Manual of Clinical Microbiology, 4th Edition. E. H. Lennette. etal eds. ASM, Wash., DC. pp 485-489.

2. Larsen, S. A., L. L. Bradford. 1986. Serodiagnosis of Syphilis. In: Manual of Clinical Laboratory Immunology, 3rd Edition. N. R. Rose, H. Friedman, J. L. Fahey. eds. ASM, Wash., DC. pp 425-434.

3. Neimeister, R. P. and J. T. Bartola. 1985. How accurate is today's syphilis testing. Diagnostic Medicine. March: 25-28.

4. U.S. Dept. of Public Health, Ed., and Welfare, National Commun. Disease Cntr., Venereal Disease Program. Manual of tests of syphilis, 1969. Center for Disease Control, Atlanta.

5. Bradford, L. L. and S. A. Larsen. 1986. Serologic Tests for syphilis. In: Manual of Clinical Microbiology, 4th Edition. E. H. Lennette. etal eds. ASM, Wash., DC. pp 910-919.

6. Hunter, E. F., W. E. Deacon, and P. E. Meyer. 1964. An improved test for syphilis-the absorption procedure (FTA-ABS). Public Health Rep. 79: 410-412.

P/N 6300-29 Rev B 2/95



Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA

Phone: (703) 266-5667, FAX: (703) 266-5664

http://www.atlaslink-inc.com, info@atlaslink-inc.com




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