E0610

8°C
Syphilis Ab ELISA KIT
2°C
(SERUM / PLASMA)
INTENDED USE 3. Bring all reagents to room temperature (18°C-28°C) before use.
4. Do not use the components in any other type of test kit as a substitute for the
The Aria Syphilis Ab ELISA Kit is a solid phase enzyme linked immunoabsorbent assay for the components in this kit.
qualitative detection of antibodies (IgG, IgM, IgA) against Treponema pallidum in human serum 5. Do not use hemolized blood specimen for testing.
or plasma. It is intended for professional use only as an aid in the diagnosis of infection with 6. Do not ingest the reagents. Avoid contact with eyes, skin and mucose. Wear protective
Tp. Any reactive specimen with the Aria Syphilis Ab ELISA Kit must be confirmed with clothing and disposable gloves while handling the kit reagents and clinical specimens.
alternative testing method(s) and clinical findings. Wash hands thoroughly after performing the test.
7. Do not smoke, drink, or eat in areas where specimens or kit reagents are being
INTRODUCTION handled.
Tp, a spirochete bacterium, is the causative agent of the venereal disease syphilis. Although 8. Users of this test should follow the US CDC Universal Precautions for prevention of
syphilis rates are declining in the United States after an epidemic outbreak between 1986 and transmission of HIV, HBV and other blood-borne pathogens.

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19901, the incidence of syphilis in Europe has increased since 1992, especially in the countries 9. Dispose of all specimens and materials used to perform the test as biohazardous waste.
of the Russian Federation, where peaks of 263 cases per 100,000 have been reported . In
2 10. In the beginning of each incubation and after adding Stopping Solution, gently rocking
1995, WHO reported 12 million new cases of syphilis3. Currently, the positive rate of syphilis the microwells to ensure thorough mixing. Avoid the formation of air bubbles as which
serological tests in HIV-infected individuals has been rising recently. results in inaccurate absorbance values. Avoid splash liquid while rocking or shaking

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the wells.
Serological detection of anti-Tp antibody has been long recognized in the diagnosis of syphilis 11. Don’t allow the microplate to dry between the end of the washing operation and the
since the natural course of the infection was characterized by periods without clinical reagent distribution.
manifestations. Both IgM and IgG antibodies were detected in sera from patients with primary 12. The enzyme reaction is very sensitive to metal ions. Thus, do not allow any metal
and secondary syphilis. The IgM antibody may be detectable towards the second week of element to come into contact with the conjugate or substrate solution.
infection, while IgG antibody appears later, at about 4 weeks4. These antibodies could last for 13. The substrate solution must be colorless. The appearance of color indicates that the
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several years or even decades in the serum of a patient with untreated latent syphilis . reagent cannot be used and must be replaced. The Substrate B must be stored in the

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dark.
Antigens such as Rapid Plasma Cardiolipin antigen (RPR) and Tp bacterial extracts have been 14. Use a new distribution tip for each specimen. Never use the specimen container to
used in the syphilis serological tests for decades. However, RPR antigen is a non-treponema distribute conjugate and substrate.
antigen, derived from bovine heart. Antibody to RPR antigen does not develop until 1-4 weeks 15. The wash procedure is critical. Wells must be aspirated completely before adding the
after the appearance of the chancre, thus this antigen lacks of sensitivity to primary syphilis. Washing Solution or liquid reagents. Insufficient washing will result in poor precision and
The Tp extracts are prepared from inoculated rabbit testis and contain a certain amount of falsely elevated absorbance.
contaminated materials such as flagella, which can lead to cross reactions with borreliae and 16. Avoid strong light during color development.
leptospires in the serological test. In addition, the composition of extracts may vary from lot to SPECIMEN COLLECTION AND PREPARATION
lot.

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 Serum should be prepared from a whole blood specimen obtained by acceptable
In contrast, the Aria Syphilis Ab ELISA Kit utilizes Tp specific recombinant antigens6-9, which venipuncture technique.
redeems the test highly specific, sensitive, and reproducible.  This kit is designed for use with serum specimen without additives only.
 If a specimen is not tested immediately, refrigerated at 2°C-8°C. If storage period
TEST PRINCIPLE greater than three days are anticipated, the specimen should be frozen (-20°C). Avoid
The Aria Syphilis Ab ELISA Kit is a solid phase enzyme linked immunoabsorbent assay based

The Aria Syphilis Ab ELISA Kit is composed of two key components:

1) Solid microwells pre-coated with recombinant Tp antigens;

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on the principle of the double antigen sandwich technique for the detection of antibodies to Tp
in human serum or plasma. 


repeated freezing-thawing of specimens. If a specimen is to be shipped, pack in
compliance with federal regulation covering the transportation of etiologic agents.
Specimens containing precipitants may give inconsistent test results. Clarify such
specimens by centrifugation prior to assaying.
Do not use serum specimens demonstrating gross lipemia, gross hemolysis or turbidity.
Do not use specimens containing sodium azide.

PREPARATION OF THE REAGENTS

2) Liquid conjugates composed of recombinant Tp antigens conjugated with horse reddish 1. Bring all reagents, controls to room temperature (18°C-28˚C).
peroxidase (HRP-Tp conjugates).
2. Dilute concentrated Washing Buffer 30 fold with water as following:
During the assay, the test specimen and HRP-Tp conjugates are incubated simultaneously
with the coated microwells. Antibodies (IgG, IgM, or IgA) to Tp if present in the specimen, Plate DI water 30 X wash buffer Final volume
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reacts to the Tp antigens coated on the microwell surface as well as the HRP-Tp conjugates, Full plate 580 mL 20 mL 600 mL
forming sandwich complex conjugates. Half plate 290 mL 10 mL 300 mL
A quarter plate 145 mL 5 mL 150 mL
Unbounded conjugates are then removed by washing. The presence of the complexed
conjugates is shown by a blue color upon additional incubation with TMB substrate. The Warm up the concentrated Washing Buffer at 37˚C to dissolve the precipitant if it
reaction is stopped with Stop Solution and absorbances are read using a spectrophotometer at appears.
450 /620-690 nm.
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REAGENTS AND MATERIALS PROVIDED 3. Mix each reagent before adding to the test wells.

Item Description Quantity Catalog 4. Determine the number of microwells needed and mark on the ELISA Working Sheet
1. Microwells coated with Tp antigens 8 wells x12 strips E0610W with the appropriate information. Positive and Negative Controls require to be run in
2. Syphilis Ab negative control 1 mL E0610N duplicate to ensure accuracy.
3. Syphilis Ab positive control 1 mL E0610P
4. HRP–Tp conjugates 6 mL E0610H ASSAY PROCEDURE
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5. Wash buffer (30 x concentrate) 20 mL WE3000

6. TMB substrate A 6 mL TME2000A 1. Remove the desired number of strips and secure them in the microwell frame. Reseal
7. TMB substrate B 6 mL TME2000B un-used strips.
8. Stop solution 6 mL SE1000
9. ELISA Working Sheet 2 sets E0001ES 2. Add specimens according to the designation on the ELISA Working Sheet
10. Product insert 1 set PI-E0610
2.1 Blank well: Leave the blank well alone. Don’t add any reagents.
MATERIALS REQUIRED BUT NOT PROVIDED
2.2 Control wells: Add 50 µL of Syphilis Ab Positive, Negative Control into the
1. Pipette capable of delivering 50 µL and 100 µL volumes with a precision better than designated control wells, respectively.
1.5%.
2. Microplate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 2.3 Test wells: Add 50 µL of test specimen into each test well, respectively.
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OD or greater at 450 nm wavelength is acceptable.

3. Absorbent paper for blotting the microplate wells. 3. Add 50 µL of HRP-Tp Conjugate solution into each well, but not the blank well.
4. Distilled or de-ionized water
5. Timer. 4. Gently rock the wells for twenty second, then cover the wells.
6. Parafilm or other adhesive film sealant for sealing plate.
5. Incubate the wells at 37°C for 60 minutes.
STORAGE AND STABILITY
6. Carefully remove the incubation mixture by empting the solution into a waste container.
 Test components are stable up to their expiration data when stored at 2°C-8°C. Do not Fill each well with diluted wash buffer and shake gently for 20-30 second. Discard the
freeze. wash solution completely by inverting and tapping the plate on absorbent paper.
 Return all reagents requiring refrigeration immediately after use. Repeat above procedure 4 more times.
 Reseal the microwells immediately after removing the desired number of wells.
 Do not mix or use components from the kits with different lot numbers. Do not use 7. Drain the wells by firmly tapping the plate on a clean paper towel to remove excess
reagents after their expiration date. washing solution.
WARNINGS AND PRECAUTIONS
8. Add 50 µL (1 drop) of TMB substrate A and 50 µL (1 drop) of TMB substrate B into each
For in Vitro Diagnostic Use well.
1. This package insert must be read completely before performing the test. Failure to
follow the insert gives inaccurate test results. 9. Incubate at 37°C in the dark for 10 minutes.
2. Do not use expired devices.

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 10. Stop the reaction by adding 50 µl (or 1 drop) of stop buffer to each well. Gently mix for 8. Bailey MJ, Thomas CM, Cockayne A, Strugnell RA, Penn CW. Cloning and expression
30 seconds. It is important to make sure that all the blue color changes to yellow of Treponema pallidum antigens in Escherichia coli. J Gen Microbiol 1989; 135 ( Pt
color completely. 9):2365-78.
9. Sambri V, Marangoni A, Simone MA, D'Antuono A, Negosanti M, Cevenini R.
11. Set the microplate reader wavelength at 450 nm and measure the absorbance of each Evaluation of recomWell Treponema, a novel recombinant antigen-based enzyme-
well against the blank well within 15 minutes after adding Stop Solution. A filter of 620 - linked immunosorbent assay for the diagnosis of syphilis. Clin Microbiol Infect 2001;
690 nm can be used as a reference wavelength to optimize the assay result. 7(4):200-5.
10. Rufli T. Syphilis and HIV infection. Dermatologica 1989; 179:113-117.
INTERPRETATION OF RESULTS

A. Set up the cut-off value Index of CE Symbols

Consult For in vitro
The cut-off value = 0.15 + N instructions for use diagnostic use only Use by
N: Mean OD of the negative control. Use 0.05 for calculation of the cut-off value if the
mean OD is less than 0.05. Tests per kit
REF Catalog # Lot Number N

B. Calculation of specimen OD ratio Store between 2-8°C Authorized

Do not reuse
Representative

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Calculate an OD ratio for each specimen by dividing its OD value by the Cut-off Value Manufacturer Date of manufacture
as follows:

Specimen OD

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Specimen OD ratio =  CTK Biotech, Inc.
Cut-off Value 10110 Mesa Rim Road
C. Assay validation MDSS GmbH
San Diego, CA 92121, USA
Schiffgraben 41, 30175 Hannover Germany
Tel: 858-457-8698
The mean OD value of the Syphilis Ab positive controls should be > 0.80. Fax: 858-535-1739
The mean OD value of the Syphilis Ab negative controls should be < 0.10. E-mail: [email protected]

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Check the procedure and repeat assay if above conditions are not met.
PI-E0610-ARIA Rev D
D. Interpretation of the results Effective date: 2015-03-03
English version
Specimen OD ratio
For Export Only, Not For Re-sale In the USA.
Negative < 1.00
Positive  1.00

1. The negative result indicates that there is no detectable anti-Tp antibodies in the

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specimen.
2. Results just below the cut-off value (Lower than 10% of the cut-off value) should
be interpreted with caution (it is advisable to re-test in duplicate the
corresponding specimens when it is applicable).
3. Specimens with cut-off > 1.00 are initially considered to be positive by the Aria
Syphilis Ab ELISA Kit. They should be retested in duplicate before final
interpretation.
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If after retesting of a specimen, the absorbance value of the 2 duplicates are less
than the cut-off value, the initial result is non repeatable and the specimen is
considered to be negative with the Aria Syphilis Ab ELISA Kit.
Non repeatable reactions are often caused by:
 Inadequate microwell washing,
 Contamination of negative specimens by serum or plasma with a high
antibody titer,
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 Contamination of the substrate solution by oxidizing agents (bleach, metal
ions, etc.)
 Contamination of the stopping solution
If after retesting the absorbance of one of the duplicates is equal or greater than
the cut-off value, the initial result is repeatable and the specimen is considered to
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be positive with the Aria Syphilis Ab ELISA Kit, subject to the limitation of the
procedure, described below.

LIMITATION OF THE TEST

1. The Assay Procedure and the Assay Result Interpretation must be followed closely
when testing the presence of anti-Tp antibodies in serum or plasma from individual
subjects. Failure to follow the procedure may give inaccurate results.
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2. The Aria Syphilis Ab ELISA Kit is limited to the qualitative detection of anti-Tp antibodies
in human serum or plasma. The intensity of the color does not have linear correlation
with the antibody titer in the specimen.
3. A negative result for an individual subject indicates absence of detectable anti-Tp
antibodies. However, a negative test result does not preclude the possibility of exposure
to or infection with Tp.
4. A negative result can occur if the quantity of anti-Tp antibodies present in the specimen
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is below the detection limit of the assay, or the antibody that are detected are not
present during the stage of disease in which a specimen is collected.
5. Some specimens containing unusually high titer of heterophile antibodies or rheumatoid
factor may affect expected results.
6. The results obtained with this test should only be interpreted in conjunction with other
diagnostic procedures and clinical findings.
REFERENCES

1. Centers for Disease Control and Prevention. Chlamydia trachomatis infections: policy
guidelines from prevention and control. Morbid. Mortal. Weekly Rep. 1995; 34:53S-
74S.
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2. Tichonova, L., K. Borisenko, H.Ward, A.meheus, et al. Epidemics of syphilis in the

Russian Federation: Trends, origins, and priorities for control. Lancet 1997; 350:210-
213.
3. Gerbase, A. C., J. T. Rowley, D. H. Heymann, S. F. Berkley, and P. Piot. Global
prevalence and incidence estimates of selected curable STDs. Sex. Transm. Infect
1998; 74:S12-S16.
4. Luger AFH. Serological Diagnosis of Syphilis: Current methods. In: Young H, McMillan
A, eds. Immunological diagnosis of sexually transmitted diseases. New York: Marcel
Decker, 1988: 249-274.
5. Baker-Zander SA, Hook EW 3rd, Bonin P, Handsfield HH, Lukehart SA. Antigens of
Treponema pallidum recognized by IgG and IgM antibodies during syphilis in humans. J
Infect Dis. 1985; 151(2):264-72.
6. Norgard MV, Chamberlain NR, Swancutt MA, Goldberg MS. Cloning and expression of
the major 47-kilodalton surface immunogen of treponema pallidum in Escherichia Coli.
Infect Immun 1986; 54:500-506.
7. Purcell BK, Chamberlain NR, Goldberg MS, Andrews LP, Robinson EJ, Norgard MV,
Radolf JD. Molecular cloning and characterization of the15-kilodalton major immunogen
of Treponema pallidum. Infect Immun. 1989; 57(12):3708-14.

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