99.90% Sensitivity Elisa Test Kit , Syphilis Rapid Test Kit Enzyme Immunoassay

Syphilis ELISA test , high accuracy, Elisa Sandwich method, for quantitative measurement

Intended Use

Syphilis ELISA test is a qualitative enzyme immunoassay for the in vitro diagnostic detection of Treponema pallidum (syphilis) antibodies in human serum or EDTA and citrated plasma. This product can be used as an initial screening test or as a confirmatory diagnostic test.

Summary:

Syphilis is still a common sexually transmitted disease in many areas of the world. In 1999 the WHO estimated that the worldwide annual incidence of sexually acquired syphilis was 12 million cases. Venereal syphilis is divided into: early syphilis subdivided into primary, secondary and early latent stages; late syphilis that may occur after extended periods of latent syphilis. Serological tests for syphilis are subdivided into: non treponemal tests that measure IgM and IgG antibodies to lipoidal material released from damaged host cells and antibodies to lipoprotein‐like material and cardiolipin released from the treponemes. The most commonly used are RPR card and VDRL. The tests are used for screening and for determining the efficacy of threatment. They lack sensitivity in early primary syphilis and in late syphilis and it can appear a prozone reaction or false positive results. Treponema tests use T. pallidum subsp. pallidum or its derivates (recombinant proteins). They are used as confirmatory tests and in stablishing the diagnosis of late latent or late syphilis. The most commonly used tests are: FTA‐ABS, TP‐PA (T. pallidum particle agglutination) and MHA‐IP (micro hemagglutination assay to T. pallidum). Several tests using enzyme immunoassays (EIA) have being used as confirmatory test for syphilis. they have sensitivities and specificities similar to those of the other treponemal tests.

Principle of Test:

The Treponema pallidum Screen ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA). Microtiter wells as a solid phase are coated with specific, recombinant treponemal antigens. Specimens and ready-for-use controls are pipetted into these wells. During incubation Treponema pallidum-specific antibodies of positive specimens and controls are bound to the immobilized antigens. After a washing step to remove unbound sample and control material horseradish peroxidase conjugated treponemal antigens are dispensed into the wells. During a second incubation this conjugate binds specifically to antibodies resulting in the formation of enzyme-linked immune complexes. After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid. The intensity of this color is directly proportional to the amount of Treponema pallidum-specific antibody in the specimen. Absorbance at 450 nm is read using an ELISA microtiter plate reader.

Kit Components

1.  Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with Treponema pallidum antigen. (incl. 1 strip holder and 1 cover foil)
2.  Pos. Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.
3.  Neg. Control ***, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.
4.  Cut-off Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, black cap.
5.  Enzyme Conjugate **, 1 vial, 13 mL, ready to use,
6.  Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).
7.  Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/l H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.
8.  Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 7.2 ± 0.2 see „Preparation of Reagents“.

* contains 0.03 % ProClin 300

** contains 0.03 % ProClin 300 + 0.01 % Gentamicin sulphate

*** contains 0.03 % ProClin 300 + 0.015 % 5-bromo-5-nitro-1,3-dioxane (BND) + 0.010 % 2-methyl-2H-isothiazol-3-one (MIT)

ASSAY PROCEDURE

All materials should be at room temperature prior to starting the assay.

Bring all reagents to room temperature (15-30^oC) before use.

Remove the amount of strips being used for the day’s testing and replace the remainder in a re-sealable plastic bag with the desiccant and store at 2-8°C.

Allow samples to come to room temperature (15-30^oC).

1.  Dispense 100µL per well of Negative Control in the second and third wells;
2.  Dispense 100µL per well of Cut off Calibrator in wells four, five and six;
3.  Dispense 100µL per well of Positive Control in wells seven and eight
4.  Dispense 100µL per well of patient samples into respective wells.
5.   Incubate plate for 60 (±5) min at 37°C (cover the plate)
6.  Discard or aspirate the contents of the plate and wash 4 times with 300µL per well of Wash Buffer Working Solution.
7.  Add 100µL per well enzyme Conjugated Antigens to all wells
8.  Incubate plate for 30 (±3) minutes at 37°C (cover the plate)
9.  Discard or aspirate the contents of the wells and wash 4 times with 300µL per well of Wash Buffer Working Solution.
10.  Add 100µL per well of TMB substrate solution to all wells.
11.  Incubate plate for 15 (±2) minutes at 37°C, protected from light.
12.  Add 100µL of Stop Solution to each well
13.  Read within 15 minutes with optical density at 450 nm and calculate results. Bi-chromatic measurement with a reference filter at 600-690 nm is recommended if available.

INTERPRETATION OF RESULTS

Validation of the Assay

1.  O.D. of Negative control must be < 0.20
2.  O.D. of Positive control must be > 1.0
3.  O.D. of Cut-off Calibrator must be between 0.15 and 0.50 O.D.
4.  O.D. of Negative control < O.D. of Cut-off Calibrator < O.D. positive control.

If any of these conditions are not met, the assay is invalid and needs to be repeated.

Calculation of Results

1. Subtract O.D. of Blank (“Bl”) from all wells.

If the resulting O.D. value has a negative value, it is to be taken as 0.001.

1.  Determine the mean of the Cut-off Calibrator. Each value should be within 20% of the mean.
2.  To obtain the Index Values, divide the O.D. of samples (patients) or controls by the mean Cut-off Calibrator value (CO).

OD Sample/Control

Index =

OD Cut-off Calibrator

The following is intended as a guide to interpretation of the EIA results; each laboratory should establish their own criteria for test interpretation based on its sample population.

Samples with values in the equivocal range (0.8 to 1.2 ratios) should be retested. If the sample remains equivocal on retest, the patient should be considered suspect for infection with T. pallidum since a low level of antibody is detected. A second sample should be collected

2 – 4 weeks later and tested. An equivocal result indicates that a low level of antibody is detected, and the patient should be monitored for antibody status.

A negative sample and/or the negative control will have an index value < 0.8. A negative result indicates that no, or very low levels of antibody are present in the sample, but does not rule out a recent or current infection.

A positive sample and/or the positive control will have an index value > 1.2. A positive result indicates that antibody is present in the sample as a result of previous or present infection with T. pallidum. The magnitude of the measured result above the cut-off is not indicative of the total amount of antibody present.

Consult “Quality Control Certificate” for typical results obtained when run manually.

The following Table provides an algorithm to aid in interpreting and reporting syphilis serology results for diagnosis of T. Pallidum infection status.