AntibodyToTreponema Pallidum (Syphilis) ELISA Kit (EX.)

This kit is anenzyme-linked immunosorbent assay (ELISA) for qualitative determination ofthe antibodies to Treponema pallidum (TP) in human serum or plasma. It isintended for screening of blood donors and as an aid for the diagnosis andmanagement of clinical conditions known as syphilis.

Treponema pallidum

With this kit, thedetection of anti-TP antibodies is achieved by antigen “sandwich” enzyme-linkedmethod (ELISA) where polystyrene microwell strips are pre-coated withrecombinant Treponema pallidum antigens expressed in E. coli. The sample isincubated in the microwells together with recombinant TP antigens conjugatedto horseradish peroxidase (HRP-Conjugate). The pre-coated antigens expressthe same epitopes as the HRP-Conjugate antigens, but are expressed indifferent hosts. In case of presence of anti-TP in the sample, duringincubation the pre-coated and conjugated antigens will be bound to the twovariable domains of the antibody and the specific antigens-antibodyimmunocomplex is captured on the solid phase. After washing to remove sampleand unbound conjugates, Chromogen solutions containing tetramethylbenzidine(TMB) and urea peroxide are added into the wells. In presence of theantigen-antibody-antigen “sandwich” complex, the colorless Chromogens arehydrolyzed by the bound HRP conjugate to a blue-colored product. The bluecolor turns yellow after stopping the reaction with sulfuric acid. The amountof color can be measured and is proportional to the amount of antibody in thesample. Wells containing samples negative for anti-TP remain colorless.

1.Validated microplate reader. 2.Eppendorf Tubes for dilution for samples and standards. 3.Deionized or distilled water. 4.Validated adjustable micropipettes, single and multi-channel. 5.Automatic microtiter plate washer or manual vacuum aspiration equipment. 6.37C incubator.

Centrifugethe serum, plasma or cell culture supernatant samples for 10 minutes at1, 000×g. Remove particulates and assay immediately or aliquot and storesamples at -20C or -80oC. Avoid repeated freeze-thaw cycles.

Eachmicroplate should be considered separately when calculating and interpretingresults of the assay, regardless of the number of plates concurrentlyprocessed. The results are calculated by relating each sample’s opticaldensity (OD) value to the Cut-off value (C.O.) of the plate. If the Cut-offreading is based on single filter plate reader, the results should becalculated by subtracting the Blank well OD value from the print reportvalues of samples and controls. In case the reading is based on dual filterplate reader, do not subtract the Blank well OD from the print report valuesof samples and controls. 1. Calculation of Cut-off value: Cut-offvalue (C.O.) = *Nc + 0.18 *Nc= the mean absorbance value for three negative controls. Ifone of the Negative control values does not meet the Quality control rangespecifications, it should be discarded and the mean value is calculated againusing the remaining two values. If more than one negative control OD valuedoes not meet the Quality control range specifications, the test is invalidand must be repeated. 2. Quality control range: Thetest results are valid if the Quality Control criteria are verified. It isrecommended that each laboratory must establish appropriate quality controlsystem with quality control material similar to or identical with the patientsample being analyzed 1)The OD value of the Positive control must be equal to or greater than 0.800at 450/630nm, or at 450nm after blanking. 2)The OD value of the Negative control must be less than 0.100 at 450/630nm orat 450nm after blanking. 3)The OD value of the Blank well, which contains only Chromogen and Stopsolution, is less than 0.080 at 450 nm. 3. Interpretations of the results: (S= the individual optical density (OD) of each specimen) Negative Results (S/C.O.<1): Samples givingabsorbance less than the Cut-off value are negative for this assay, whichindicates that no anti-TP antibodies have been detected with this kit.Therefore, the patient is probably not infected and there are no serologicalindications for past infection with TP. Positive Results (S/C.O.>=1): Samplesgiving an absorbance greater than or equal to the Cut-off value areconsidered initially reactive , which indicates that TP antibodies have beendetected using this anti-TP ELISA kit. Retesting in duplicates of any initiallyreactive sample is recommended. Repeatedly reactive samples can be consideredpositive for antibodies to Treponema pallidum and therefore there areserological indications for current or past infection with TP. Any blood unitcontaining antibodies to Treponema pallidum should be immediately discarded. Borderline (S / C.O. = 0.9-1.1): Sampleswith absorbance to Cut-off ratio between 0.9 and 1.1 are consideredborderline and retesting of these samples in duplicates is recommended toconfirm the results. Repeatedly positive samples could be considered positivefor antibodies to TP. Retestingof these samples in duplicates is recommended. Repeatedly positive samplescould be considered positive for Treponema pallidum. Follow-up andsupplementary testing any positive with other analytical system is required.

Eachmicroplate should be considered separately when calculating and interpretingresults of the assay, regardless of the number of plates concurrentlyprocessed. The results are calculated by relating each sample’s opticaldensity (OD) value to the Cut-off value (C.O.) of the plate. If the Cut-offreading is based on single filter plate reader, the results should becalculated by subtracting the Blank well OD value from the print reportvalues of samples and controls. In case the reading is based on dual filterplate reader, do not subtract the Blank well OD from the print report valuesof samples and controls. 1. Calculation of Cut-off value: Cut-offvalue (C.O.) = *Nc + 0.18 *Nc= the mean absorbance value for three negative controls. Ifone of the Negative control values does not meet the Quality control rangespecifications, it should be discarded and the mean value is calculated againusing the remaining two values. If more than one negative control OD valuedoes not meet the Quality control range specifications, the test is invalidand must be repeated. 2. Quality control range: Thetest results are valid if the Quality Control criteria are verified. It isrecommended that each laboratory must establish appropriate quality controlsystem with quality control material similar to or identical with the patientsample being analyzed 1)The OD value of the Positive control must be equal to or greater than 0.800at 450/630nm, or at 450nm after blanking. 2)The OD value of the Negative control must be less than 0.100 at 450/630nm orat 450nm after blanking. 3)The OD value of the Blank well, which contains only Chromogen and Stopsolution, is less than 0.080 at 450 nm. 3. Interpretations of the results: (S= the individual optical density (OD) of each specimen) Negative Results (S/C.O.<1): Samples givingabsorbance less than the Cut-off value are negative for this assay, whichindicates that no anti-TP antibodies have been detected with this kit.Therefore, the patient is probably not infected and there are no serologicalindications for past infection with TP. Positive Results (S/C.O.>=1): Samplesgiving an absorbance greater than or equal to the Cut-off value areconsidered initially reactive , which indicates that TP antibodies have beendetected using this anti-TP ELISA kit. Retesting in duplicates of any initiallyreactive sample is recommended. Repeatedly reactive samples can be consideredpositive for antibodies to Treponema pallidum and therefore there areserological indications for current or past infection with TP. Any blood unitcontaining antibodies to Treponema pallidum should be immediately discarded. Borderline (S / C.O. = 0.9-1.1): Sampleswith absorbance to Cut-off ratio between 0.9 and 1.1 are consideredborderline and retesting of these samples in duplicates is recommended toconfirm the results. Repeatedly positive samples could be considered positivefor antibodies to TP. Retestingof these samples in duplicates is recommended. Repeatedly positive samplescould be considered positive for Treponema pallidum. Follow-up andsupplementary testing any positive with other analytical system is required.

1.Values of the Positive or Negative controls, which are out of the indicatedQuality control range, are indicator of possible deterioration of thereagents and/or operator or equipment errors. In such case, the resultsshould be considered as invalid and the samples must be retested. In case ofconstant erroneous results classified as due to deterioration or instabilityof the reagents, immediately substitute the reagents with new ones, orcontact our technical support for further assistance. 2.If after mixing of the TMB Solution A and B into the wells, the color of themixture turns blue within few minutes, do not continue carrying out thetesting and replace the reagents with fresh ones.