Limitations of the ELISA, Possible Test Interference
Mistakes in handling the test can cause errors.Possible sources for such errors can be:
Inadequate storage conditions of the test kit,wrong pipetting sequence or inaccurate volumes of the reagents, too long or too short incubation times during the immune and/or substrate reaction,extreme
temperatures during the test performance (lower than 10°C or higher than 30°C).
Swine and cattle muscle samples have been tested using the extraction procedure listed in the sample preparation section and found to give recoveries between 85
120%.The ELISA kit provides screening results.As with any analytical technique (GC,HPLC, etc.) positive samples requiring some action should be confirmed by an alternative method.
ELISA procedures:
- Bring test kit to the room temperature (20 - 25 °C ) for at least 30 min,note that each reagent must be shaken evenly before use;put the required micro-well strips into plate frames.Re-sealed the unused microplate,stored at 2 - 8 °C ,not frozen .
- Numbering: number the micro-wells according to samples and standard solution;each sample and standard solution should be performed in duplicate;record their positions .
- Add 20 µ L of the sample or the standard solution into separate duplicate wells,then add enzyme conjugate,50 µL/well; antibody working solution,80 µ L/ well,Mix gently by shaking the plate manuall y,seal the microplate with the cover membrane,incubate at 25 °C for 30 min .
- Pour liquid out of microwell,flap to dry on absorbent paper;add 250 µ L/well of tri-deionized water,wash for 4-5 times,marinate for 10 s every time,then take out and flap to dry with absorbent paper.
- Coloration: add 50 µ L of substrate A solution and 50 µ L B solution into each well.Mix gently by shaking the plate manuall y,and incubate at 25 °C for 15 min in the dark for coloration .
- Determination: add 50 µ L of the stop solution into each well.Mix gently by shaking the plate manuall y.Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well.(Recommend to read the OD value at the dual-wavelength 450/630 nm within 5min ).
Read the result
Negative: only one pink colored band appears on the control region.No apparent band on the test region.
Positive: in addition to a pink color band,a distinct pink colored.band will also appear in the test region.
Invalid: a total absence of color in both regions is an indication of procedure error and/or that test reagent deterioration has occurred.
More rapid diagnostic products supplied:
HCG,LH,FSH,HIV,HBSAG,HCV,HBV,HEV,SYPLILIS,TB,HP,NGH,Chlamydia,Malaria,PSA,CEA,
FOB,Dengue,Troponin I,Toxoplasma,Drug of Abuse test,saliva alcohol test and so on.
