产品名称：Diphtheria ELISA Kit

Limitations of procedure:
This assay determines the relative amount of DAT antibodies in serum. It cannot be used to diagnose active disease or conclusively determine immune/non-immune status.

Preparation Wash Buffer - Remove cap and add contents of bottle to 475 mL of reagent grade water. Place diluted wash buffer into a squeeze bottle with a narrow tip opening. Note: Washings consist of filling to the top of each well, shaking out the contents and refilling. Avoid generating bubbles in the wells during the washing steps.

Coagulate blood and remove serum. Freeze sample at -20 °C or lower if not used immediately. Do not heat inactivate serum and avoid repeated freezing and thawing of samples. Test samples: Make a 1:100 and a 1:1,000 dilution of patients' sera using the dilution buffer. to any antibodies present. Before the third incubation step, more washings are necessary. Then a chromogen (tetramethylbenzidine or TMB) is added. With the presence of Enzyme Conjugate and the peroxidase causing the consumption of peroxide, the chromogen changes to a blue color. The blue color turns to a bright yellow color after the addition of the stop solution, which ends the reaction. ELISA readers can be used to obtain results. Test Diphtheria IgG ELISA Method Enzyme Linked Immunosorbent Assay Principle Sandwich Complex Detection Range Quantitative : Positive, Negative Control Sample 5 μL serum Total Time - 20 min.
Shelf Life
12 Months from the manufacturing date Specificity

1. Break off number of wells needed (four for calibrators plus number of samples) and place in strip holder.
   2. Add 100 μL of each calibrator to wells 1-4, then add 100 μL of the diluted test samples to the remaining wells. Note: Standards are supplied prediluted. Do not dilute further.
   3. Incubate at room temperature for 10 minutes.
   4. Shake out contents and wash 3 times with the diluted wash buffer.
   5. Add 100 μL of Enzyme Conjugate to each well.
   6. Incubate at room temperature for 5 minutes.
   7. Shake out contents and wash 3 times with wash buffer, then rinse once with DI water. Slap wells against paper towels to remove excess moisture.
   8. Add 100 μL of the Chromogen to every well. Mix by gently tapping strip holder.
   9. Incubate at room temperature for 5 minutes.
   10. Add 100 μL of the Stop Solution and mix by tapping strip holder.

ELISA Reader: Zero reader on air. Set for bichromatic readings at 450/650-620 nm. T roubleshooting Negative control has excessive color after development. Reason: inadequate washings. Correction: wash more vigorously. Remove excessive liquid from the wells by tapping against an absorbent towel. Do not allow test wells to dry out. Interpretation of Results Construct a standard curve using the absorbance (OD) results of the four controls and the controls' International Units (IU) included in the kit. All graphs should be on log-log 10 paper: Y axis for absorbance and X axis for IU's. Plot the control coordinates and determine the best fit line. Using the absorbance data and the standard curve as a guide, determine the approximate IU for each sample. Once the IU value has been determined by the graph, multiply this number by the dilution factor of the sample. Example: Sample "A" has an absorbance of 0.4 OD units at a 1:100 serum dilution. This OD value corresponds to an IU value of 0.008 IU/mL. Thus the sample has a DAT value of 0.8 IU/mL (0.008 x 100 dilution.