Tetanus Toxoid IgM ELISA Kit

Catalog No. ABIN956289

The Rat Tetanus Toxoid IgM ELISA Kit (ABIN956289) is a Colorimetric ELISA Kit designed to quantify Rat Tetanus Toxoid IgM.

Quick Overview for Tetanus Toxoid IgM ELISA Kit (ABIN956289)

Target: Tetanus Toxoid IgM

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Product Details

Analytical Method: Quantitative

Characteristics: The Rat Anti-TT IgM ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses tetanus toxoid for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-rat IgM antibodies for detection. Test serum or plasma samples are diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. Anti-TT IgM molecules are thus sandwiched between immobilized TT antigens and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies and TMB reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of stop solution, changing the color to yellow, and optical density is measured spectrophotometrically at 450 nm. The concentration of anti-TT IgM is proportional to the optical density of the test sample.

Components: Microtiter Plate: TT coated 96-well plate (12 strips of 8 wells)
Enzyme Conjugate Solution: 11 mL
Calibrator: Lyoph. x 3 Store -20°C
Diluent Buffer: 30 mL
TMB Solution: 11 mL
Stop Solution: 11 mL, 1N HCl
Wash Buffer (20x): 50 mL.

Material not included: Plate reader (450 nm)
Micropipette and tips
De-ionized water
Graph paper (PC software is optional)
Paper towels
Polypropylene or glass tubes
Vortex mixer
Plate shaker/incubator
Plate washer.

Application Details

Plate: Pre-coated

Reagent Preparation:

Wash Buffer: The wash solution is provided as 20x stock. Prior to use dilute the contents of the bottle (50 mL) with 950 mL of distilled of deionized water. Calibrator
1. The rat anti-tetanus toxoid IgM calibrator is provided as a lyophilized stock. Reconstitute one vial with distilled or deionized water as described on the vial label and mix gently until dissolved. (The reconstituted calibrator is stable for at least one day if stored at 4°C.
2. Label 6 polypropylene or glass tubes as 100, 50, 25, 12.5, 6.25, and 3.125 u/mL.
3. Into the tube labelled 100 u/mL, pipette the volume of diluent indicated on the stock vial label. Then add the volume of reconstituted calibrator stock detailed on the vial label and mix. This provides the 100 u/mL calibrator.
4. Pipette 250 µL of diluent into the tubes labelled 50, 25, 12.5, 6.25, and 3.125 u/mL.
5. Into the tube labelled 50 u/mL pipette and mix 250 µL of the 100 u/mL reconstituted calibrator. This provides the 50 u/mL calibrator.
6. Similarly prepare the 25, 12.5, 6.25, and 3.125 u/mL calibrators by serial dilution.

Sample Preparation:

Note: Studies indicate that anti-TT IgM is present in rat serum or plasma at concentrations of approximately 2,500 u/mL 5 days after immunization with TT. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 50 fold using the following procedure for each sample tested. Optimal dilutions may need to be determined empirically.
1. For each test sample dispense 343 µL diluent into tubes.
2. Pipette and mix 7 µL of the serum/plasma sample into the tube containing 343 µL of diluent. This provides a 50 fold diluted sample.
3. Repeat this procedure for each sample to be tested.

Assay Procedure:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators, and diluted samples into the wells (we recommend that samples be tested in triplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1x wash solution using a plate washer (400 µL/well). The entire wash procedure should be performed as quickly as possible.
   5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer.
   6. Add 100 µL of enzyme conjugate reagent into each well.
   7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   8. Wash as detailed in 4 and 5 above.
   9. Dispense 100 µL of TMB reagent into each well.
   10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature for 20 minutes.
   11. Stop the reaction by adding 100 µL of Stop Solution to each well.
   12. Gently mix. It is important to make sure all the blue color changes to yellow.
   13. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.

Calculation of Results:

1. Calculate the average absorbance values for each set of calibrators, and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each calibrator against its concentration in u/mL on linear graph paper, with absorbance values on the vertical or Y axis and concentrations on the horizontal or X axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of anti-TT IgM in u/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-TT IgM in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD values of samples fall outside the calibration curve when tested at a dilution of 100, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Handling Advice: Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of and in accordance with the instructions detailed above.
The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

Storage: 4 °C/-20 °C

Storage Comment: Store at 4°C. The calibrator stock should be stored at or below -20°C. Microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. The kit is stable until the expiration date when stored as noted in this section.

Expiry Date: The expiry date is stated on the label.

Target Details

Target: Tetanus Toxoid IgM

Target Type: Antibody