IgM ELISA Kit

Catalog No. ABIN956142

Product Details IgM ELISA Kit

Target: IgM

Reactivity: Monkey

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: Cross-reactivity with immunoglobulins from other species has not been investigated.

Characteristics: The monkey IgM ELISA kit is designed for measurement of IgM in old world monkey serum or plasma. The assay uses goat anti-monkey IgM for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated goat anti-monkey IgM antibodies for detection. Both capture and detection antibodies were cross-absorbed on monkey IgG and IgA agarose columns, thereby ensuring specificity for IgM.Test samples are diluted and incubated in the microtiter wells for 45 minutes alongside prepared monkey IgM calibrators. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. IgM molecules are thus sandwiched between the immobilization and detection antibodies. 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 IgM is proportional to the optical density of the test sample and is derived from a calibration curve.

Components: Anti-monkey IgM -coated microtiter wells, 96 wells
Monkey IgM Calibrator (lyophilized)
Diluent (10X), 50 mL
HRP Conjugate, 11 mL
Wash Solution (20X), 50 mL
TMB Reagent (One-step), 11 mL
Stop Solution (1N HCl), 11 mL.

Material not included: Precision pipettes and tips
Distilled or de-ionized water
Polypropylene or glass tubes
Vortex mixer
Absorbent paper or paper towels
Micro-Plate incubator/shaker mixing speed of ~150 rpm
A microtiter plate reader capable of measuring absorbance at 450 nm, with a bandwidth of 10 nm or less and an OD range of 0-4 OD
Graph paper (PC graphing software is optional)

Application Details

Plate: Pre-coated

Sample Preparation:

General Note: IgM is typically present in monkey serum or plasma at concentrations of ~0.3 mg/mL. In order to obtain values within range of the calibration curve, we suggest that samples initially be diluted 10,000 fold using the following procedure for each sample to be tested:
1. Dispense 247.5 µL of 1x diluent into two tubes.
2. Pipette and mix 2.5 µL of the serum/plasma sample with 247.5 µL of 1x diluent in the first tube. This provides a 100 fold diluted sample.
3. Mix 2.5 µL of the 100 fold diluted sample with the 247.5 µL of diluent in the second tube. This provides a 10,000 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested Tissue extracts and body fluids other than serum or plasma will likely have lower IgM levels than those found in serum. Optimal dilutions of such samples should be determined empirically.

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 duplicate).
   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 to 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 (18-25°C) 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 that 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 mean absorbance values (A450) for each set of reference calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference calibrator against its concentration in ng/mL on linear graph paper, with absorbance values on the vertical or Y-axis and concentration on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of IgM in ng/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration of IgM in the sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of the sample fall outside the calibration curve, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store kit at 4°C. Keep the microtiter plate in a sealed bag with desiccant to minimize exposure to damp air. The expiration date of the kit is indicated on the box label.

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

Target Details for IgM

Target: IgM

Abstract: IgM Products

Synonyms: IgM constant region ELISA Kit, IGM ELISA Kit

Target Type: Antibody