ELISA for the Quantitative Determination of

Mouse IgG in Serum or Plasma

INTRODUCTION

The mouse IgG ELISA kit is designed for measurement of IgG in mouse serum or plasma. The assay uses goat anti-mouse IgG for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibodies for detection. Both capture and detection antibodies were crossabsorbed

on mouse IgM and IgA agarose columns, thereby ensuring specificity for IgG. Cross-reactivity with immunoglobulins from other species has not been investigated.

PRINCIPLE OF THE TEST

Test samples are diluted and incubated in the microtiter wells for 45 minutes alongside prepared mouse IgG standards. The microtiter wells are subsequently washed and HRP conjugate is added and

incubated for 45 minutes. IgG 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 IgG is proportional to the optical density of the test sample and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

· Anti mouse IgG coated 96-well plate (12 strips of 8 wells)

· HRP Conjugate Reagent, 11 ml

· Reference standard (lyophilized)1

· 20x Wash Solution, 50 ml

· 10x Diluent (50 ml)

· TMB Reagent (One-Step) 11 ml

· Stop Solution (1N HCl), 11 ml

Materials required but not provided:

· Precision pipettes and tips

· Distilled or deionized water

· Polypropylene or glass tubes

· Vortex mixer

· Absorbent paper or paper towels

· Micro-Plate incubator/shaker mixing speed of ~150 rpm

· Plate washer

· Plate reader with an optical density range of 0-4 at 450nm

· Graph paper (PC graphing software is optional)

SAMPLE PREPARATION

General Note: IgG is typically present in mouse serum or plasma at concentrations of ~15 mg/ml. In order to obtain values within range of the standard curve, we suggest that samples initially be diluted 200,000 fold using the following procedure for each sample to be tested:

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1. Dispense 998 ml and 798 ml of 1x diluent into separate tubes.

2. Pipette and mix 2 ml of the serum/plasma sample into the tube containing 998 ml of diluent. This provides a 500 fold diluted sample.

3. Mix 2 ml of the 500 fold diluted sample with the 798 ml of diluent in the second tube. This provides a 200,000 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 IgG 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 ml of standards 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 ml/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 ml 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 ml 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 ml 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.