RBC IgG ELISA Kit

Catalog No. ABIN956284

Sheep RBC IgG ELISA Kit, Colorimetric assay for quantification of Sheep RBC IgG.

Quick Overview for RBC IgG ELISA Kit (ABIN956284)

Target: RBC IgG (Anti-Red Blood Cell IgG (RBC IgG))

Reactivity: Sheep

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Product Details RBC IgG ELISA Kit

Analytical Method: Quantitative

Characteristics: The mouse anti-SRBC IgG ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses detergent solubilized SRBC ghosts for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-mouse IgG 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-SRBC IgG molecules are thus sandwiched between immobilized SRBC 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-SRBC IgG is proportional to the optical density of the test sample.

Components: SRBC coated 96-well plate (provided as 12 strips of 8 wells)
Enzyme Conjugate Reagent: 11 mL
Reference calibrator stock: lyophilized
Diluent: 30 mL
TMB Reagent (One-Step): 11 mL
Stop Solution (1N HCl): 11 mL
20X Wash Solution: 50 mL.

Material not included: Plate reader with an optical density range of 0-4 at 450 nm
Precision pipettes and tips
Distilled or de-ionized water
Graph paper (PC graphing software is optional)
Absorbent paper or paper towels
Polypropylene or glass tubes
Vortex mixer
Micro-Plate incubator/shaker mixing speed of ~150 rpm
Plate washer.

Application Details

Plate: Pre-coated

Sample Preparation:

Note: Studies indicate that anti-SRBC IgG is present in mouse serum or plasma from SRBC immunized animals at concentrations in excess of 500 u/mL. In order to obtain values within the range of the calibration curve, we suggest that samples initially be diluted 50 fold using the following procedure for each sample tested:
1. For each test sample dispense 294 µL of diluent into separate tubes.
2. Pipette and mix 6 µL of the serum/plasma sample into a tube containing 294 µL of diluent. This provides a 50 fold diluted sample.
3. Repeat this procedure for each sample to be tested. Important: Do not use dilutions lower than 15 fold.

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 average absorbance values (A450) for each set of calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference 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-SRBC IgG in u/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-SRBC IgG in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of samples fall outside the calibration curve when tested at a dilution of 50, samples should be diluted appropriately and re-tested (do not use dilutions lower than 15 fold).

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: The kit should be stored at 4°C and the microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. Test kits will remain stable until the expiration date provided that the components are stored as described above.

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

Target Details for RBC IgG

Target: RBC IgG (Anti-Red Blood Cell IgG (RBC IgG))

Alternative Name: Red Blood Cell (SRBC) IgG

Target Type: Antibody, Antibody

Background: Recent studies have demonstrated that suppression of anti-SRBC IgG levels by therapeutic agents serves as a useful indicator of immunosuppression. This ELISA allows rapid and quantitative measurement of mouse anti-SRBC IgG levels in serum or plasma samples