IgG1 ELISA Kit

Catalog No. ABIN956264

Product Details IgG1 ELISA Kit

Target: IgG1

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: This ELISA kit is designed for measurement of IgG1 in old world monkey serum or plasma. The assay uses specific polyclonal anti-monkey IgG for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated mouse monoclonal anti-monkey IgG1 antibody for detection. Studies at have demonstrated that the kit recognizes old world monkey IgG1 and shows no reactivity with rhesus monkey IgG2, IgG3 or IgG4. The ELISA does not recognize human IgG.Test samples are diluted and incubated in the microtiter wells for 45 minutes alongside monkey IgG1 calibrators. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. IgG1 molecules are thus sandwiched between the immobilization and detection antibodies. The wells are then washed to remove unbound HRP-conjugate 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 IgG1 is proportional to the optical density of the test sample and is derived from a calibration curve.

Components: Anti monkey IgG coated 96-well plate (12 strips of 8 wells)
Monkey IgG1 stock (lyophilized)
10X Immunoglobulin diluent, 25 mL
HRP Conjugate Reagent, 11 mL
20X Wash Solution, 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
Vortex mixer
Absorbent paper or paper towels
Graph paper (PC graphing software is optional)
Polypropylene or glass tubes
Plate reader with an optical density range of 0-4 at 450 nm.
Micro-Plate incubator/shaker mixing speed of ~150 rpm
Plate washer

Application Details

Plate: Pre-coated

Sample Preparation:

General Note: IgG1 is present in monkey serum or plasma at concentrations of ~10 mg/mL, In order to obtain values within range of the calibration curve, we suggest that samples initially be diluted 250,000 fold using the following procedure for each sample to be tested:
1. Dispense 999 µL and 498 µL of 1X diluent into separate tubes.
2. Pipette and mix 1.0 µL of the serum/plasma sample into the tube containing 999 µL of diluent. This provides a 1,000 fold diluted sample.
3. Mix 2.0 µL of the 1,000 fold diluted sample with 498 µL of diluent in the second tube. This provides a 250,000 fold dilution of the sample. 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 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 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 concentrations on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of IgG in ng/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration of IgG in the sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of the samples fall outside the calibration curve, samples should be diluted appropriately and re-tested.
   7. Do not extend or extrapolate the calibration curve beyond 6.25 and 100 ng/mL. Studies at have demonstrated the ELISA provides reliable and reproducible results only within this range.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: When the kit is received place the lyophilized IgG1 stock vial in a -20°C to -80°C freezer. Store all remaining kit components in a refrigerator at 4°C. The test kit will remain stable until the expiration date provided that the components are stored as described. The microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air.

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

Target Details for IgG1

Target: IgG1

Abstract: IgG1 Products

Synonyms: IgG1 ELISA Kit, Igh-4 ELISA Kit, VH7183 ELISA Kit, immunoglobulin heavy constant gamma 1 (G1m marker) ELISA Kit, Ighg1 ELISA Kit

Target Type: Antibody