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Mouse IgG ELISA Kit   (DEIA8704)  


Datasheet

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Size
96T
Sample
serum, plasma, hybridoma cell supernatants, ascites, other biological fluids
Species Reactivity
Mouse
Intended Use
This Mouse Immunoglobulin G (IgG) antigen assay is intended for the quantitative determination of total mouse IgG antigen in serum, plasma, hybridoma cell supernatants, ascites or other biological fluids. This assay does not distinguish IgG subclasses. For research use only.
Contents of Kit
1. 96-well antibody coated microtiter strip plate (removable wells 8x12) containing anti-mouse IgG antibody, blocked and dried.
2. 10X Wash buffer: 1 bottle of 50 mL
3. 5X Diluent: 1 bottle of 50 mL
4. Mouse IgG standard: 1 vial lyophilized standard
5. Anti-mouse horseradish peroxidase antibody: 1 vial concentrated HRP labeled antibody
6. TMB substrate solution: 1 bottle of 10 mL
Storage
Store all kit components at 4°C upon arrival. Return any unused microplate strips to the plate pouch with desiccant. Reconstituted standard may be stored at -80°C for later use. Do not freeze-thaw the standard more than once. Store all other unused kit components at 4°C. This kit should not be used beyond the expiration date.
Precision
Intra-assay Precision: Inter-assay Precision:
Sensitivity
Sensitivity: The minimum detectable dose (MDD) was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates (range OD450: 0.05-0.059) and calculating the corresponding concentration. The MDD was 0.119 ng/ml.
General Description
IgG is the most abundant immunoglobulin in serum and is predominately involved in the secondary immune response. The IgG subclasses are designated 1, 2, 3 and 4 based on their relative prevalence in human serum.
Standard Curve
Reference Values
The concentration of IgG in normal mouse serum ranges from 5 to 12 mg/mL.
Principles of Testing
Mouse IgG will bind to the affinity purified capture antibody coated on the microtiter plate. After appropriate washing steps, horseradish peroxidase labeled polyclonal anti-mouse IgG antibody binds to the captured protein. Excess antibody is washed away and TMB substrate is used for color development at 450nm. A standard calibration curve is prepared along with the samples to be measured using dilutions of mouse IgG. Color development is directly proportional to the concentration of total IgG in the samples.
Reagents And Materials Provided
1. 96-well antibody coated microtiter strip plate (removable wells 8x12) containing anti-mouse IgG antibody, blocked and dried.
2. 10X Wash buffer: 1 bottle of 50 mL
3. 5X Diluent: 1 bottle of 50 mL
4. Mouse IgG standard: 1 vial lyophilized standard
5. Anti-mouse horseradish peroxidase antibody: 1 vial concentrated HRP labeled antibody
6. TMB substrate solution: 1 bottle of 10 mL
Materials Required But Not Supplied
1. Microtiter plate shaker capable of 300 rpm uniform horizontally circular movement
2. Manifold dispenser/aspirator or automated microplate washer
3. Microplate reader capable of measuring absorbance at 450 nm
4. Pipettes and Pipette tips
5. Deionized or distilled water
6. Polypropylene tubes for dilution of standard
7. Paper towels or laboratory wipes
8. 1N H2SO4 or 1N HCl
Assay Procedure
Perform assay at room temperature. Vigorously shake plate (300rpm) at each step of the assay.

Preparation of Standard
Reconstitute standard by adding 1ml of diluent directly to the vial and agitate gently to completely dissolve contents. This will result in a 1,000ng/ml standard solution.
Dilution table for preparation of mouse IgG standard:

NOTE: DILUTIONS FOR THE STANDARD CURVE AND ZERO STANDARD MUST BE MADE AND APPLIED TO THE PLATE IMMEDIATELY.

Standard and Unknown Addition
Remove microtiter plate from bag and add 100μL IgG standards (in duplicate) and unknowns to wells. Carefully record position of standards and unknowns. Shake plate at 300rpm for 30 minutes. Wash wells three times with 300μL wash buffer. Remove excess wash by gently tapping plate on paper towel or kimwipe.
NOTE: The assay measures IgG antigen in the 0.5-500 ng/ml range. If the unknown is thought to have high IgG levels, dilutions may be made in diluent. A 1:1,000,000 dilution for normal mouse plasma is suggested for best results.

Antibody Addition
Briefly centrifuge vial before opening. Dilute 1μL of conjugated antibody in 10 mL of diluent and add 100μL to all wells. Shake plate at 300rpm for 30 minutes. Wash wells three times with 300μL wash buffer. Remove excess wash by gently tapping plate on paper towel or kimwipe.

Substrate Incubation
Add 100μL TMB substrate to all wells and shake plate for 2-10 minutes. Substrate will change from colorless to different strengths of blue. Quench reaction by adding 50μL of 1N H2SO4 or HCl stop solution to all wells when samples are visually in the same range as the standards. Add stop solution to wells in the same order as substrate upon which color will change from blue to yellow. Mix thoroughly by gently shaking the plate.

Measurement
Set the absorbance at 450nm in a microtiter plate spectrophotometer. Measure the absorbance in all wells at 450nm. Subtract zero point from all standards and unknowns to determine corrected absorbance (A450).
Precautions
1. FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE.
2. Do not mix any reagents or components of this kit with any reagents or components of any other kit. This kit is designed to work properly as provided.
3. Always pour peroxidase substrate out of the bottle into a clean test tube. Do not pipette out of the bottle as contamination could result.
4. Keep plate covered except when adding reagents, washing, or reading.
5. DO NOT pipette reagents by mouth and avoid contact of reagents and specimens with skin.
6. DO NOT smoke, drink, or eat in areas where specimens or reagents are being handled.

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