Total Human IgG  ELISA Assay

Intended Use:

                 To quantitate total human Immunoglobulin G (IgG)

Principle of Procedure:

                 Solid phase capture sandwich ELISA assay using a microwell format.

Shelf Life:

                 The expiration date for the package and each component is stated on the label(s). Store all components at 2°- 8°
                 degrees C with the exception of the standard, which should be stored at -20^oC.

Patient and Standard Dilutions: 

Dilute each serum or plasma specimen to be tested initially 1:100 in phosphate buffered saline (PBS), e.g. 10ul of specimen into 990ul of PBS, then subdilute 1:100 with the IgG specimen diluent provided as before. Finally dilute 1:10 in the IgG specimen diluent provided. The final dilution factor will be 1:100,000.  Prepare serial two fold dilutions of the human IgG standard: Neat (N), 1:2, 1:4, 1:8 etc. with the specimen diluent provided. Use the specimen diluent alone as the blank control well.

Materials Supplied:

Anti-Human IgG coated microwell strips 12 x 8 with plastic frame

HRP conjugated goat anti-human IgG - 12mL

IgG standard (pre-diluted 1:100,000) - 1 ml

TMB/peroxide substrate color developer - 12mL

IgG specimen diluent - 60mL

Sulfuric acid termination reagent (0.5N) - 12mL

15 X Wash buffer concentrate – 60mL

Limitations of the Procedure:

No single assay should be used as the only basis for arriving at a diagnostic conclusion.
For research use only.

Dynamic Range:

0.156 mg/mL to 10.0 mg/mL.

Reproducibility:

C.V. 6% - 10% depending upon the region of the standard curve.

Assay Procedure:

                 *Allow each reagent to reach room temperature before use

 1.    Add 100uL of diluted specimen or standard to each microwell.

 2.    Incubate at room temperature for 45 minutes

 3.    Decant and wash each microwell four times with wash buffer (dilute buffer 1:15 with reagent grade water.)

 4.    Add 100uL of HRP conjugated goat anti-human IgG to each well.

 5.    Incubate at room temperature for 45 minutes.

 6.    Decant and wash as in step 3.

 7.    Add 100uL of TMB/peroxide substrate and incubate at room temperature for 15 minutes.

 8.    erminate the reaction with 100uL of 0.5N sulfuric acid.

 9.    Zero the microwell reader at 450nm using the specimen diluent zero control well.

 10.  Determine the optical density (O.D.) of the remaining wells.

 11.  Construct a standard curve using the O.D. values obtained for each of the standards.

 12.  Interpolate the unknowns from the standard curve.

Typical Standard Curve:

This assay has been demonstrated to recover 100% of the International Federation of Clinical Chemistry (IFCC) Standard