Total Human IgE ELISA Assay 

Intended Use:

                  To quantitate total human Immunoglobulin E (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° C. 
                  with the exception of the standard, which should be stored at -20^oC.

Patient and Standards:

 The Specimen diluent alone is used for a blank.

Materials Supplied:*

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

HRP conjugated mouse monoclonal anti-human IgG - 12mL

IgG standards (Low, Medium, High) - 200 uL each

TMB/peroxide substrate color developer - 12mL

               IgG Specimen diluent - 60mL

Sulfuric acid termination reagent (0.5N) - 12mL

 15 X Wash buffer concentrate - 60mL

 * Quantities are representative of single kits - for double kits, multiply amounts by two.

Limitations of the Procedure:

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

Dynamic Range:

                  0.0 IU/mL to 400 IU/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 10uL of specimen or 10ul of standard to each microwell.  Use 100uL of specimen diluent alone for negative or blank well.

2.      Add 90ul of Specimen diluent to each specimen or standard well.

 3.      Incubate at room temperature for 2 hours.

 4.      Decant and wash each microwell four times with wash buffer (diluted buffer 1:15 with reagent grade water). Firmly grip and pound the microwell strips upside down on dry folded paper towels to remove residual wash buffer after the last decanted wash.

 5.      Add 100uL of HRP conjugated mouse monoclonal  anti-human IgG to each well.

 6.      Incubate at room temperature for 2 hours.

 7.      Decant and wash as in "Step 3."

 8.      Add 100uL of TMB/peroxide substrate and incubate at room temperature for 30 minutes.

 9.      Terminate the reaction with 100uL of 0.5N sulfuric acid.

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

 11.     Determine the optical density (O.D.@450nm) of the remaining wells.

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

 13.     Interpolate the unknowns from the standard curve.

Typical Standard Curve: