Human Allergen Specific IgE ELISA Assay

 

Intended Use:

                  To qualitate and quantitate allergen specific human Immunoglobulin E

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. Do not freeze all or in part.

Materials Supplied:

Allergen coated microwell strips 8 x 12 with plastic frame

HRP conjugated mouse monoclonal anti-human IgE - 12mL

TMB/peroxide substrate color developer - 12mL

Sulfuric acid termination reagent (1.0N) - 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.

Dynamic Range:

0.35 - 0.70 PRU/mL, depending on the type of allergen used

Reproducibility:

                  C.V. 6% - 10%

Assay Procedure:

*Caution: All human fluids should be treated as infectious agents that could carry HIV.

For each specimen, place one strip of allergen coated microwells in the plastic frame and label the corresponding schematic with the patient’s names or ID numbers.

1.           Bring all reagents to 25°C.

2.           Wash and decant microwell strips 4 times with reagent grade water. After the last decanted wash, firmly tamp upside down on a clean paper towel to remove residual water.

3.           Add 100uL of human serum or plasma to each microwell respectively.

4.           Incubate for 2 hours > 25°C but not greater than 30°C.

5.           Decant and wash 5 times with previously diluted wash buffer (see "preparation of reagents.")  Remove any residual wash buffer by tamping plates upside down on clean paper towel.   

6.           Add 100uL of HRP Conjugated Anti–Human IgE to each well respectively.

7.           Incubate for 2 hours  > 25°C but not greater than 30°C.

8.           Decant and wash as in step 5.

9.           Add 100uL of TMB/Peroxide Substrate (ColorBurst^tm Blue) to each well respectively.

10.       Incubate for 30 minutes at > 25°C but not greater than 30°C.

11.       Add 100uL of termination reagent (1.0N Sulfuric Acid) to each well respectively.

12.       Read the optical density (O.D.) at 450nm using a standard microwell plate reader zeroed on the negative control well.

        flipSCREEN QAT Results:

0.000 - 0.250 = Negative    

0.251 - 0.350 = Class I

                  0.351 - 0.450 = Class II

                  0.451 - 0.550 = Class III

                     >       0.550 = Class IV

Preparation of Reagents:

Wash Buffer:

Prior to performing the assay, dilute 1 part of 15X Wash Buffer Concentrate in 14 parts of reagent grade water. Mix thoroughly and dispense into a squeeze wash bottle as needed.