Total Human IgM Assay

Intended Use:

                 To quantitate total human Immunoglobulin M (IgM)

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 except for 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 IgM specimen diluent provided for a final dilution of 1:10,000. Prepare serial two fold dilutions of the human IgM 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 IgM coated microwell strips 12 x 8 with plastic frame

HRP conjugated goat anti-human IgM - 12mL

IgM standard (pre-diluted 1:10,000) - 1 ml

TMB/peroxide substrate color developer - 12mL

               IgM specimen diluent (Specimen Diluent Green II) - 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:

0.03 mg/mL to 2.0 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.    ncubate at room temperature for 60 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 IgM to each well

 5.    Incubate at room temperature for 60 minutes

 6.    Decant and wash as in step 3

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

 8.    Terminate 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