The IMMUNO-TEK Human IgM EIA Kit is for Research Purposes Only.

• Microplate, (1x96 well): Strips coated with purified goat anti-human IgM

• Detector Antibody (12 ml): Contains conjugated goat anti-human IgM peroxidase

• Human IgM Standard (5 ml): Contains human IgM and assay diluent

• Assay Diluent (100 ml): Contains PBS, Triton X-100^® and 2-chloroacetamide

• Plate Wash Buffer (125 ml): Contains PBS, Tween 20^® and 2-chloroacetamide

• Substrate (12 ml): Contains Tetramethyl Benzidine (TMB)

• Stop Solution (12 ml): Proprietary formulation

• Microtiter Plate Sealers (1 pk): 10 sealers per pack

• Plastic Bag (1 bag): For storage of unused microtiter plate strips

® Triton X-100 is a registered trademark of Rohm and Haas. Tween 20 is a registered trademark of Imperial Chemical Industries.

Storage:

Store all kit reagents at 2-8 ° C. Do not freeze.

Materials Required but not Supplied:

• Disposable gloves
• Test tubes and racks for preparing specimen and IgM standard dilutions
• Validated adjustable micropipettes, single and multi-channel
• Distilled or deionized water
• Validated incubator capable of maintaining 37 + 1° C
• Graduated cylinders and assorted beakers
• Validated microtiter plate reader
• Automatic microtiter plate washer or manual vacuum aspiration equipment
• Timer

FOR RESEARCH USE ONLY. Not For in vitro Diagnostic Use.

• Prior to Performing the assay, carefully read all instructions

• Use universal precautions when handling kit components and test specimens*

• To avoid cross-contamination, use separate pipette tips for each specimen.

• When testing potentially infectious specimens, adhere to all applicable local, state and federal regulations regarding the disposal of biohazardous materials.

• Stop Solution contains hydrochloric acid which may cause severe burns. In case of contact with eyes or skin, rinse immediately with water and seek medical assistance. Wear protective clothing and eyewear.

*MMWR, June 24, 1988, Vol. 37, No. 24, pp. 377-382, 387-388

Dilute 10X Plate Wash Buffer 1:10 in distilled or deionized water prior to use. Mix thoroughly. Prepared 1X Plate Wash Buffer can be stored at 2-8° C for up to one week. Additional 10X Plate Wash Buffer (ZMC Catalog #: 0801060) may be ordered if needed.

Human IgM Standard Curve

Label 6 test tubes as shown below. The Human IgM Standard is provided at 125 ng/ml. This should be diluted in Assay Diluent as follows to prepare a standard curve.

Hybridoma supernatants from stationary cell cultures will typically contain between 1 µg/ml and 30 µg/ml of monoclonal antibody. Therefore, we recommend preparing a 1:250 dilution of cell culture supernatants in Assay Diluent for initial testing.

When using cell culture supernatants from bioreactors, a further dilution may be necessary since many bioreactors are capable of producing much higher concentrations of monoclonal antibodies than standard stationary cell cultures. Refer to the technical literature provided with the bioreactor to determine a dilution that will yield a monoclonal antibody concentration between 125 ng/ml and 7.8 ng/ml.

After initial testing, it may be necessary to adjust the concentration of the antibody solution to be tested in order to obtain a concentration between 125 ng/ml and 7.8 ng/ml for accurate quantification.

Ascites

Human serum will typically contain between 1 mg/ml and 10 mg/ml of monoclonal antibody. Because of this, we recommend preparing a 1:250,000 dilution of ascites in Assay Diluent for initial testing.

After initial testing, it may be necessary to adjust the concentration of the antibody solution to be tested in order to obtain a concentration between 125 ng/ml and 7.8 ng/ml for accurate quantification.

Step 2: Designate one well on the plate and leave empty. This well will serve as a substrate blank.

Step 3: Pipette 200 µl of standards #1-6 into duplicate wells.

Step 4: Pipette 200 µl of each specimen into duplicate wells.

Step 5: Cover the microplate with a plate sealer and incubate the plate for 30 minutes at 37° C.

Step 6: Aspirate the contents of each well and wash the wells 4 times with 1X Plate Wash Buffer. To wash, fill the wells with 300 µl of 1X plate wash buffer and aspirate. Perform 4 fill/aspirate cycles. After the final wash cycle, thoroughly blot the plate by carefully striking the plate on a pad of absorbent paper towels. Continue until no visible droplets of Plate Wash Buffer are observed.

Step 7: Pipette 100 µl of Detector Antibody into each standard and specimen well.
Do not add Detector Antibody to the substrate blank well.

Step 8: Cover the plate with a plate sealer and incubate for 30 minutes at 37° C.

Step 9: Wash the plate 4 times with Plate Wash Buffer as described in Step 6.

Step 10:  Pipette 100 µl of Substrate into each well including the substrate blank well.

Step 11: Incubate the plate for 30 minutes at room temperature. A blue color will develop in wells containing human IgM.

Step 12: Pipette 100 µl of Stop Solution into each well. A color change from blue to yellow will occur.        

Step 13: Within 15 minutes, read the optical density of each well at 450 nm using a microtiter plate reader.

Using linear graph paper or a computer program, plot the optical densities of each standard on the Y-axis versus the corresponding concentration of the standards on the X-axis.

The concentration of human IgM in each diluted specimen may then be determined manually using a ruler to extrapolate, by linear regression using a computer program or pocket calculator with a linear regression function, or by point to point calculation again using a computer or calculator.

Correct the diluted specimen values by the dilution factor used to obtain the final concentration of human IgM in the original specimen.

Typical Standard Curve

Below is an example of a typical standard curve. Variations will occur laboratory to laboratory due to pipetting, incubator temperatures, plate readers, etc.

PREPARE REAGENT DILUTIONS

PIPETTE SPECIMENS AND STANDARDS

INCUBATE 30 MINUTES AT 37^°+ 1^°C

WASH PLATE

PIPETTE DETECTOR ANTIBODY

INCUBATE 30 MINUTES AT 37^°+ 1^°C

WASH PLATE

PIPETTE SUBSTRATE SOLUTION

INCUBATE 30 MINUTES AT ROOM TEMPERATURE

ADD STOP SOLUTION AND READ AT 450 NM