ZMC Catalog # :

0801182

INTENDED
USE

The Immuno-Tek Human IgG EIA Kit is for Research Purposes Only.

PRINCIPLE OF
THE TEST

Microwells coated with polyclonal antibodies to human IgG form the capture phase of the assay. These antibodies bind uniformly to all subclasses of human IgG. Captured human IgG then reacts with detector antibody which is a polyclonal anti-human IgG conjugated with horseradish peroxidase. This reagent also reacts uniformly to all subclasses of human IgG. Enzyme activity in the wells are then quantified using tetramethyl benzidine as a substrate.

REAGENTS

Materials Supplied

• Microplate, (1x96 well): Strips coated with purified Goat Anti-Human IgG

• Detector Antibody (12 ml): Contains conjugated Goat Anti-Human IgG peroxidase

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

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

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

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

• Stop Solution (12 ml):  Contains 2M Sulfuric Acid

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

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

(R) 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 ｰ C. Do not freeze.

Materials Required but not Supplied

• Test tubes and racks for preparing specimen and IgG standard dilutions

• Adjustable micropipets, single and multi-channel

• Distilled or deionized water

• Incubator capable of maintaining 37 + 1ｰ C

• Graduated cylinders and assorted beakers

• Automatic microtiter plate washer or manual vacuum aspiration equipment

PRECAUTIONS

PREPARATION
OF REAGENTS

Plate Wash Buffer

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ｰ C for up to one week. Additional 10X Plate Wash Buffer (ZMC Catalog #: 0801060) may be ordered if needed.

Human IgG Standard Curve

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

SPECIMEN
DILUTIONS

Hybridoma Supernatants

Hybridoma supernatants from stationary cell cultures will typically contain between 1 μg/ml and 30 μg/ml of monoclonal antibody. Because of this, 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.

TEST
PROCEDURE

To avoid cross contamination, use separate pipet tips for each specimen.

Step 1: Label each strip on its end tab to ensure identity should the strips become detached from the plate frame during the assay.

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

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

Step 4: Pipet 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ｰ 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 exists.

Step 7: Pipet 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ｰ C.

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

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

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

Step 12: Pipet 100 μl of Stop Solution (2M Sulfuric Acid) 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.

TEST
VALIDITY

For the test to be valid, the mean optical density of the 0 pg/ml standard and the substrate blank must be below 0.200.

CALCULATIONS
AND
INTERPRETATION
OF RESULTS

The concentration of human IgG 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 IgG 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.

Caution: This kit is for Research Use Only. It is not to be used as an in vitro Diagnostic

PROCEDURAL
FLOW CHART

PREPARE REAGENT DILUTIONS

PIPET SPECIMENS AND STANDARDS

INCUBATE 30 MINUTES AT 37⁰+ 1⁰C

WASH PLATE

PIPET DETECTOR ANTIBODY

INCUBATE 30 MINUTES AT 37⁰+ 1⁰C

WASH PLATE

PIPET SUBSTRATE SOLUTION

INCUBATE 30 MINUTES AT ROOM TEMPERATURE

ADD STOP SOLUTION AND READ AT 450 NM