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This Package
Insert is provided for product evaluation purposes only and is
not intended to be used in place of the Package Insert shipped with the
product.
IMMUNO-TEK
Quantitative
Human IgG Antigen ELISA
FOR RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC USE.
ZMC Catalog # : |
0801182 |
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INTENDED
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The Immuno-Tek Human IgG EIA Kit is a rapid, easy to use enzyme
linked immunosorbant assay (EIA) designed for the measurement of
human IgG in cell culture supernatants, ascites or other
biological fluid. The kit is especially useful in monitoring the
production and purification of mouse monoclonal antibodies. The
kit contains premixed reagents and takes less than two hours to
obtain results. The microplate and detector antibody in the kit
have been specifically balanced to react uniformly with all
subclasses of human IgG. The Immuno-Tek Human IgG EIA Kit is for Research Purposes Only. |
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PRINCIPLE OF |
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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. |
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REAGENTS |
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Materials Supplied
(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
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PRECAUTIONS |
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FOR RESEARCH USE ONLY. Not For in vitro Diagnostic Use. | |||||||||||||||||||||||||||||
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PREPARATION |
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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.
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SPECIMEN |
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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. |
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TEST
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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. 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. |
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TEST |
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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. |
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CALCULATIONS |
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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 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 |
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PROCEDURAL |
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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 |
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Rev. 09/00
Send mail to [email protected] with questions or comments about this web site.Copyright � 2009 GENTAUR Last modified: 03/02/10 |