Horse IgG ELISA
The IMMUNO-TEK Horse IgG ELISA Kit is a rapid, easy to use enzyme linked immunosorbent assay (ELISA) designed for the measurement of horse IgG in horse serum, plasma or other biological fluids. The assay contains ready-to-use reagents and takes less than two hours to perform. The microplate and detector antibody in the kit have been specifically balanced to react uniformly with all subclasses of horse IgG.
The IMMUNO-TEK Horse IgG ELISA Kit is for Research Purposes Only.
PRINCIPLE OF THE TEST
Microplate wells coated with polyclonal antibodies to horse IgG form the capture phase of the assay. These antibodies bind uniformly to all subclasses of horse IgG. Captured horse IgG then reacts with detector antibody, which is a polyclonal anti-horse IgG, conjugated with horseradish peroxidase. This reagent also reacts uniformly with all subclasses of horse IgG.
Enzyme activity in the wells is then quantified using tetramethyl benzidine as a substrate.
- Microplate, (1x96 well): Strips coated with purified goat anti-horse IgG
- Detector Antibody (12 ml): Contains conjugated goat anti-horse IgG peroxidase
- Horse IgG Standard (7 ml): Contains horse IgG 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
- Microplate Sealers (1 pk): 10 sealers per pack
- Plastic Bag (1 bag): For storage of unused microplate strips
® Triton X-100 is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc.
Tween 20 is a registered trademark of Imperial Chemical Industries.
Materials required but not supplied:
- Disposable gloves
- Test tubes and racks for preparing specimen and IgG standard dilutions
- Validated adjustable micropipettes, single and multi-channel
- Distilled or deionized water
- Validated incubator capable of maintaining 37C+ 1C
- Graduated cylinders and assorted beakers
- Validated microtiter plate reader
- Automatic microtiter plate washer or manual vacuum aspiration equipment Timer
Store all kit reagents at 2-8°C. Do not freeze.
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
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 2-8C for up to one week. Additional 10X Plate Wash Buffer (ZMC Catalog #: 0801060) may be ordered if needed.
Horse IgG Standard Curve
Label 6 test tubes as shown below. The Horse IgG Standard is provided at 125 ng/ml. This should be diluted in Assay Diluent as follows to prepare a standard curve.
Horse serum will normally contain 10-15 mg/ml of IgG antibody. Because of this, we recommend preparing a 1:250,000 dilution of horse serum 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.
Allow all reagents to reach room temperature before use. Label test tubes to be used for the preparation of standards and specimens. If the entire 96 well plate will not be used, remove surplus strips from the plate frame and place into the resealable Plastic Bag with desiccant. Seal bag and store at 2-8°C.
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: Pipette 200 ul of standards 1-6 into duplicate wells.
Step 4: Pipette 200 ul 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 ul 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 ul 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 ul 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 horse IgG.
Step 12: Pipette 100 ul of Stop Solution into each well. A color change from blue to yellow will occur.
Below is an example of a typical standard curve. Variations will occur from laboratory to laboratory due to pipetting, incubator temperatures, plate readers, etc.
CALCULATION OF RESULTS
For the test to be valid, the mean optical density of the 0 ng/ml standard and the substrate blank must be below 0.200.
1. 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.
2. The concentration of horse 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.
3. Correct the diluted specimen values by the dilution factor used to obtain the final concentration of horse IgG in the original specimen.
PROCEDURAL FLOW CHART
PREPARE REAGENT DILUTIONS
PIPETTE SPECIMENS AND STANDARDS
INCUBATE 30 MINUTES AT 37º + 1ºC
PIPETTE DETECTOR ANTIBODY
INCUBATE 30 MINUTES AT 37º + 1ºC
PIPETTE SUBSTRATE SOLUTION
INCUBATE 30 MINUTES AT ROOM TEMPERATURE
ADD STOP SOLUTION AND READ AT 450 NM
For Research Use Only NOT for in vitro Diagnostic Use