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.

•  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 (5 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 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.

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

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 ° C 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.

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 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 horse IgG.

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.

Test Validity:

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.

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.

Correct the diluted specimen values by the dilution factor used to obtain the final concentration of horse 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.

PREPARE REAGENT DILUTIONS

PIPETTE SPECIMENS AND STANDARDS

INCUBATE 30 MINUTES AT 37º + 1ºC

WASH PLATE

PIPETTE DETECTOR ANTIBODY

INCUBATE 30 MINUTES AT 37º + 1ºC

WASH PLATE

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