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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

IMMUNO-TEK
Horse IgG ELISA

FOR RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC USE.

ZMC Catalog # : 0801221
   
INTENDED
USE
 

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.
   
REAGENTS   Materials Supplied:

  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 2-8C. Do not freeze.

   
PRECAUTIONS  

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 2-8 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.

Tube Number

Concentration of Horse IgG

Volume of Horse IgG Standard

Volume of Assay Diluent

1

125 ng/ml

1000 m l

0 m l

2

62.5 ng/ml

500 l of #1

500 l

3

31.25 ng/ml

500 l of #2

500 l

4

15.6 ng/ml

500 l of #3

500 l

5

7.8 ng/ml

500 l of #4

500 l

6

0 ng/ml

0 l

500 l

   
SPECIMEN
DILUTIONS
  Horse Serum

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.

   
TEST
PROCEDURE
  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 2-8C.

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 l of standards 1-6 into duplicate wells.

Step 4: Pipette 200 l 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 l 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 l 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 l 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 l 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.

   
CALCULATIONS
AND
INTERPRETATION
OF RESULTS
  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 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.

Horse IgG Standard Concentration

Optical Density
at 450 nm

125 ng/ml

1.924

62.5 ng/ml

1.239

31.25 ng/ml

0.786

15.6 ng/ml

0.457

7.8 ng/ml

0.265

0 ng/ml

0.065

Substrate Blank

0.055

 

Standard Curve

   
PROCEDURAL
FLOW CHART
 

PREPARE REAGENT DILUTIONS

PIPETTE SPECIMENS AND STANDARDS

INCUBATE 30 MINUTES AT 37 + 1C

WASH PLATE

PIPETTE DETECTOR ANTIBODY

INCUBATE 30 MINUTES AT 37 + 1C

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

   

Rev.: 04/05

 

For more information :: Bioxys and Gentaur BVBA :: Av. de l'Arme 68 B4 :: BE-1040 BELGIUM

Email: info@gentaur.com
 

International 

+32 (0) 16 58 90 45

+32 (0) 16 50 90 45

France

01 43 25 01 50

01 43 25 01 60

Italy

02 36 00 65 93

02 36 00 65 94

Germany

0241 6085 13140

0241 6085 33033

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Last modified: feb-07