General ELISA Procedure for the detection of antibody (titration)

Notes:

1 This ELISA procedure is applicable for the qualitative detection of antibodies produced to purified antigens (proteins or peptides).  This is not designed to measure antibody concentration in serum or other fluids.

2 The procedure assumes that the primary antibodies were made in rabbits.  If other host animals were used then it will be necessary to use appropriate second antibodies.  

COATING: 

1. Make 1-10 ug/ml soln. of antigen (peptide or protein) in Coating buffer (do not store diluted antigen).

2. Coat High Binding ELISA Strip Plates (Cat # 80112) by dispensing 100 ul of antigen soln. per well. Cover the plates with Adhesive  Films. Incubate at RT for 2-6 h or overnight at 4oC with gentle orbital shaking.

3. Aspirate solution and wash plates 2X with Wash Buffer.

BLOCKING: 

1. Prepare Blocking Buffer by diluting from the stock.  Dispense 300 ul per well and Incubate plates for 2-6 h at RT or overnight at 4oC with gentle mixing.

2 Aspirate blocking buffer soln. and tap over paper towels to remove all solution. The plates can be used directly for the assay or kept at 4oC in sealed bag for future use.

ANTIBODY ASSAY:

1. Dilute preimmune and immune sera in Antibody/Conjugate Diluent (Serial dilutions of 1:100, 1:1K, 1:10K, 1:100K). Dispense 100 ul of each antibody and preimmune dilution to antigen-coated wells.

2. Cover the plates with Adhesive Films. Incubate at RT for 30 min with gentle orbital shaking.

3. Aspirate solution and wash plates 5X with Wash Buffer (200-300 ul wash buffer should be used for each washing; an improper washing will lead to high background).

4. Dilute Anti-Rabbit IgG-HRP Conjugate (1:5K-1:20K) in Antibody Conjugate diluent. Dispense 100 ul of conjugate per well (An improper dilution of conjugate may give too low color or high background).

5. Cover the plates with Adhesive Films. Incubate at RT for 30 min with gentle orbital shaking.

6. Aspirate plates and wash plates 5X with Wash Buffer. Tap over paper towels to remove all solution.

7. Dispense 100 ul/per well of ready-to-use TMB Substrate Soln.

8. Cover the plates with Adhesive Films. Incubate at RT for up to 15 min with gentle orbital shaking. A blue color will develop in positive wells.

9 Dispense 100 ul diluted soln. per well. The blue color will turn into yellow.

10. Read absorbance at 450 nm.  

NOTE: ALL reagents must be dispensed using fresh tips and Reagent reservoir. All of the required reagents for ELISA (except the specific antigen or antibody) can be conveniently purchased in a kit.   

ELISA Kits for the titration of various antibodies-Ordering information  

Cat#       Product Description  

80150       ELISA Kit for the detection of Mouse Antibodies in serum, 75 min

80155       ELISA Kit for the detection of Rat Antibodies in serum, 75 min

80160       ELISA Kit for the detection of Rabbit Antibodies in serum, 75 min

80165       ELISA Kit for the detection of Goat Antibodies in serum, 75 min

80166       ELISA Kit for the detection of Sheep Antibodies in serum, 75 min

80170       ELISA Kit for the detection of Human Antibodies in serum, 75 min

80171       ELISA Kit for the detection of G. Pig Antibodies in serum, 75 min

80175       ELISA Kit for the detection of Monkey Antibodies in serum, 75 min

80176       ELISA Kit for the detection of Chicken Antibodies in serum, 75 min

80180       ELISA Kit for the detection of Hamster Antibodies in serum, 75 min

80185       ELISA Kit for the detection of Bovine Antibodies in serum, 75 min

80186       ELISA Kit for the detection of Pig Antibodies in serum, 75 min

80187       ELISA Kit for the detection of Turkey Antibodies in serum, 75 min

The Kit contains all reagents including coating buffer, wash buffer, antibody/conjugate diluent.  TMB substrate, and stop solution, and Anti-rabbit IgG-HRP Conjugate; Sufficient reagents are provided to perform 1000-2000 tests or 10-20 ELISA plates). Please obtain the kit depending upon the host for primary antibodies.  The above kits can be used to determine the antibody titer to a given antigen. They are not suitable to determine the antibody (IgG, IgA or IgM) concentration in serum. We have other ELISA kits that are suitable to determine antibody levels in various animal sera (please see details at the web site or contact ADI).

ADI also supplies commonly used secondary antibody-enzyme conjugates, ELISA Reader and ELISA Washer.