Antibody Capture ELISA Teaching Kit
Cat# BB-ITK060 (4 Reactions)
Aim: To determine the antibody concentration by Antibody Captured ELISA. It involves the following experiments:
- Coating the wells with
- Blocking & Incubation with Primary and Secondary
Principle: Enzyme-linked immunosorbent assay, commonly known as ELISA is a popular format of analytical biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. In this technique, an enzyme conjugated with an antibody reacts with a colourless substrate to generate a coloured product. Such a substrate is called a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphatase, (AP) horseradish peroxidase (HRP) etc.
Bio Bharati Antibody Capture ELISA kit is HRP based. The antigen is first coated on the plate followed by incubation with primary and HRP-tagged secondary antibody. The HRP-tagged secondary antibody is detected using hydrogen peroxide as a substrate and 3,3’,5,5’- Tetramethyl Benzidine (TMB) as a chromogen. TMB acts as a hydrogen donor for the reduction of hydrogen peroxide to water by horseradish peroxidase. The TMB oxide is deposited wherever enzyme is present and appears as bluish coloured chromogen. The intensity of the colour is measured using a spectrophotometer at 450 nm. The developed colour is directly proportional to the amount of antibody present in sample.
Kit Description: In this kit, antigen, primary antibody and their respective diluent buffers have been provided. Students will dilute the antigen and the primary antibody with their respective diluents as mentioned in the procedure just prior to initiating the experiment.
Additionally, Horse Radish Peroxidase (HRP) conjugated Secondary antibody and Developing reagent (consists of three components) along with the detachable ELISA plate and Blocker have been also provided in this kit.
#BB-ITK060 is designed to perform 4 set of reactions.
Duration of experiment: Experiment is carried out over a span of two days, approximate time taken on each day is indicated below.
Day 1: 30 minutes; to coat the well of the ELISA plate with antigen provided in the kit.
Day 2: 6 hours; to carry out blocking, primary, secondary antibody incubation, development, and data analysis.
Quantity for 4 reactions
1X Diluent buffer for Antigen
10X Diluent buffer for Antibodies
Test Antibody (T1, T2, T3) in 4 aliquots, ready to use
1.0ml each Ab
HRP-conjugated Secondary Antibody
10X Wash Buffer
Microtiter plate (detachable)
Developing Solution A
Developing Solution B
Developing Solution C
Materials required but not provided:
Consumables: Micro Pipettes, Tips, Buffer
reservoir, Paper tissue, Ultra-pure water.
Instrument: ELISA reader
Bio Bharati Western Blotting Teaching kit is stable for 3 months from the date of receipt without showing any change in performance if stored properly.
Once received, store both the diluent buffers, all the components of developing reagents, stop solution and Secondary Ab at 4°C, Wash buffer, Blocker and ELISA plates at room temperature and other items at -20°C.
- Read the instructions carefully before starting the
- Developing solution C contains hydrogen peroxide and Stop Solution contains sulfuric acid, take utmost care while handling these chemicals.
- Prepare 1X Diluent buffer for Antibodies and 1X Wash buffer from respective 10X stock by adding 9 parts of ultra-pure water with 1 part of 10X stock.
Day: 1: Coating of wells with antigen
- Dilute 50µl of concentrated antigen with 2950µl of 1X diluent buffer for antigen
- Pipette 100µl of diluted antigen in each well.
- Pipette the antigen in all 24
- Tap or shake the plate well to ensure that there are no bubbles inside the wells and the antigen solution is evenly distributed within the well.
- Cover the plate with an aluminium foil and incubate the micro titer plate at 4°C for overnight.
Day 2: Blocking, Antibody Incubation and Detection
Remove the coating solution and wash the plate thrice with 200µl of 1X wash buffer. The washes are removed by flicking the plate over a sink; the remaining drops are removed by patting the plate on a paper towel.
Block the antigen coated plate by adding 100µl of blocking solution/well (make the blocking solution by adding 0.25gm of blocker to 5ml of wash buffer provided in the kit)
Cover the plate with an aluminium foil and incubate for at least 1hour at room temperature (This incubation step can be changed to overnight at 4°C if necessary)
Wash the plate thrice with 1X Wash buffer. The washes are removed by flicking the plate over a sink; the remaining drops are removed by patting the plate on a paper towel.
Incubation with Primary and Secondary Antibody:
- Add 100µl of different dilutions of primary antibody in all the wells as stated Table No 1:
Table No. 1:
Calculation for Dilution
Dilutions of Standard Antibody
Concentration of Antibody
2µl of Primary Ab + 998µl of 1 X Wash Buffer
500µl of (a) + 500µl of 1X Wash Buffer
1000ng/ µl (b)
500µl of (b) + 500µl of 1X Wash Buffer
500ng/ µl (c)
500µl of (c) + 500µl of 1X Wash Buffer
250ng/ µl (d)
500µl of (d) + 500µl of 1X Wash Buffer
125ng/ µl (e)
500µl of (e) + 500µl of 1X Wash Buffer
62.5ng/ µl (f)
500µl of (f) + 500µl of 1X Wash Buffer
31.25ng/ µl (g)
500µl of (a) + 500µl of 1X Wash Buffer
15.5ng/ µl (h)
- Wash the plate thrice with 1X Wash Buffer.
- Add 100µl of conjugated secondary antibody (just before use, dilute the antibody provided in the kit in Blocking buffer at 1:5000 dilution)
- Cover the plate with an adhesive plastic and incubate at RT for 1h.
- Wash the plate four times with1X PBS. The washes are removed by flicking the plate over a sink; the remaining drops are removed by patting the plate on a paper towel.
- Mix 4.5 ml of Solution A, 0.5 ml of Solution B, 1µl of Solution C provided in the kit to get the final developing
Note: Remember Solution B is light sensitive and Solution C contains Hydrogen peroxide (H2O2) which is very toxic. Always carry out the detection procedure at dark and wear gloves while using.
- Add 100 µl of developing reagent in each
- Wait until a bluish green colour appears in each well according to the concentration of the primary (Approximately 5–10 mins).
- Add 100µl of stop solution provided in the kit, the bluish-green colour changes to yellowish-orange colour.
Note: Stop solution contains sulfuric acid, therefore it is advisable to take utmost care while handling).
- Take the reading at 450nm in an ELISA
Figure 1: Standard curve showing different concentrations of primary antibody: Different dilutions of Primary Antibody used in the ELISA are – 1:500; 1:1000; 1:2000; 1:4000; 1:8000; 1:16000; 1:32000 and 1:64000.
Calculation of antibody concentration:
- Derivation of Straight Line Equation from MS Excel:
- Put the OD450 value on MS Excel sheet representing different concentrations of
- Calculate the average value of two readings for each of the Ab
- Subtract the average value of each amount from blank value.
- Select all the values; click on “Insert” and select “Scatter” option and you will get the “Scatter Graph”
- Select any point on graph; right click and select “Add Trend line” option
- Select “Display equation on chart” and “Display R square value on chart” situated at the end of the dialogue
- You will get the straight line equation on graph along with the R2 -value*
# R2 value should be greater than 0.9. If it is less than 0.9, there has been major pipetting error issues. Repeat the experiment.
- Determination of Ab concentration of test antibody from Standard Curve
- Put the subtracted final OD value in place of Y
- Deduce the value of X from equation which represents the dilution of the test antibody
- Derive the concentration of Test Abs with their corresponding dilution from the Table No
Reason for Problem
The steps are not followed step by step.
Go through the manual thoroughly and repeat the experiment.
Repeat the experiment. Wash plates thoroughly after every incubation.
Technical Assistance: At BioBharati, we always promise to deliver the best products for quality research to our customers. All the reagents are made at our In-house R&D facility with utmost care to give optimum result. We are always happy to serve you at your research help. For any assistance please write to us at [email protected]
Adapted from internet