实验概要
The Sandwich ELISA measures the amount of antigen between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich.
Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows fine detection and quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible.
The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect).
主要试剂
Carbonate Coating Buffer
8.4 g NaHCO3
3.56 g Na2CO3
Add ddH2O up to 1.0L, pH to 9.5
Phosphate Buffered Saline (PBS):
80.0 g NaCl
14.4 g Na2HPO4
2.4 g KH2PO4
2.0 g KCl
Add ddH2O up to 10 L, pH to 7.2 with HCl
PBS/Tween:
0.5 ml of Tween-20 in 1 L PBS
Blocking Solution:
10% fetal bovine serum or 1% BSA in PBS. Filter before use to remove particulates.
ABTS Substrate Solution:
150 mg 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, Cat. No. A-1888)
Add to 500 ml of 0.1M citric acid in ddH2O
Adjust pH to 4.35 with NaOH
Aliquot 11 ml per vial and store at -200 C.
Avoid light exposure during preparation and storage.
ABTS Stop Solution:
Combine 50 ml dimethylformamide (DMF; Pierce, Cat. No. 20672) with 50 ml ddH2O
Add 20 g sodium dodecyl sulfate
TMB (tetramethylbenzidine) Substrate Reagent Set:
BioLegend Cat. No. 421101
TMB Stop Solution:
1M H3PO4 or 2N H2SO4
实验步骤
1. Coat the Plate:
1) Dilute unlabeled capture antibody to a final concentration of 0.5 – 8 µg/ml in Coating Buffer and transfer 100 µl to each well of a high affinity, protein-binding ELISA plate.
2) Seal plate to prevent evaporation. Incubate at 4°C overnight.
2. Block the Plate:
1) Bring the plate to room temperature, flick off the capture antibody solution, wash 3 times with PBS/Tween, and block non-specific binding sites by adding 200 µl of Blocking Solution to each well.
2) Seal plate and incubate at room temperature for ≥ 1 hour.
3) Wash 3 times with PBS/Tween. Firmly blot plate against clean paper towels.
3. Add Standards and Samples:
1) Dilute standards and samples to desired concentrations in Blocking Solution (perform dilutions in polypropylene tubes or plate) and add 100 µl per well to the ELISA plate.
2) Seal the plate and incubate at room temperature for 2-4 hours or at 4°C overnight.
3) Wash ≥ 3 times with PBS/Tween. Washes can be effectively accomplished by filling wells with either a squirt bottle, carboy, manifold dispenser, multi- channel pipettor or automatic plate washer. For increased stringency, after each wash, let the plate stand briefly, flick off the buffer, and blot plates on paper towels before refilling.
4. Add Detection Antibody:
1) Dilute the biotin-labeled detection antibody to 0.25 – 2 µg/ml in Blocking Solution. Add 100 µl of diluted antibody to each well.
2) Seal the plate and incubate at room temperature for 1 hour.
3) Wash ≥ 3 times with PBS/Tween.
5. Add Avidin-Horseradish Peroxidase (Av-HRP):
1) Dilute the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate to its pre-determined optimal concentration in Blocking Buffer (usually be- tween 1/500 – 1/2000). Add 100 µl per well.
2) Seal the plate and incubate at room temperature for 30 min.
3) Wash ≥ 5 times with PBS/Tween.
6. Add Substrate (ABTS for slower color development):
1) Thaw ABTS Substrate Solution within 20 min of use. Add 11 µl of 30% H2O2 per 11 ml of substrate and vortex. Immediately dispense 100 µl into each well and incubate at room temperature (4-60 min) for color development. To stop the color reaction, add 50 µl of ABTS Stop Solution.
2) Read the optical density (OD) for each well with a microplate reader set to 405 nm.
注意事项
Poor signal-to-noise ratio
1) Try Capture Antibody at 1 – 10 µg/ml (general 2µg/ml).
2) Try Detection Antibody at 0.25 – 2 µg/ml (generally 1 µg/ml).
3) Titrate against each other to obtain optimal dilutions.
Low Sensitivity
1) Try overnight incubations of standards and samples at 4°C.
Poor Signal
1) If using HRP, avoid sodium azide in wash buffers and diluents, as sodium azide inhibits HRP.
2) Verify that appropriate antibody pairs were used and the activity of samples and/or standards.
3) Check activity of enzyme and substrate by coating with Detection Antibody (1 µg/ml), adding biotinylated avidin and revealing with appropriate substrate. If enzyme/substrate is active, a strong signal should be observed.
Poor Standard Curve
1) Handling Instructions for standards are lot-specific. Refer to product information for proper handling.
2) Recombinant protein vials should be quick-spun for maximum recovery.
3) BioLegend suggests that cytokines be stored in a concentrated format (>100 ng/ml) and in the presence of a protein carrier.
High Background
1) Increase stringency of washing steps by soaking plates for ~1 minute during washes.
2) Determine optimum Capture and Detection Antibody dilutions.
3) Increase the dilution of Detection Antibody and/or increase number of washes after Av-HRP incubation.