SandwichELISAProtocol

實驗概要

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).

主要試劑

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

實驗步驟

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.

注意事項

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.