ELISA PROTOCOL
Enzyme Linked Immunosorbent Assay (ELISA) is the most widely used immunological assay technique, which combines the specificity of antigen-antibody reaction with the high efficiency of enzyme catalysis, and determines the result by the color reaction of the enzyme on the substrate. Generally, the absorbance (OD value) of an enzyme marker is measured to reflect the antigen or antibody content, and the sensitivity can reach the level of nanograms (ng) or even picograms (pg) per milliliter. Due to the high catalytic efficiency of the enzyme, the results of the immune response are indirectly amplified, making the assay highly sensitive.
Currently, the commonly used ELISA methods are direct, indirect, double antibody sandwich and competitive ELISA, and the procedure of double antibody sandwich ELISA is described here.
ELISA Experimental Steps:
I. Equipment/Materials
l Coating Solution
l ELISA Wash Solution:
ELISA Wash Solution: 19.38 mM Na2HPO4·12H2O 69.4 g, 1.8 mM KH2PO4 2.4 g, 121.24 mM NaCl 70.9 g, 0.2% Tween 20 20ml. PH 7.4 to 10L.
l Sequestering solution and dilution: PBST+5% skimmed milk powder
l Primary antibody
l Secondary antibody
l TMB color development solution:
Anhydrous sodium acetate 27.2 g, citric acid 3.2 g, 30% hydrogen peroxide 0.6 ml, and make up to 1 L.
l 1.7 TMB Color Developing Solution:
Anhydrous sodium acetate 27.2 g, citric acid 3.2 g, 30% hydrogen peroxide 0.6 ml, and make up to 1 L.
II. Operational Process
Allow each reagent to equilibrate to room temperature before starting the ELISA (do not dissolve reagents directly at 37°C). When diluting reagents or samples, be sure to mix well and avoid foaming.
1. Coated Antibody: Dilute the coated antibody to a certain dilution with carbonate buffer solution (CBS) or phosphate buffer solution (PBS) according to the experimental needs, 100 μL/well coated, overnight at 37℃, 2 h or 4℃.
2. Wash the plate: Discard the liquid in the wells, shake dry, 10 mM PBST (10 mM PBS+0.05% Tween-20) wash the plate twice, each time submerged for 1-2 min, 350 μL/well, shake dry (you can also tap the liquid in the wells to dry them).
3. Sealing: 10 mM PBST (10 mM PBS+0.05% Tween-20) containing 1% BSA or 5% skim milk was used as sealing solution, 350-400 μL/well, 37℃ for 2 h.
4. The plate was washed as in step 2. (The plate was washed twice, each time soaking for 1-2 min, 350 μL/well.
(Note: Commercial kits usually have the encapsulated antibody pre-coated on the enzyme-labeled plate and do not need to perform steps 1-4, please be sure to check before using).
5. Sample addition: Prepare zero wells, standard wells, and sample wells to be tested. Add 100 μL of Specimen Diluent to the blank wells and 100 μL of Standard or specimen to be tested to the remaining wells. (Take care to avoid air bubbles, add the sample to the bottom of the wells of the enzyme plate, trying not to touch the walls of the wells, and complete the sample on a plate within 10 minutes). The plate should be capped or laminated and allowed to react at 37°C for 60-120 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment.
6. Wash the plate: discard the liquid in the wells, shake dry, 10 mM PBST (10 mM PBS + 0.05% Tween-20) wash the plate 4 times, each time submerged for 1-2 min, 350-400 μL/well, shake dry (you can also tap the liquid in the wells to dry them).
7. Add detection antibody: according to the experimental needs, dilute the detection antibody to a certain dilution with PBST, 100 μL/well, 37 ℃, 1 h.
8. Wash the plate: as in step 6.
9. Add secondary antibody: according to experimental needs, dilute secondary antibody with 10 mM PBST to a specific dilution, 100 μL/well, 37℃, 1 hour.
10. Wash plate: same as step 6.
11. Enzyme Marker Reading: Take 630 nm as the calibration wavelength, use the enzyme marker to measure the absorbance (OD value) of each well sequentially at 450 nm, and read the result within 5 minutes after adding the termination solution.
12. Evaluate the results:
A. The OD value of each standard and sample is subtracted from the OD value of the zero well, and the average value is taken when compound wells are set;
B. Take the concentration of the standard as the horizontal coordinate and the OD value as the vertical coordinate, and use professional four-parameter fitting (4-PL) software such as Origin, ELISACalc, etc., and derive the corresponding fitted concentration from the standard curve according to the OD value of the sample, and multiply it by the dilution factor, which is the concentration of the sample to be measured.