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ELISA_Protocols

ELISA Protocols

ELISA Workflow-Enzyme-linked Immunosorbant Assays

Sandwich ELISA Method

1.Before the assay,both antibody preparations should be purified and one must be

labeled.

2.For most applications,a polyvinylchloride(PVC)microtiter plate is best;however,

consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

3.Bind the unlabeled antibody to the bottom of each well by adding approximately50

μL of antibody solution to each well(20μg/mL in PBS).PVC will bind approximately 100ng/well(300ng/cm2).The amount of antibody used will depend on the individual assay,but if maximal binding is required,use at least1μg/well.This is well above the capacity of the well,but the binding will occur more rapidly,and the binding solution can be saved and used again.

4.Incubate the plate overnight at4°C to allow complete binding.

5.Wash the wells twice with PBS.A500mL squirt bottle is convenient.The antibody

solution washes can be removed by flicking the plate over a suitable container.

6.The remaining sites for protein binding on the microtiter plate must be saturated by

incubating with blocking buffer.Fill the wells to the top with3%BSA/PBS with0.02% sodium azide.Incubate for2hrs.to overnight in a humid atmosphere at room

temperature.

Note:Sodium azide is an inhibitor or horseradish peroxidase.Do not include sodium azide in buffers or wash solutions if an HRP-labeled antibody will be used for

detection.

7.Wash wells twice with PBS.

8.Add50μL of the antigen solution to the wells(the antigen solution should be titrated).

All dilutions should be done in the blocking buffer(3%BSA/PBS).Incubate for at

least2hrs.at room temperature in a humid atmosphere.

9.Wash the plate four times with PBS.

10.Add the labeled second antibody.The amount to be added can be determined in

preliminary experiments.For accurate quantitation,the second antibody should be used in excess.All dilutions should be done in the blocking buffer.

11.Incubate for2hrs.or more at room temperature in a humid atmosphere.

12.Wash with several changes of PBS.

13.Add substrate as indicated by manufacturer.After suggested incubation time has

elapsed,optical densities at target wavelengths can be measured on an ELISA plate reader.

Note:Some enzyme substrates are considered hazardous,due to potential

carcinogenicity.Handle with care and refer to Material Safety Data Sheets for proper handling precautions.

Note:Some enzyme substrates are considered hazardous,due to potential

carcinogenicity.Handle with care and refer to Material Safety Data Sheets for proper handling precautions.

For quantitative results,compare signal of unknown samples against those of a standard curve.Standards must be run with each assay to ensure accuracy.

Competitive ELISA Method

1.For most applications,a polyvinylchloride(PVC)microtiter plate is best;however,

consult manufacturer guidelines to determine the most appropriate type of plate for

protein binding.

2.Add50μL of diluted primary antibody(capture)to each well.The appropriate dilution

should be determined using an checkerboard titration prior to testing samples.PVC

will bind approximately100ng/well(300ng/cm2).The amount of antibody used will

depend on the individual assay,but if maximal binding is required,use at least1

μg/well.this is well above the capacity of the well,but the binding will occur more

rapidly,and the binding solution can be saved and used again.Allow to incubate for4 hrs.at room temperature or4°C overnight.

Note:If a purified capture antibody is not available,the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody

according to the following procedure:

a.Bind the unlabeled secondary antibody to the bottom of each well by adding

approximately50μL of antibody solution to each well(20μg/mL in PBS).

b.Incubate the plate overnight at4°C to allow complete binding.

c.Add primary capture antibody(as above).

1.Wash the wells twice with PBS.A500mL squirt bottle is convenient.The antibody

solution washes can be removed by flicking the plate over a suitable container.

2.The remaining sites for protein binding on the microtiter plate must be saturated by

incubating with blocking buffer.Fill the wells to the top with3%BSA/PBS with0.02% sodium azide.Incubate for2hrs.to overnight in a humid atmosphere at room

temperature

3.Wash wells twice with PBS.

4.Add50μL of the standards or sample solution to the wells.All dilutions should be

done in the blocking buffer(3%BSA/PBS with0.05%Tween-20)

Note:Sodium azide is an inhibitor or horseradish peroxidase.Do not include

sodium azide in buffers or wash solutions,if an HRP-labeled conjugate will be used

for detection.

5.Add50μL of the antigen-conjugate solution to the wells(the antigen solution should

be titrated).All dilutions should be done in the blocking buffer(3%BSA/PBS with

0.05%Tween-20).Incubate for at least2hrs.at room temperature in a humid

atmosphere.

6.Wash the plate four times with PBS.

7.Add substrate as indicated by manufacturer.After suggested incubation time has

elapsed,optical densities at target wavelengths can be measured on an ELISA

reader.

Note:Competitive ELISAs yield an inverse curve,where higher values of antigen in the samples or standards yield a lower amount of color change.

Competitive ELISA Method