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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

ANTIBODIES: ELISA

ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA)

Enzyme-Linked Immunosorbant Assay (ELISA)
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Procedure
1. Add 50 μl of antigen diluted in ELISA Coat Buffer to 96-well Polyvinyl Chloride (PVC) plates.

2. Incubate for 2 hr to overnight at 37°C.

3. Aspirate the antigen solution from the sample wells (see Hint #1).

4. Add 100 μl of PBS-Tween Buffer to each well, mix the solution, and aspirate the PBS-Tween Buffer from the well.

5. Add 200 μl of 3% BSA-ELISA Coat Buffer to each well and incubate the plate at room temperature for 2 hours.

6. Wash sample wells with 100 μl of PBS-Tween Buffer (as in Step #4).

7. Repeat the BSA-ELISA Coat Buffer wash (as in Step #5).

8. Add 50 μl of Undiluted Monoclonal Supernatant or Diluted Serum to each well. The appropriate dilution in PBS-Tween buffer is empirically determined (see Hint #2).

9. Incubate plate at room temperature for 4 hr to overnight at 4°C.

10. Wash sample wells three times with 100 μl of PBS-Tween Buffer (as in Step #4).

11. Add 50 μl of 1:2,500 (v/v) diluted (in PBS-Tween buffer) Urease-Conjugated Rabbit Anti-Mouse IgG Antibody to each well (see Hint #3).

12. Incubate the plate at room temperature for 2 hours.

13. Wash the sample wells three times with 100 μl of PBS-Tween buffer (as in Step #4).

14. Wash the sample wells three times with 100 μl 0.15 M NaCl.

15. Add 100 μl of substrate to each well and watch for color development (see Hint #4).

Solutions
0.15 M NaCl
Urease Substrate Solution   Store at 4°C
pH to 4.8 with 0.001 M HCl (color will change from purple to yellow)
ddH2O to 100 ml final volume
1.48 of ml 0.01 M NaOH
8 mg of Bromocresol Purple
80 ml of 0.25 M EDTA
100 mg of Urea
0.001 M HCl
0.25 M EDTA
0.01 M NaOH
PBS Buffer   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
PBS-Tween Buffer   in PBS Buffer
0.05% (v/v) Tween-20
3% BSA-ELISA Coat Buffer   in ELISA Coat Buffer
3% (w/v) Bovine Serum Albumin Fraction V
ELISA Coat Buffer   Bring the final volume to 1 liter (with ddH2O)
pH approximately 9.6
Store at 4°C
2.93 g of Sodium Bicarbonate (NaHCO3)
1.59 g of Sodium Carbonate (Na2CO3)
 
BioReagents and Chemicals
Hydrochloric Acid
Tween 20
Antibody urease-conjugated Rabbit anti-mouse IgG
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Sodium Carbonate
Sodium Bicarbonate
Bovine Serum Albumin
Urea
EDTA
Sodium Phosphate Monobasic
Sodium Hydroxide
Bromocresol Purple
Primary Antibody
 
Protocol Hints
1. Plates can be stored at -20°C after incubation.

2. Optimal dilution of primary antibody requires empirical testing. Typically, a titration of serum from 1:1000 (v/v) to 1:32,000 diluted in PBS-Tween Buffer will identify the optimal dilution. Monoclonal supernatant does not require dilution.

3. The optimal dilution of Secondary Antibodies may vary. Performing a titration is suggested. Other reactions involving enzyme-conjugated secondary antibodies and different substrates can be used in place of the urease-conjugated antibodies used here. A proper titration will need to be performed to determine optimal dilution with alternate detection systems.

4. Positive antibody-antigen reactions should progress from yellow to green to intense purple in color. Development time can vary from experiment to experiment. Subjectively determine color development. If background is suitably low, weak signals can be seen even 1 to 2 days later