125:315 Biomedical Measurements and Analysis Lab

Quantitation of Proteins by ELISA

Faculty Advisor: Charles Roth

Background Reading:

P. Tijssen, Practice and Theory of Immunoassays, Chapter 2, Elsevier, Amsterdam, 1985.

Overview

In this exercise, you will be introduced to the principles of the enzyme linked immunosorbent assay (ELISA). Specifically, you will use the technique to measure levels of human albumin in cell culture supernatants. Human albumin ELISA assay is based on competitive binding of human albumin from samples and albumin coated on the microwell plate to the enzyme labeled monoclonal anti-human albumin antibody. Due to this competition, higher concentrations of albumin in the samples result in decreased binding of enzyme labeled antibody to the microwell plate. After a washing step, chromogenic substrate is added and colors developed. The enzymatic reaction (color) is inversely proportional to the amount of albumin present in the sample. Adding stopping solution terminates the reaction. Absorbance is then measured on a 96-well ELISA reader at 490 nm and the concentration of albumin in samples and control is read off the standard curve.

Protocol

1. Coating plates

• Make concentrated stock solution in PBS of approx. 1 mg/mL.

Determine concentration via absorbance reading using  = 0.59 cm2/mg.

• Prepare 10 mL/plate of albumin at 5 µg/mL in PBS + 1 extra mL (i.e.,

11 mL for one plate, 21 for two plates, etc.).

• Add 100 µL of the albumin solution to each well of the plate.

• Cover with adhesive plate sealer.

• Incubate at 4 ˚C overnight.

This step has been performed in advance by the Lab Assistants.

2. Prepare standards

• Dilute albumin stock to 50 µg/mL in culture medium (make perhaps 2 mL)

• Dilute 50 µg/mL solution to make 1 ml each of {25, 10, 5, 2.5, 1.0, 0.5} µg/mL in culture medium.

Instruction in pipetting and dilution will be provided at this stage.

3. Prepare antibody solution

• Thaw one vial of albumin antibody from -80 ˚C freezer.

• Prepare needed volume of solution at dilution of 1:10000 in PBS-Tween.

5 mL per plate is needed, plus a little extra.

One vial can make enough dilute solution for several groups.

4. Prepare plate

• Empty plate and wash 4x 100 µL with PBS-Tween.

• Add 50 µL of sample or standard per well. Each plate must have standards. Both samples and standards should be done in triplicate.

• Add 50 µL of antibody solution to each well using multi-pipettor.

• Cover with adhesive plate sealer.

• Incubate 60 minutes at 37 ˚C.

In this interval, student will be introduced: 1) the principles used in data analysis;

2) safe problems in the lab.

5. Develop plates

• Substrate is hazardous. Wear gloves!

• Dissolve one pill OPD (10 mg) in 25 mL substrate buffer at room temp.

• Meanwhile, wash plate 4x 100 µL with PBS-Tween.

• Add 10 µL 30% H2O2 to OPD solution

• Add 100 µL/well of OPD solution wells one column at a time using

multi-pipettor. Fill columns at regular intervals (e.g., every 10 seconds).

• At t = 5 min. (from initial start), begin adding 50 µL/well of 8 N H2SO4(note: 8 N ≠ 8 M H2SO4), again one column at a time at the same regular interval as before). If intensity is low, develop longer.

• Read on plate reader.

• turn on plate reader

• set up Instrument to read 490 nm

• record data

• analyze data and print report

Equipment

Rocker/incubator; 490 nm plate reader.

Materials

Materials
96-well plate
Reagent reservoir
Cover
Human albumin
Conjugated antibody
OPD

Report (to be turned in following week)

Data Analysis

  1. Create a standard curve using two different methods (regressions) in the Microplate Manager software. Which is better and why?
  2. Using the better standard curve, calculate the albumin concentration in each of the unknown samples.
  3. Average the values for replicate measurements of each sample and report the values for each unknown as a mean ± s.d.
  4. Use a t-test to determine whether the control vs. treated unknowns are statistically different at the p < 0.05 level.

Questions

  1. Why is this assay called a competitive ELISA (Hint: it’s not because it’s a race to finish first!).
  2. Why is the wash step needed after incubation with antibody?
  3. Why is the plate rocked while incubating with antibody?
  4. What do you think would happen if you used 100x too much conjugated antibody?
  5. Instead of incubating at 37 ˚C for 60 minutes, many people incubate overnight at 4 ˚C. What is likely to be different in the two methods in terms of the molecular-level biochemical events occurring.
  6. If you performed the whole assay and saw no signal, what might be the possible cause (give 3 reasons)?
  7. Suppose you are working in a company, the results of this experiment are very important to a client, and the data are very scattered. You can repeat the assay tomorrow, but this is your last shot. List several precautions you would take to maximize your chances of getting good (i.e., internally consistent) data?