Method for determining monoclonal antibody titer by ELISA - Master's thesis - Dissertation

One

Experimental Principle

ELISA, or Enzyme-Linked Immunosorbent Assay, is a powerful and sensitive technique that combines the specificity of antigen-antibody interactions with the catalytic efficiency of enzymes. This method relies on immunological reactions where an enzyme is labeled onto an antibody or antigen to create an enzyme-conjugated molecule. After an immune reaction occurs, the enzyme in the conjugate catalyzes a substrate to produce a color change, which allows for both the detection and quantification of antigens or antibodies based on the presence or intensity of the color. The key feature of ELISA is that it immobilizes antigens or antibodies on a polystyrene microplate, enabling the immune and enzymatic reactions to take place within the wells. Washing steps are performed after each reaction to maintain accuracy and eliminate unbound components, reducing the need for complex separation procedures.

Several common ELISA methods include the indirect method for detecting antibodies, the sandwich method for measuring antigens, and the competitive method for determining antigen concentration. In this experiment, we used the indirect method to determine the titer of monoclonal antibodies. The process involved coating the microplate with a known quantity of antigen, incubating with the test antibody, washing to remove unbound material, adding an enzyme-labeled secondary antibody, and then developing the color using a substrate. After stopping the reaction, the absorbance was measured at 450 nm using a plate reader, and the highest dilution showing a positive result was considered the titer of the sample.

Two

Materials, Instruments, and Reagents

Instruments:

Polystyrene 96-well microtiter plate, micropipette, ELISA reader, water bath.

Materials:

Monoclonal antibodies

Reagents:

Antigen and enzyme-labeled antibody

1. Antigen: Rabbit anti-human IgG (Code No. A0423)

2. Enzyme-labeled anti-antibody: Rabbit anti-mouse IgG-HRP (Code No. P0260)

3. Washing solution (PBS + 0.05% Tween 20): 500 μL Tween-20 in 1 L PBS

4. Blocking solution (PBS + 1% BSA + 0.1% Tween 20): 100 mg BSA, 10 μL Tween-20 in 10 mL PBS

5. Substrate solution

- Sodium phosphate buffer (0.1 mol/L, pH 6.0): 2.2 g Na₂HPO₄·12H₂O, 6.84 g NaH₂PO₄·2H₂O in 500 mL distilled water

- TMB stock solution: 60 mg TMB in 10 mL DMSO, stored at 4°C

- Substrate working solution: 10 mL sodium phosphate buffer + 10 μL TMB stock + 15 μL 30% H₂O₂

6. Stop solution: 2 M Hâ‚‚SOâ‚„

Three

Procedure

1. Antigen Coating: Rabbit anti-human IgG was diluted 1:8000 in coating buffer and added 100 μL per well. Incubate overnight at 4°C.

2. Washing: Remove liquid and wash 3 times with washing solution.

3. Blocking: Add 100 μL blocking solution per well, incubate at room temperature for 30 minutes.

4. Washing: Wash 3 times with washing solution.

5. Sample Addition: Dilute the test antibody (cell culture supernatant) in PBS (1:2 or 1:10), add 100 μL per well. Include negative and positive controls. Incubate at 37°C for 1–2 hours.

6. Washing: Wash 3 times with washing solution.

7. Enzyme-Labeled Antibody Addition: Rabbit anti-mouse IgG-HRP, diluted 1:8000 in blocking solution, add 100 μL per well. Incubate at 37°C for 1 hour.

8. Washing: Wash 5 times with washing solution, then 2 times with distilled water.

9. Color Development: Add 100 μL freshly prepared substrate solution per well. Incubate in the dark at room temperature for 5–30 minutes until blue color appears.

10. Stop Reaction and Read: Add 50 μL stop solution per well. The color turns yellow. Measure absorbance at 450 nm using a microplate reader. The highest dilution showing a positive signal represents the titer of the sample.