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

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Experimental principle

ELISA is a highly sensitive test technique that combines the specific reaction of antigens and antibodies with the efficient catalysis of enzymes on substrates based on immunological reactions. The immunoenzyme technique involves labeling an enzyme on an antibody/antigen molecule to form an enzyme-labeled antibody/enzyme-labeled antigen, called an enzyme conjugate. After the immune reaction, the enzyme of the enzyme conjugate acts on the substrate to make it color, and locates or quantifies the antigen/antibody according to the presence or absence of color and depth. The ELISA method is a kind of immunoenzyme technology, which is characterized in that an antigen/antibody is adsorbed by a polystyrene microplate (or a sphere) to immobilize it, and an immune reaction and an enzymatic reaction are carried out therein. Washing is repeated after each reaction, which not only ensures the quantitative relationship of the reaction, but also avoids the separation step of the free antibody/antigen of the final reaction. In the ELISA method, the enzymatic reaction is carried out only once, and the immune reaction of the antigen and the antibody can be carried out once or several times, and the secondary antibody (anti-antibody) and the third antibody can be used for the immune reaction again.

Several ELISA methods commonly used at present include: an indirect method for measuring an antibody, a double antibody sandwich method for measuring an antigen, and a competition method for measuring an antigen. In this experiment, the indirect method was used to determine the titer of monoclonal antibodies. The main process is as follows: firstly, the known quantitative antigen is adsorbed in the concave hole of the polystyrene micro-reaction plate, and the antibody to be tested is added, and after washing, the unbound heteroprotein is removed to remove the unbound heteroprotein, and the enzyme-labeled anti-antibody is added, and the sample is heated and washed. After the substrate was incubated for 30 minutes, the enzymatic reaction was terminated by addition of an acid or a base, and the antibody content was determined by visual or photoelectric colorimetry.

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, instruments, materials and reagents

instrument

Polystyrene 96-well microtiter plate, micropipette, enzyme-linked immunosorbent reader, water bath.

raw material

Monoclonal antibodies

Reagent

Antigen and enzyme-labeled antibody

1 antigen: rabbit anti-human IgG (Rabbit Aanti-human IgG, Code No. A0423);

2 enzyme-labeled anti-antibody: rabbit anti-mouse IgG-HRP (Rabbit Anti-mouse IgG-HRP, Code No. P0260); 4. Washing solution (PBS containing 0.05% Tween 20): Tween-20 500 μL was added to 1 L of PBS.

5. Blocking solution (PBS containing 1% bovine serum albumin, 0.1% Tween 20): 100 mg BSA, 10 μ LTween-20 was added to 10 mL PBS.

6. Substrate solution

1 sodium phosphate buffer (0.1 mol / L pH 6.0): said Na2HPO4 · 12H2O2.2g, NaH2PO4 · 2H2O 6.84g, dissolved in distilled water, to a volume of 500mL.

2 TMB stock solution: Weigh 60mg TMB dissolved in 10mL dimethyl sulfoxide, stored at 4 ° C protected from light.

3 substrate application solution: the current use, sodium phosphate buffer 10mL, TNB stock solution 10μL. Mix 30% H2O2 15μL.

7. Stop solution: 2mol/L H2SO4

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,Steps

1 Antigen coating: Rabbit anti-human IgG was used as an antigen, diluted with a coating solution of 1:8000, and 100 μL/well was added to a polystyrene 96-well reaction plate. Leave at 4 ° C overnight.

2 Washing: The liquid in the recessed hole was poured out the next day, and the washing liquid was washed 3 times.

3 Blocking: Add l00μL/well blocking solution and leave it at room temperature for 0.5h.

4 Washing: Wash 3 times with washing solution.

5 plus sample to be tested (primary antibody): The cell culture supernatant containing the single-gram-down antibody was serially diluted with PBS on a separate plate (according to 1:2 or 1:10), and 100 μL/well was added to the coated On the plate, each sample was made in parallel, PBS or blank medium was used as a negative control, and the sample was known as a positive control. Cover with a 37 ° C incubator for 1~2h.

6 Washing: Wash 3 times with washing solution.

7 plus enzyme-labeled anti-antibody: rabbit anti-mouse IgG-HRP, diluted with blocking solution 1:8000, 100 μL / well, incubated with 37 ° C incubator for 1 h.

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

9 color development: add freshly prepared substrate solution 100μL / hole, placed in the dark at room temperature for 5 ~ 30min, showing blue.

10 termination reaction, colorimetric: add 50 μL / well stop solution. The color turns yellow; the absorbance of each well at 450 nm is measured by a microplate reader, and the maximum dilution of the positive reaction is the titer of the sample to be tested.