Polyclonal antibody preparation service

Polyclonal antibody preparation service

The general preparation process of multiple antibodies: * antigen preparation -+rabbit immunization - → potency detection and final release -+antibody affinity purification -+antibody concentration and preservation.

The specific experimental steps are as follows:

1. Preparation of rabbits

Select two healthy 6-week-old New Zealand white rabbits (approximately 2Kg) to adapt to the new living environment. After stabilizing for at least a few days, perform * blood collection and pre blood collection (for negative reference)

1.1 Carefully place the rabbit into a fixed rack to calm it down;

1.2 Carefully shave off the hair on the rabbit ear to make the blood vessels clearly visible (or not shaved);

1.3 If necessary, a small cotton ball dipped in alcohol can be used to apply to the vascular area to expand the blood vessels;

1.4 Extract approximately 10ml of blood (approximately 5ml of serum) from the ear vein using a syringe;

1.5 Carefully remove the needle, press the wound appropriately to prevent bleeding, and then disinfect the wound with an alcohol swab;

1.6 Place the collected blood at 37 ° C for 30 minutes to inactivate, and then leave it overnight at 4 ° C to coagulate and release serum;

1.7 Centrifuge the coagulated blood at 10000r/min for 10 minutes; h. Collect the supernatant, which is the serum.

Rabbit immunity

2.1 Inject 1ml of antigen into two rabbits, and the antigen buffer solution must not contain any harmful chemical reagents to the rabbits. It is more suitable for each rabbit to be immunized with 400ug of antigen for the first time, or it can be reduced appropriately to obtain better results. Subsequent immunizations should be administered with 100ug each time.

2.2 Mix 1m of Freund's adjuvant thoroughly with the prepared 1m antigen, resulting in a milky white color; .

2.3 Carefully remove the rabbit from the cage and immunize it in 4 different areas (including the back and thigh base), each with 250ul. Insert the needle into the subcutaneous tissue at a 45 degree angle for 1-2cm and wait for a few seconds after injection to prevent antigen leakage.

The rabbit immunization cycle is 20 days, and blood samples are taken 7-10 days after immunization (including mid-term testing and final bleeding), for a total of 4-5 immunizations.

3. Potency testing

3.1 Two reference negative samples are pre collected blood serum, both diluted with the initial concentration of 1 antibody (blocking dilution solution); Positive reference is from the past

Positive serum

3.2 Add 100ul, 1ug/m antigen dropwise to each well in a 96 well plate, overnight at 49C, or incubate at 37 ° C for 2 hours.

3.3 Discard the antigen solution, add 200u of blocking solution to each well, and incubate overnight at 49C or 379C for 2 hours.

3.4 Pour out the sealing solution and pat it on absorbent paper to absorb as much residual liquid as possible. Wash the board three times with detergent and pat the residual liquid as much as possible each time. If it needs to be stored for a period of time, dry it at 37 ° C and seal it in a sealed bag at 20 ° C.

3.5 Take the packaged 96 well plate,第---Add 100ul of negative reference solution to the well, 100ul of positive reference solution to the second well, and 1:500 of the test serum to the third well. Dilute the subsequent wells in multiple ratios based on this, and incubate at 37 ° C for 1 hour.

3.6 Pour out the liquid, wash the board three times with detergent, and pat dry. Add 100u of HRP labeled mouse anti rabbit lgG (diluted 1:2000) to each well and incubate at 37 ° C for 45 minutes.

3.7 Pour out the liquid, wash the board three times with detergent, and pat dry. Add 100u of TMB substrate (Sigma single component TMB) to each well and incubate at 37 ° C for 5-20 minutes (depending on the color depth).

Add 50u of termination solution (2N H2SO4) to each well and read the absorbance value at a wavelength of 450nm on an enzyme-linked immunosorbent assay (ELISA) reader.

After the antibody titer test reaches over 1 million, rabbits can be subjected to final bloodletting.

Purification of antibodies

4. Affinity of Antibodies - Preparation of Affinity Columns

4.1 Weigh 1mg of CNBr - Sepherose 4B agarose gel and add it into 2mmol/L hydrochloric acid. Allow it to fully dissolve at 4 'C overnight to obtain 3m swelling gel.

4.2 Transfer the gel into the purification column, and wash the medium three times with about 20ml 2mmol/L hydrochloric acid.

4.3 Wash the medium with coupling buffer solution - times, because the activating groups are easily hydrolyzed at the pH value of the coupling buffer solution, so this stepComplete within a few minutes.

4.4 Add 5-10mg antigen dissolved in 5ml coupling buffer into gel

Gently mix and shake at room temperature for 2-4 hours, or overnight at 4 ° C. If binding efficiency needs to be measured, take a small amount of solution after this step is completed for testing. Wash the medium once with 20ml of coupling buffer solution.

4.6 Add 15ml of 1% BSA solution and incubate at room temperature for 2 hours or overnight at 4 ° C.

Wash the medium with phosphate buffer solution for at least 3 times, each time for at least 15m

4.8 Antigen solid-phase is completed and can be used for purification.

4.9 If not used immediately or after use, seal with 20% ethanol.

5. Purification and preservation of anti serum

5.1 Dilute the serum with equal volume of PBS, centrifuge at 5000-10000/min for 15 minutes, and take the supernatant

5.2 Wash the antigen affinity column with 10 times the volume of PBS to balance the column.

5.3 Add 10ml of diluted anti serum to the equilibrated column.

5.4 Gently mix and shake at room temperature for 2-4 hours, or overnight at 4 'C

5.5 Wash the antigen column with 10 times the volume of PBS to remove impurities bound to the column

Wash the column with twice the volume of antibody eluent to obtain specific antibodies

5.7 Balance the column with 10 times the volume of PBS

Seal the column with 20% alcohol and store at 4 ° C.

After obtaining the eluted antibody, it was concentrated with sucrose or polyethylene glycol and dialyzed in PBS to remove salt. The OD value of the antibody was measured using a UV visible spectrophotometer at a wavelength of 280nm. The OD value obtained was divided by 1.35 to obtain the concentration of the measured antibody. 40-50% glycerol was added and stored at 20 ^ C for a long time.

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