CN101343322A - A rapid immunization method for preparation of domoic acid monoclonal antibody - Google Patents

Abstract

An immunization method for rapidly preparing domoic acid monoclonal antibody, which involves the preparation of monoclonal antibody in the immune safety detection technology. The steps are as follows: take 8-week-old male BALB/C mice and use Freund's complete adjuvant on the first day Emulsify the complete antigen, the antigen-adjuvant ratio is 6:4, inject 18-22 μl/foot on the footpad of the hind limb, and wait for the stress state to decrease on the third or fourth day, emulsify with Freund's incomplete adjuvant, the antigen-adjuvant ratio is 6:4, Inject 27-33 μl/foot into the foot pads of the hind limbs, and test the serum titer on the 14th to 16th day, and the titer is above 1:3200, and the popliteal lymph nodes of the two hind legs are taken for cell fusion. Compared with the traditional method, this immunization scheme has a faster immunization speed, saves at least one month of time, and uses less antigen, thereby effectively reducing the cost of monoclonal antibody preparation.

Description

A rapid immunization method for preparation of domoic acid monoclonal antibody

[technical field]

The invention relates to the preparation of a monoclonal antibody in immune safety detection technology, and further relates to the preparation of a domoic acid monoclonal antibody applicable to aquatic products and water environments.

[Background technique]

Domoic acid (DA) is an analogue of glutamic acid and kainic acid mainly produced by diatoms of the genus Pseudomonas. After being enriched in the food chain, it will have a serious impact on the ecological environment of the area. And cause animal disease and death, clams, mussels and fish in the water can be enriched in DA, human consumption of contaminated aquatic products can cause poisoning. The molecular formula of DA is C ₅ H ₂₁ NO ₆ , the molecular weight is 311.34, the pure product is a colorless crystal, stable to heat, and the melting point is 223-224°C. Soluble in water, dilute acid and alkali solution, slightly soluble in methanol and ethanol, insoluble in petroleum ether and benzene. Pseudonitzia producing DA is a ubiquitous algae in the coastal areas of China, and is an important algae that causes red tides. Red tides have been caused in Dalian, Qingdao, the Yangtze River Estuary of the Yellow Sea, Xiamen Port and various harbors in the South China Sea.

DA is the pathogenic factor causing amnesia poisoning in humans. Poisoning symptoms include gastrointestinal symptoms and neurological symptoms, especially the temporary loss of memory is a unique symptom of this poisoning. Therefore, DA poisoning is also called "memory loss poisoning". At present, there is no safety standard for DA in water and aquatic products in my country. The safety standard stipulated by Canada is □20μg/g shellfish tissue, and this standard has also been adopted by some other countries.

The detection methods of DA are mainly instrumental analysis methods, including high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). HPLC has been regulated by Australia as a national standard detection method, and countries such as Canada, Spain, Denmark, New Zealand and the United States also use HPLC to detect metabolized or accumulated DA in plankton and shellfish. Capillary electrophoresis is one of the techniques for the separation and detection of algal toxins developed in recent years. Immunological detection method has been widely used in the detection of some algae toxins due to its stability, high sensitivity and fast speed.

The preparation of monoclonal antibodies is one of the key steps in the establishment of immunological detection methods. However, due to the high price and difficulty in obtaining algae toxin standard products, there have been no reports on the preparation of DA monoclonal antibodies in China so far.

At present, the preparation of monoclonal antibodies mostly adopts intraperitoneal and subcutaneous injection immunization schemes. After the serum titer of the immunized mice reached 4× ¹⁰⁴ or more, the splenocytes of the mice were fused with sp2/0 cells, cloned by limiting dilution, and the positive hybridoma cell lines were screened by ELISA. After the positive cells were expanded and cultured, the mouse ascites Preparation of monoclonal antibodies. This method is already a very mature technology, widely used in the preparation of monoclonal antibodies in food and environmental immune safety detection technology. However, the immunization time required by this immunization method is relatively long, requiring at least 4 times of immunization before cell fusion can take place, and at least 1.5 months; the demand for antigen is large, generally 200-475 μg/mouse. This greatly increases the cost of monoclonal antibody preparation, especially for expensive antigens, such as marine biotoxins, etc., tens of thousands of yuan is required to prepare a monoclonal antibody.

【Content of invention】

The technical problem to be solved by the present invention is to provide a rapid immunization method for preparing domoic acid monoclonal antibody, which has the characteristics of fast immunization speed and small amount of antigen required, thereby effectively reducing the cost of monoclonal antibody preparation.

The solution adopted by the present invention to solve technical problems adopts the following steps:

(1) The immunized animals were 8-week-old male BALB/C mice;

(2) On the first day, the complete antigen was emulsified with Freund's complete adjuvant, the antigen-adjuvant ratio was 6:4, and 18-22 μl/foot was injected into the hindlimb foot pad, a total of 36-44 μl/mouse, and the antigen amount was 36-44 μg/mouse;

(3) On the third or fourth day, when the stress state is reduced (the state of the testis can be observed, until the swelling returns to normal), emulsify with Freund's incomplete adjuvant, the ratio of antigen and adjuvant is 6:4, and inject 27-33 μl of the foot pad of the hind limb /foot, a total of 54-66μl/mouse. The amount of antigen is 54-66μg/mouse;

(4) Serum titer was detected on the 14th to 16th day, and the titer reached more than 1:3200, and the popliteal lymph nodes of both hind legs were collected for cell fusion.

The above injection method is to use a 1ml syringe with a 4.5-gauge needle, insert the needle at the second or third meat shop of the rear foot pad, move parallel to the sole of the foot to the center of the sole of the foot for injection, stop for 4-6 seconds after the injection, turn the needle 30 degrees and then inject. The needle is removed so as to prevent the antigen from being pressed out of the skin.

On the 14th to 16th day of the first administration, the popliteal lymph nodes were obviously swollen, the size of rice grains, white and transparent in color, and soft in texture. Grinding and crushing the lymph nodes yielded 2×10 ⁸ cells. The fusion rate is 10 times that of conventional methods.

The ascites titer of the monoclonal antibody prepared by this immunization method can reach 4.8×10 ⁵ , and the affinity constant can reach 2.9×10 ⁸ M ^-1 .

Compared with the traditional method, this immunization scheme has a faster immunization speed and saves at least one month; the amount of antigen required is less (only 100 μg/mouse).

The method of the invention has short immunization time and less antigen consumption, thereby effectively reducing the cost of monoclonal antibody preparation.

[Example 1] Reagent source and complete antigen preparation

Polyethylene glycol (polythylene glycol-4000, PEG), RPMI1640 medium, dialysis bag, HAT selective medium, DA standard, human IgG, bovine serum albumin (OVA), N-hydroxysuccinimide, N, N-dicyclohexylcarbodiimide (N, N-dicyclohexylcarbodiimide, DCC), N, N-dimethylformamide (N, N-dimethylformamide, DMF), Freund's complete adjuvant (FCA), Freund's Incomplete adjuvant (FICA) and o-phenylenediamine (OPD) were purchased from Sigma; centrifugal ultrafiltration tubes were purchased from MILLPORE; horseradish peroxidase Jiji goat anti-mouse IgG was purchased from Dingguo Biotechnology Co. Beijing), and other reagents used were of domestic analytical grade.

The complete antigen was prepared by the active ester method. The specific preparation method of coupled antigen DA-IgG (for immunization) and DA-OVA (for detection) is as follows: 1mgDA, 0.25mg N-hydroxysuccinimide, 0.6mg N, N dicycloethanecarbodiimide Mix well in 80μL DMF, stir at room temperature for 2.5h, add 4mgIgG (dissolved in 50μL 0.1mol/L borate buffer) to 40μL reaction solution, and add 4mgOVA (dissolve in 50μL 0.1mol/L borate buffer solution) to the other 40μL reaction solution, at room temperature Continue to incubate for 2.5 hours, remove unreacted substances by ultrafiltration and centrifugation, dissolve the conjugated antigen with an appropriate volume of borate buffer (pH 8.2) to a concentration of 1 g/L, and store at -80°C for future use.

[Example 2] Recovery and expanded culture of SP2/0 cells

Take out the SP2/0 cell cryopreservation tube, immediately put it into a thermos bucket filled with 40°C water, and shake gently until the dynamic storage solution is completely dissolved, which should be completed within 1-2 minutes. Move the moving solution to a 15mL centrifuge tube, add 5mL culture medium, blow gently evenly, 1000rpm, 5min, discard the supernatant. Add 1 tube of culture solution with a Pasteur pipette, blow evenly, transfer it into a 50mL culture bottle, then add 2 pipette culture solution, blow evenly, and divide into two new 50mL culture bottles, so that the cells in one cryopreservation tube are divided into three 50mL culture flasks. Place them in an incubator containing 5% carbon dioxide at 37°C. Change the medium after 24h, 4 Pasteur tubes per bottle. It can be used for fusion after 48h. If not fused, change the medium every 2-3 days. Or according to color (turning yellow). After the cell wall is covered with a single layer, it must be subcultured, that is, the old culture medium is discarded, and new culture medium is added with 3 pipettes.

Suspend the SP2/0 cultured for 72 hours with 1640 culture medium, centrifuge at 1000 RPM for 10 minutes, and wash the cells, a total of 3 times (20 mL each time). Suspend with 1640 culture medium to make the cell concentration 5×10 ⁶ /mL, and the volume is more than 2mL for fusion.

[Example 3] Preparation of feeder cells

Take 1-2 mice over 12 weeks old, preferably of the same strain as myeloma. One of the eyeballs was gouged out and the blood was let out until the blood dripped out. Kill by pulling the neck, and immerse the rat in 75% alcohol for 5 minutes for disinfection. Under aseptic conditions, use sterile forceps and scissors to cut the outer skin of the mouse abdomen (do not cut the peritoneum to cause fluid loss in the peritoneal cavity). The skin of the mouse was then pulled away in the direction of the upper and lower sides to expose the peritoneum. Draw 3 mL of serum-free HT1640 culture solution with a sterile syringe, lift the peritoneum with tweezers, and inject the solution into the mouse abdominal cavity. Fix the syringe with one hand, keep the needle in the abdominal cavity, massage the abdominal wall for 1-2 minutes (to make the macrophages swim out), then withdraw the intraperitoneal fluid with a syringe, and perform 3 flushes. Collect the washing solution, centrifuge at 1000rpm for 5min, and discard the supernatant. The centrifuged pelleted cells were diluted with HAT medium (20%), and the cell concentration was adjusted to 1×10 ⁵ cells/mL. Plate the cell suspension at 100ul (about two drops) per well. Place them in an incubator containing 5% carbon dioxide at 37°C. The cells were prepared 24 h before fusion.

[Example 4] Animal immunization

Take 8-week-old male BALB/C mice, use Freund's complete adjuvant to emulsify the complete antigen on the first day, the antigen-adjuvant ratio is 6:4, inject 20 μl/foot of the hind limb foot pad, a total of 40 μl/mouse, the amount of antigen is 40 μg/ Rats; on the third day, the stress state was reduced (the state of the testis can be observed, and the swelling returned to normal). The complete antigen was emulsified with Freund's incomplete adjuvant, the antigen-adjuvant ratio was 6:4, and 30 μl/foot was injected into the foot pad of the hind limb. A total of 60 μl/mouse. The amount of antigen was 60 μg/mouse; the serum titer was detected on the 15th day, and the titer reached above 1:3200. The injection method is to use a 1ml syringe with a 4.5-gauge needle, insert the needle at the second or third buttock of the rear foot pad, move parallel to the sole of the foot to the center of the sole of the foot for injection, stop for 5 seconds after the injection, turn the needle 30 degrees and then withdraw the needle .

[Example 5] Acquisition of Popliteal Lymphocytes

The eyeballs of the immunized mice in Example 4 were removed to obtain serum and stored at -20°C as a positive control, and the mice were sterilized by immersing them in 75% alcohol for 2 minutes. Under aseptic conditions, a small incision was made at the base of the back of the mouse, and the blunt-tip scissors were used to support the two sides to separate the skin from the muscle, and the popliteal lymph nodes were removed. Add 5mL of 1640 culture medium to the plate, put the popliteal lymph nodes into it, and grind the popliteal lymph nodes with a syringe core and copper mesh to obtain popliteal lymphocytes. After pipetting evenly, put the cell suspension into the test tube, and wash the plate once with 1640 culture medium. Centrifuge at 1000rpm for 10 minutes to wash the popliteal lymphocytes, a total of 3 times. Suspend the cells with 5mL1640 medium and count the cells. The cell concentration is 5×10 ⁷ cells/mL, and the volume is more than 2 mL.

[Example 6] Cell fusion and culture of cells after fusion

The popliteal lymphocytes obtained in Example 5 and the SP2/0 cells obtained in Example 2 (the ratio of the number of cells is 10:1) were transferred to a round-bottomed glass centrifuge tube, mixed evenly, and centrifuged at 1000 rpm for 10 min. After discarding the supernatant, flick the bottom of the test tube slightly with your fingers to make the precipitate a paste. Add 50% PEG preheated at 37°C quickly (0.2 mL PEG per 1.0×10 ⁸ lymphocytes). Quickly pipet 5-6 times with the Barnyard pipette. 30 seconds after adding PEG, centrifuge at 700rpm for 3min. 4 minutes after adding PEG, add 5 mL of RPMI1640 culture solution, pay attention to gently add along the tube wall, do not disperse the cells. Move the lower part of the test tube in a circular motion for 2 minutes, taking care not to disperse the cells. Centrifuge at 1000rpm for 10min, discard the supernatant. Repeat washing the cells one more time. Carefully add 5mL of HT culture solution and place at room temperature for 30min. Add RPMI1640-HAT culture medium containing 20% fetal bovine serum to make SP2/0 cells 3.3×10 ⁵ cells/mL, carefully pipette several times with a Pasteur pipette to suspend the cells evenly. Add 0.1 mL (about 2 drops) to four 96-well culture plates (0.1 mL of feeder cells per well), and place the culture plates in a 37°C 5% CO ₂ incubator for culture.

During the cultivation process, the half-volume liquid replacement method was adopted, that is, 0.1 mL of culture liquid was aspirated from each small well each time, and 0.1 mL of fresh culture liquid was added. Change to 20% calf serum HAT solution on the 5th, 8th and 11th day, and change to 20% calf serum HT solution on the 14th, 18th, 22nd and 25th day

After 3-5 days of culture, a single hybridoma cell begins to grow into a colony of several or more than a dozen cells, most of which are formed in 8-10 days, and some can only be seen in about two weeks. When the size of the hybridoma cell colony reaches 1/10 of the bottom area of the well, the antibody in the supernatant can be detected, and the hybridoma cells secreting the antibody can be screened out and cloned. The first detection of antibody was carried out two days after the second change of medium, that is, on the 10th day after fusion. If the OD value of the tested well is more than 2 times higher than the OD value of the tumor cell supernatant control, it is regarded as positive.

[Example 7] Re-cloning and expanded culture of positive hybridoma cells

When the cells in the positive wells are nearly full, gently blow up the cells with RPMI1640 medium containing 20% fetal bovine serum 1, suck them into a sterile vial, count the cells, and dilute to a density of 0.5-1 cells per 100 μl . Then drop the diluted cell suspension into the feeder cell culture plate prepared by the method in Example 2, 100 μl per well. Place the plate in a 5% _CO2 incubator at 37°C, observe once a day, change the medium once every 3 days, observe the number of clones in each well, and mark the wells with two or no cells respectively. When growing to 8-10 days, detect the antibody, mark the positive wells, change the medium for all the clonal cell wells, and test again after 2 days of changing the medium, and compare the wells with high OD values detected twice. Positive wells were cloned again. Until all cloned cell wells are positive, finally select hybridoma cells with high OD value and good cell viability for expansion culture.

[Example 8] Preparation, purification and affinity determination of ascites antibody

Take 8-week-old BALB/C mice, intraperitoneally inject 0.4 mL/mouse of liquid paraffin and 0.2 mL/mouse of Freund's incomplete adjuvant, 8 days later, intraperitoneally inject 2×10 ⁶ hybridoma cells obtained in Example 7/mouse, and inoculate for 7 days Observe the occurrence of ascites in the mice every day. If the abdominal cavity is obviously swollen, the mice have difficulty moving and the skin feels tense, collect the ascites with a 16-gauge needle, centrifuge the ascites at 1000g/min for 10min, take the supernatant, and use octanoic acid-saturated sulfuric acid Preliminary purification by ammonium method and frozen at -20°C for future use.

Ascites (after purification, the ascites was diluted to the same volume as before purification) were serially diluted 2 times starting from 4000 times, and the same dilution of ascites inoculated with SP2/0 cells was used as a control.

Antibody affinity was determined by non-competitive enzyme immunoassay. The coating agent (OVA-DA) was coated with 1, 0.5, 0.25, 0.125 μg/ml, 100 μl/well, 37°C, 2h; after blocking with 1% gelatin, the monoclonal antibody was multiplied from 4000 times dilution. Taking the logarithmic value of the antibody concentration (mol/L) as the abscissa and the corresponding OD value as the ordinate, four S-shaped curves can be drawn in one coordinate system. Find the top of the S-curve and set it as ODmax. Find the antibody concentrations corresponding to 50% ODmax of each of the four curves in the curves. The 4 concentrations were paired together, and the affinity constant of the monoclonal antibody was calculated according to the formula.

Ka=(n-1)/2(n[Ab`]t-[Ab]t)

Note: n is the multiple of the two coating concentrations in each group, [Ab`]t and [Ab]t are the two antibody concentrations (mol/L) corresponding to 50% ODmax in each group, and Ka is the affinity constant .

Claims (4)

1, a kind of immunization method of quick preparation domoic acid monoclone antibody is characterized in that: adopt following steps:

(1) getting immune animal is male BALB/C mice in 8 ages in week;

Used Freund's complete adjuvant emulsification complete antigen in (2) first days, antigen adjuvant was than 6: 4, and injection hind leg foot pad 18-22 μ l/ pin is total to 36-44 μ l/ mouse, and the antigen amount is a 36-44 μ g/ mouse;

Treated that stress situation reduced in (3) the 3rd or four days, use Freund's incomplete adjuvant emulsification, antigen adjuvant was than 6: 4, and injection hind leg foot fills up 27-33 μ l/ pin, 54-66 μ l/ mouse altogether.The antigen amount is a 54-66 μ g/ mouse;

Detected serum titer in (4) 14-16 days, tiring reaches more than 1: 3200, gets two Hou Tui lymphonodi popliteis and carries out cytogamy.

2, immunization method according to claim 1 is characterized in that: adopt the 1ml syringe, No. 4.5 syringe needles, at metapedes pad second or place, three butcher's shops inserting needle, parallel sole moves to the injection of sole centre, stops 4-6 second after injection finishes, and rotates syringe needle 30 degree and goes out pin again.

3, immunization method according to claim 1 is characterized in that: get male BALB/C mice in 8 ages in week, first day use Freund's complete adjuvant emulsification complete antigen, antigen adjuvant was than 6: 4, injection hind leg foot pad 20 μ l/ pin, totally 40 μ l/ mouse, the antigen amount is 40 μ g/ mouse; Treated on the 3rd day that stress situation reduced, use Freund's incomplete adjuvant emulsification complete antigen, antigen adjuvant was than 6: 4, and injection hind leg foot fills up 30 μ l/ pin, totally 60 μ l/ mouse.The antigen amount is 60 μ g/ mouse; Detected serum titer on the 15th day, tiring reaches more than 1: 3200, gets two back leg rouge lymphoglandula and carries out cytogamy.

4, according to claim 1 or 3 described immunization methods, it is characterized in that: rouge is lymphocytic obtain be with won by immune mouse eyeball obtain serum in-20 ℃ of preservations as positive control, mouse is immersed in 75% alcohol 2min sterilization, under the aseptic condition mouse is retreated base portion and cut off an osculum, with blunt nosed scissors to two side stays, skin is separated with muscle, extract the rouge lymphoglandula, the RPMI-1640 that adds 5mL in the plate, the rouge lymphoglandula is gone into wherein, grind the rouge lymphoglandula with piston and copper mesh, to obtain the rouge lymphocyte.After the piping and druming evenly, cell suspension is complied with in test tube, and wash a plate with RPMI-1640.The centrifugal 10min of 1000rpm washes quiet rouge lymphocyte, and totally 3 times, with 5mL1640 nutrient solution suspension cell, counting cells, making cell concn is 5 * 10 ⁷Individual/mL, more than the volume 2mL.