CN105838680A - Estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof - Google Patents

Abstract

The invention provides an estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof and belongs to the technical field of food safety immune detection. The estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 is preserved in General Microbiology Center, Microbial Preservation Management Committee, China, and the preservation number is CGMCC No. 12016. An estriol-specificity-resistant monoclonal antibody is generated by secretion of the hybridoma cell strain NaN-2. The monoclonal antibody secreted by the cell strain has good specificity to estriol (the IC 50 value is 0.25 ng/mL) and can be used for estriol specificity detection in food safety. The obtained estriol-resistant monoclonal antibody cell strain has good detection sensitivity and appetency to estriol, a novel method for synthesizing estriol immunogen is further provided, the synthesis step of hapten is more simplified and effective, and a thought and method for synthesizing immunogen are provided for people to conduct research in the future.

Description

An anti-estriol specific monoclonal antibody hybridoma cell line NaN-2 and its application

technical field

The invention relates to an anti-estriol-specific monoclonal antibody hybridoma cell line NaN-2 and an application thereof, belonging to the technical field of food safety immunoassay.

Background technique

Estriol (E ₃ ) is an endogenous steroid hormone, which is a metabolite of estradiol (E ₂ ) and estrone (E ₁ ). E ₃ in pregnant women mainly comes from the placenta. It is an important indicator of fetal development and placental function, and it is also a common indicator of estrogen detection in cosmetics. Estriol is used in animal husbandry to promote the growth and development of livestock. Improper use of estriol may lead to residual pollution of estriol in aquatic products, meat, eggs and milk. At present, my country has explicitly prohibited the addition of estriol in the feeding of food animals.

At present, the detection of estriol mostly adopts high performance liquid chromatography, liquid chromatography and mass spectrometry, gas chromatography, radioimmunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay, and capillary electrophoresis. In China, GB/T 21981-2008 "Detection method for hormone residues in food of animal origin: liquid chromatography-mass spectrometry-mass spectrometry" is generally used for detection, which requires homogenization, enzymatic hydrolysis, extraction, solid phase extraction enrichment and purification, and instrumental determination , internal standard quantification and other steps. Clinically, radioimmunoassay is mainly used to detect estriol, which has the disadvantages of radioisotope waste disposal and short shelf life of markers. The above-mentioned detection methods can be used for quantitative analysis and have a lower detection limit, but usually require expensive instruments and complicated operations, and the pretreatment and detection time is long, which seriously restricts the promotion of these detection methods. The immunoassay method has the characteristics of low cost, high throughput, high sensitivity, and relatively low requirements for technicians, so it is suitable for rapid screening of a large number of samples. The purpose of the present invention is to provide a method for preparing a monoclonal antibody hybridoma cell line with higher affinity and detection sensitivity to estriol. It laid the foundation for the development and promotion of indirect competition ELISA kits and colloidal gold test strips.

Contents of the invention

The object of the present invention is to provide an anti-estriol specific monoclonal antibody hybridoma cell line, the antibody prepared by the cell line has good affinity and detection sensitivity to estriol, and can be used to establish estriolase Linked immunoassay method, or establish colloidal gold immunochromatography rapid detection method.

The technical scheme of the present invention, an anti-estriol-specific monoclonal antibody hybridoma cell line NaN-2, has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, referred to as CGMCC, and the preservation number is CGMCC No.12016.

The anti-estriol-specific monoclonal antibody is secreted and produced by the anti-estriol-specific monoclonal antibody hybridoma cell line NaN-2 with the storage number CGMCC No.12016.

Application of the anti-estriol-specific monoclonal antibody: its application in the detection of estriol residues in food safety.

The basic steps for the preparation of the NaN-2 cell strain provided by the invention are:

(1) Preparation and identification of immunogen: react estriol with 6-bromohexanoic acid to prepare a carboxyl-containing hapten, which is connected to the amino group of the protein carrier by the carbodiimide method. The coupled small molecule haptens and complete antigens are identified by UV absorption scanning method;

(2) Immunization of mice: After emulsifying the immunogen with Freund's adjuvant completely, BALB/c mice were immunized by subcutaneous multi-point injection. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunization. The immunization dose during the sprint immunization was half of the previous immunization dose, and it was mixed with normal saline and injected directly into the intraperitoneal cavity; the interval between each immunization was three weeks . After the third immunization, blood was collected one week apart to detect serum titer and inhibition;

(3) Cell fusion and cell line establishment: fused mouse splenocytes and mouse myeloma cells by polyethylene glycol (PEG 4000) method, cultured in HAT medium, detected positive cell wells by indirect ELISA, and further The inhibitory effect of positive cell wells was determined by indirect competitive ELISA method, and the positive cell wells with the best inhibition were subcloned three times by limiting dilution method, and the hybridoma cell line NaN-2 was finally screened;

(4) Identification of the properties of hybridoma cell lines: the enzyme-labeled secondary antibody kit for identification of mouse monoclonal antibody Ig class/subclass was used to determine; IC ₅₀ value, cross-reactivity rate and affinity were determined by ELISA method.

Beneficial effects of the present invention: the anti-estriol monoclonal antibody cell line obtained by the present invention has better detection sensitivity and affinity to estriol, and also provides a new method for synthesizing estriol immunogen, semi The synthesis steps of the antigen are more simplified and effective, which provides the idea and method of synthesizing the immunogen for people's research in the future.

Preservation of biological material samples: monoclonal cell line NaN-2, which has been preserved in the General Microbiology Center of China Committee for Microbial Culture Collection, referred to as CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , the deposit number is CGMCC No.12016, and the deposit date is January 20, 2016.

Description of drawings

Figure 1 UV absorption spectrum characterization of the immunogen.

Figure 2 The standard inhibition curve of NaN-2 monoclonal antibody to estriol.

detailed description

The following examples of the present invention are only used as a further description of the content of the present invention, and cannot be regarded as the content or scope of the present invention. Below by embodiment the present invention will be further described.

The present invention immunizes mice with the complete antigen of estriol, through cell fusion, cultured in HAT selective medium, and through indirect ELISA and indirect competition ELISA to screen the cell supernatant, and finally obtains a compound with good affinity and detection sensitivity for estriol monoclonal antibody hybridoma cell lines.

Embodiment 1 Preparation of hybridoma cell line NaN-2

(1) Synthesis of complete antigen: 300 mg (1.10 mmol) of E3 was dissolved in ₆ mL of dry dimethyl sulfoxide, and 1 g of KOH powder was added. After stirring for 5 min, 300 mg of bromohexanoic acid was added. Stirring was continued, and 50 mL of ice water was added after 2 h of reaction. Unreacted E ₃ was recovered by extraction with ethyl acetate. The aqueous phase was acidified with 2mol/L HCl, and a white precipitate appeared. The sand core funnel was filtered, and the precipitate was washed with distilled water until neutral, and dried in vacuum. Methanol-chloroform recrystallization gave colorless crystals, namely the estriol hapten. Take 4.5mg of the above hapten, then add 2.0mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 1.0mg NHS (N-hydroxysuccinimide) , dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 4 hours; another 5 mg of BSA (bovine serum albumin) was dissolved in 2 mL of 0.05 M, pH 9.6 CB (carbonate buffer solution ) solution, the above activation solution was added dropwise to the BSA solution, stirred overnight at room temperature, dialyzed at 4°C for three days, and stored at -20°C.

(2) Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. After the complete antigen of estriol (1 mg/mL) was emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multi-point injection, 100 μL each. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunization. The immunization dose during the sprint immunization was half of the previous immunization dose, and it was mixed with normal saline and injected directly into the intraperitoneal cavity; the interval between each immunization was three weeks . After the third immunization, blood was collected at intervals of one week to detect the serum titer and inhibition; the mice with the best inhibition were selected and immunized 21 days after the fifth immunization to prepare for fusion.

(3) Cell fusion: Three days after the sprint immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:

a. Take the mouse spleen aseptically, grind it and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension, and perform cell counting;

b. Collect SP2/0 cells, suspend them in RPMI-1640 basal culture medium, and perform cell counting;

c. Mix splenocytes and SP2/0 cells at a count ratio of 5-10:1, centrifuge and fuse with PEG for 1 min, then add RPMI-1640 basal culture medium from slow to fast, centrifuge and suspend in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50×HAT was added to a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO ₂ .

(4) Cell screening and cell line establishment: On the third day of cell fusion, half-change the RPMI-1640 screening culture medium for the fused cells, and on the fifth day, use 20% fetal bovine serum, 1% 100×HT The RPMI-1640 transition culture medium was completely changed, and the cell supernatant was collected on the 7th day for screening. The screening is divided into two steps: in the first step, positive cell wells are screened out by indirect ELISA; in the second step, estriol is selected as a standard, and the inhibitory effect of positive cell wells is determined by indirect competitive ELISA. Select the cell wells with better inhibition to estriol, use the limited dilution method for subcloning, and use the same method for detection. Repeat three times to obtain cell line NaN-2.

(5) Preparation and identification of monoclonal antibody: 8-10 weeks old BALB/c mice were taken, and each mouse was intraperitoneally injected with 1 mL of paraffin oil; 7 days later, each mouse was intraperitoneally injected with 1×10 ⁶ hybridoma cells, from which On the seventh day, the ascites was collected, and the ascites was purified by octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody purified from ascites, and the subtype was IgG1.

Using the indirect competition ELISA method, the IC ₅₀ of the monoclonal antibody against estriol was determined to be 0.25 μg/L, which can be used for the specific and rapid detection of estriol.

(6) Antibody application

The monoclonal antibody prepared by the hybridoma cell line NaN-2 through the in vivo ascites was applied to the estriol ELISA addition and recovery test, and the specific steps were as follows:

6.1 Use 0.1 μg/mL diluted with carbonate buffer solution (CBS) as the original coating material to coat the 96-well ELISA plate, 100 μL per well, after coating for 2 hours at 37°C, wash the plate three times with PBST washing solution, each time 250 μL per well, 3 min each time, pat dry;

6.2 Block with CBS containing 0.2% gelatin, 200 μL per well, block for 2 h at 37 °C, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry;

6.3 Use phosphate buffered saline (PBS) to prepare 0, 0.2, 0.5, 1, 2, 5, 10, 20 ng/mL estriol standard solutions respectively. Add the standard solution and the extract of the sample to be tested to the sealed ELISA plate, 50 μL per well, repeat 3 wells for each sample, and then add 50 μL of 1:16000 diluted anti-estriol monoclonal to each well Antibody, after reacting at 37°C for 0.5 h, wash the plate and pat dry;

6.4 Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin 1:3000 to each well, react at 37°C for 0.5 h, wash the plate and pat dry;

6.5 Add 100 μL TMB chromogenic solution to each well, develop color at 37°C for 15 minutes, add 50 μL 2M H ₂ SO ₄ stop solution to each well, and measure the absorbance at 450 nm;

6.6 Addition recovery and sample pretreatment: Weigh 1g of milk sample into a 50mL centrifuge tube, add 0.2ng, 0.4ng and 1ng of estriol respectively. Add 25 mL of 80% methanol in PBS solution (w/v) to the sample, oscillate evenly, extract by ultrasonic for 15 min, and filter the supernatant with a 0.45 μm filter membrane after standing still. The ELISA sample extract was added to the recovery test using indirect competition ELISA, and the recovery rates were 83%, 87%, and 84%, respectively.

Solution configuration:

Carbonate buffer solution (CBS): Weigh 1.59g of Na ₂ CO ₃ and 2.93g of NaHCO ₃ , dissolve them in a small amount of double distilled water and mix, add double distilled water to about 800mL and mix well, adjust the pH to 9.6, add Dilute to 1000mL with double distilled water and store at 4°C for later use;

Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 3.62 g Na ₂ HPO ₄ 12H ₂ O, dissolved in 800 mL of pure water, adjusted to pH 7.2 with NaOH or HCl ~7.4, set the volume to 1000 mL;

PBST: PBS containing 0.05% Tween 20;

TMB chromogenic solution: A solution: Na ₂ HPO ₄ ·12H ₂ O 18.43g, citric acid 9.33g, dilute to 1000 mL with pure water; B solution: 60mg TMB dissolved in 100 mL ethylene glycol. A and B liquids are mixed according to the volume ratio of 1:5, which is the TMB chromogenic liquid, which is ready-to-use and mixed.

Table 1. Subtype Identification of NaN-2 Monoclonal Antibody

Table 2. _IC50 and cross-reactivity rate of NaN-2 monoclonal antibody to estrone and estradiol

In summary, the above are only preferred embodiments of the present invention, and are not intended to limit the implementation scope of the present invention. That is, all equivalent changes and modifications made according to the content of the patent scope of the present invention shall be within the technical scope of the present invention.

Claims (3)

1. An anti-estriol-specific monoclonal antibody hybridoma cell line NaN-2 has been preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms, referred to as CGMCC, and the preservation number is CGMCC No.12016. 2. Anti-estriol-specific monoclonal antibody, characterized in that it is secreted and produced by the anti-estriol-specific monoclonal antibody hybridoma cell line NaN-2 with the preservation number CGMCC No. 12016. 3. The application of the anti-estriol specific monoclonal antibody according to claim 2, characterized in that it is used in the detection of estriol residues in food safety.