CN116284383B - Method for rapidly obtaining 17 beta-estradiol murine IgG full-length antibody and Fab antibody - Google Patents

Abstract

The invention discloses a method for quickly obtaining 17β-estradiol mouse IgG full-length antibodies and Fab antibodies, and belongs to the technical field of genetic engineering. The DNA sequences of 17β-estradiol IgG antibodies and Fab antibodies provided by the invention are obtained through codon optimization, and the target proteins IgG and Fab are rapidly expressed in mammalian cells Expi293F cells. Based on IgG and Fab, 17β-estradiol is established. Alcohol competition ELISA standard curve belongs to the field of genetic engineering technology. In the present invention, the effects of three eukaryotic expression vectors on the expression yield of IgG target genes are compared. Among them, the eukaryotic expression vector based on pTT5 has the highest IgG expression level, which can reach 27 mg/mL. The antibodies expressed in the present invention retain the characteristics of high specificity and high affinity of traditional antibodies. The IC ₅₀ of the competitive ELISA standard curve based on IgG and Fab are 0.129ng/mL and 0.188ng/mL respectively, and can be applied to female II Development of alcohol immunoassay methods.

Description

A method to quickly obtain 17β-estradiol mouse IgG full-length antibodies and Fab antibodies

Technical field

The invention relates to a method for quickly obtaining 17β-estradiol mouse IgG full-length antibodies and Fab antibodies, and belongs to the technical field of genetic engineering.

Background technique

Environmental estrogen (E ₂ ) is the most common endocrine disruptor. When exogenous estrogen is absorbed by the human body, it will interfere with the body's hormone metabolism balance, thereby affecting its growth, development and reproduction. It is reported that in most countries, water discharge from urban sewage treatment plants is one of the main sources of estrogen pollution in surface water environments, followed by wastewater discharge from animal husbandry, hospitals, pharmaceutical factories, and aquaculture plants. This is because currently employed wastewater treatment processes are primarily designed to remove conventional pollutants, and the removal of environmental estrogens is often neglected. These environmental estrogens are released into the environment through various pathways, and inevitably enter the food chain after passing through the atmosphere and water circulation system. Therefore, it is particularly important to establish a rapid, accurate, and sensitive detection and analysis method for environmental estrogen. Researchers have developed a variety of detection methods based on large-scale analytical instruments, such as high-performance liquid chromatography and gas chromatography-mass spectrometry. However, the sample pretreatment procedures of these methods are complex and have limitations for on-site real-time detection. Since immunoassay methods have the advantages of high sensitivity and strong specificity, a large number of immunoassay methods based on antibody recognition have been developed to detect environmental estrogens.

High-affinity antibodies are the core of immunoassay methods. However, the preparation process of traditional monoclonal antibodies requires multiple subcutaneous injections of immunogen into mice, grinding their spleens to prepare hybridoma cells, and taking blood from the eyeballs to obtain positive controls. The production of serum and late-stage ascites still requires a steady stream of experimental mice. Not only does the experimental period be long and the experimental procedures are cumbersome, it also does not comply with the international community’s call to reduce the use of experimental animals through alternative experiments. Moreover, it matures in the animal body, and the monomer obtained by extraction and purification The structure of cloned antibody proteins cannot be modified in vitro. Recombinant antibodies are antibody proteins obtained by rationally designing and modifying antibody genes through DNA editing technology and protein expression technology, and then expressing, modifying, folding and secreting them in a suitable host. Compared with monoclonal antibodies prepared by hybridoma cell fusion technology, which is time-consuming and labor-intensive, genetically engineered antibodies can produce the target antibody in an expression system in just a few weeks. It can be mass-produced without animal immunization, and the process is simple and low-cost. It is the preferred strategy for preparing immune analysis recognition elements. E. coli or yeast expression systems have been widely used to produce genetically engineered antibodies; however, they often involve issues such as instability and dimerization, which are also major obstacles to their commercialization. For antibody proteins, glycosylation modification plays a very important role in their function. The mammalian cell expression system is the first system for the in vitro expression of antibody proteins. Compared with the E. coli expression system, its glycosylation modification program gives the antibody closer to the naturally mature structural properties in the body, and thus has higher performance than the antibody obtained by E. coli expression. sensitivity and specificity. Mammalian cell expression systems enable better protein folding, post-translational modification, secretory production and superior protein solubility, resulting in high-quality protein production and reduced downstream processing steps and costs. Many factors, such as the selection of vectors and cell lines, the transfection ratio of light and heavy chain plasmids, etc., may affect the expression level of the antibody.

The prerequisite for the expression of genetically engineered antibodies is to obtain the variable region gene sequence of the target antibody. Currently, many researchers have shared the prepared high-performance antibody variable region sequences and related performance data. Efficient utilization of this can greatly save subsequent researchers’ investment in experimental costs for antibody preparation, reduce resource waste, and also make Its results have been further and continuously developed. According to the research of published articles, the researchers used E ₂ -3-BSA as the immunogen to prepare monoclonal antibodies and polyclonal antibodies for estradiol, and obtained estradiol antibodies with good specificity and an affinity constant of 4×10 ⁸ M ^- Between ¹ and 5×10 ⁸ M ^-1 . In 2002, researchers shared the sequence information of two highly specific estradiol monoclonal antibodies obtained experimentally, and shared their variable region crystal structures and affinity and dissociation constants in the PDB database. Among them, the 10G6D6 antibody was specific to estradiol-6. -CMO-AP has a dissociation constant of 1.0 nM and excellent specificity for estradiol. It is the highest affinity estradiol monoclonal antibody sequence disclosed so far. In 2008, Kobayashi et al. expressed an estradiol single-chain antibody in Escherichia coli, with an affinity of 2.6×10 ⁸ M ^-1 ; Pajunen et al. expressed three estradiol antibody Fab fragments in Escherichia coli, with a yield of Between 115-2207μg/L. Compared with prokaryotic expression systems, mammalian cell expression systems are an effective means to obtain high-quality antibodies. Because of their functional properties such as glycosylation modification, the performance of the antibodies obtained is superior to products using E. coli and yeast as hosts. Compared with the cumbersome screening process of stable transfection, transient transfection is the preferred transfection method to quickly obtain expression products. The yield of current mammalian cell transient expression systems has been significantly improved, and excellent protein yields can be obtained relatively quickly. The HEK293 cell line is easy to culture and has high transfection efficiency. After modification, it has been successfully used for the transient expression of a variety of antibodies, and achieved mg/L level production.

Contents of the invention

Therefore, the present invention obtains the optimized gene sequence of the 10G6D6 estradiol antibody amino acid sequence through codon optimization, constructs heavy chain and light chain expression vectors respectively, uses the mammalian transient expression system to quickly prepare the estradiol antibody, and compares the three types The effect of expression vector on IgG expression yield is an efficient and feasible way to provide highly reproducible recognition elements for estradiol immunological detection.

In view of the above-mentioned shortcomings of the current technology, the present invention proposes a method for rapid preparation of 17β-estradiol IgG full-length antibodies and Fab antibodies in mammalian cells Expi293F. It is identified that its sensitivity is excellent and can be used for 17β-estradiol. Development of a diol immunoassay method.

The first object of the present invention is to provide an antibody against 17β-estradiol, the antibody comprising an IgG antibody heavy chain fragment and a light chain fragment, or a Fab heavy chain fragment and a light chain fragment.

In one embodiment, the nucleotide sequence of the IgG antibody heavy chain fragment is shown in SEQ ID NO.1, the nucleotide sequence of the light chain fragment is shown in SEQ ID NO.1, and the Fab The nucleotide sequence of the heavy chain fragment is shown in SEQ ID NO. 3.

In one embodiment, the N-termini of the IgG antibody heavy chain fragment, light chain fragment and Fab heavy chain fragment are all connected with a signal peptide.

In one embodiment, the signal peptide is a mouse-derived Ig heavy chain signal peptide,

In one embodiment, the amino acid sequence of the mouse-derived Ig heavy chain signal peptide is shown in SEQ ID NO. 4.

The second object of the present invention is to provide a recombinant vector containing a gene encoding the above-mentioned antibody.

In one embodiment, pTT5, pcDNA3.4 or pCEP4 is used as the expression vector.

In one embodiment, pTT5 is used as the expression vector.

The third object of the present invention is to provide cells containing genes encoding the above-mentioned antibodies or the above-mentioned recombinant vectors.

In one embodiment, the cell is a mammalian cell line.

In one embodiment, the cells are Expi293F cells.

The fourth object of the present invention is to provide a kit for detecting 17β-estradiol.

In one embodiment, the kit contains the above-mentioned antibody or the above-mentioned recombinant vector or the above-mentioned cell.

In one embodiment, the kit also contains a 17β-estradiol standard.

The fifth object of the present invention is to provide the application of the above-mentioned antibody or the above-mentioned recombinant vector or the above-mentioned cell in detecting 17β-estradiol, which application is not for the purpose of diagnosis and/or treatment of disease.

In one embodiment, the application is the preparation of a kit or reagent for detecting 17β-estradiol.

In one embodiment, the kit or reagent is used for ELISA detection of 17β-estradiol.

Beneficial effects:

1. The method provided by the invention to quickly obtain 17β-estradiol full-length IgG antibodies and Fab antibodies is to construct heavy and light chain expression vectors respectively, and co-transfect the heavy and light chain expression vectors into Expi293 according to a 2:3 mass ratio. The cells were purified using a Ni affinity column after 7 days to achieve expression and purification of IgG and Fab antibodies. SDS-PAGE verified that the molecular weight of the light chain and heavy chain of the expressed IgG full-length antibody was around 25 and 50KDa; the molecular weight of the heavy chain and light chain of the Fab antibody was around 27 and 25KDa. Based on the obtained IgG antibodies and Fab antibodies, a competitive ELISA method for detecting 17β-estradiol was constructed to achieve highly sensitive detection of 17β-estradiol. The IC ₅₀ of competitive ELISA based on IgG antibody and Fab antibody to detect 17β-estradiol are: 0.129ng/mL and 0.188ng/mL respectively.

2. The present invention compared the effects on the expression yield of 17β-estradiol IgG full-length antibody based on three different eukaryotic expression vectors pTT5, pcDNA3.4, and pCEP4. The expression yields are 27 mg/L, 7 mg/L, and 0.667 mg/L respectively. Therefore, pTT5 is more suitable as a eukaryotic expression vector for 17β-estradiol IgG full-length antibody.

3. The method of obtaining IgG and Fab antibodies in the present invention has an expression period of seven days. The antibodies obtained after purification have good affinity and can be used to identify elements in immune analysis.

3. The recombinant 17β-estradiol antibody expressed in the present invention can be genetically modified according to experimental purposes and is widely used in the rapid preparation of antibodies against other targets.

Description of drawings

In order to more clearly illustrate the specific solutions and technologies in the present invention, the technologies used in the patent and the results will be introduced below with the accompanying drawings.

Figure 1 shows the design of the heavy/light chain recombinant expression vector of 17β estradiol in the present invention;

Figure 2 is an agarose electrophoresis diagram of double enzyme digestion of plasmids pTT5-IgG H, pTT5-IgG L, and pTT5-Fab H in the present invention;

Figure 3 is an SDS-PAGE diagram of the purification process of 17βestradiol IgG antibodies and Fab antibodies expressed based on pTT5 expression vector in the present invention;

Figure 4 is an SDS-PAGE diagram of the purification process of the 17β estradiol IgG antibody expressed based on pcDNA3.4 and pCEP4 vectors in the present invention;

Figure 5 is a competitive ELISA standard curve constructed based on IgG antibodies and Fab antibodies in the present invention.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. pTT5, pcDNA3.4, and pCEP4 vectors are all commercial vectors.

LB plate medium (Amp+): yeast powder 5.0g/L, tryptone 10.0g/L, NaCl 10.0g/L, agar powder 15g/L, ampicillin 100μg/L.

LB liquid medium: yeast powder 5.0g/L, tryptone 10.0g/L, NaCl 10.0g/L.

Example 1: Antibody expression plasmid construction

This example provides a method to construct pTT5-IgG H, pTT5-IgG L and pTT5-Fab H (Figure 1), including the following steps:

(1) Use pTT5 as a vector to construct heavy chain expression plasmid, light chain expression plasmid and antibody Fab heavy chain fragment expression plasmid

The heavy chain fragment and light chain fragment of the 17β-estradiol antibody carrying the mouse Ig heavy chain signal peptide at the N-terminus were constructed between the EcoRI and BamHI digestion sites of the pTT5 vector, and the N-terminus carrying the mouse Ig heavy chain signal peptide was constructed into the pTT5 vector. The Fab heavy chain fragment of the chain signal peptide was constructed between the BamHI and XholI restriction sites of the pTT5 vector to obtain pTT5-IgG H, pTT5-IgG L, and pTT5-Fab H expression plasmids.

The glycerol bacteria expressing the three expression plasmids pTT5-IgG H, pTT5-IgG L and pTT5-Fab H were streaked into LB plate medium (Amp+) respectively and cultured at 37°C overnight. Pick a single colony from the plate and culture it in LB liquid medium at 37°C and 220 rpm for 12 hours. Use the endotoxin-free plasmid miniprep kit and follow the instructions to extract the plasmid, sterilize it with a 0.22 μm filter, and measure the plasmid concentration with Nanodrop. Take 10 μL and use specific endonucleases EcoRⅠ and BamHI or BamHI and XholⅠ to double-digest the plasmid to verify. The results in the agarose electrophoresis chart (Figure 2) are consistent with the theoretical band sizes, which preliminarily confirms that the plasmid was successfully constructed.

(2) Use pcDNA3.4 or pCEP4 as vector to construct heavy chain expression plasmid and light chain expression plasmid

Using the pTT5-IgG H and pTT5-IgG L expression plasmids constructed in step (1) as templates, the expression vectors were replaced with pcDNA3.4 and pCEP4 through seamless cloning. The seamless cloning primers are shown in Table 1. Select positive clones and send them to Sangon for sequencing to confirm that the recombinant expression vector plasmids pcDNA3.4-IgG H, pcDNA3.4-IgG L, pCEP4-IgG H and pCEP4-IgG L have no base mutations and can be used for subsequent transfection expression. .

Table 1 Primers for seamless cloning

Base sequence of estradiol antibody:

IgG heavy chain DNA sequence (SEQ ID NO.1):

GAGGTGCAGCTGCAGCAGAGTGGAGCTGAACTGGCCAGACCTGGTGCCTCTGTCAAGTTGTCTTGCCGCACGTCCGGATACAGCTTTACAACCTACTGGATGCAGTGGGTTAGACAAAGACCAGGTCAAGGCCTGGAGTGGATTGCCGCTATATACCCCGGAGACGACGATGCAAGGTACACACAAAAATTTAAAGGCAAAGCTACCCTCACCGCAGACAGGAGCTCTTCCATTGTGTACCTCCAGCTCAACTCTCTGA CAAGTGAGGACAGCGCAGTGTATAGCTGCAGCAGGACGCAGTCTCTATTACACAATGGATTACTGGGGGCAGGGGTACTTCTGTGACTGTGAGCTCTGCGAAGACCACTCCTCCTTCCGTCTATCCACTTGCCCCAGGATCTGCCGCCCAGACAAATTCAATGGTTACCCTCGGGTGTCTGGTTAAAGGATACTTTCCGGAGCCTGTGACCGTGAGCTGGAACACAGGCTCATTGAGTAGCGGGGTTTCACACTTTCCCGGCGGT TCTGCAGAGTGACTTGTACACATTGAGCAGCAGCGTGACCGTTCCCTCCTCAACATG GCCATCTGAGACTGTGACGTGTAATGTGGCACACCCAGCATCCAGCACTAAAGTGGACAAAAAAATCGTGCCCCGAGATTGTGGCTGCAAGCCATGCATTTGCACCGTGCCCGAGGTGAGCAGTGTGTTCATCTTTCCTCCGAAGCCTAAGGACGTCCTGACAATTACTCTGACCCCTAAGGTCACCTGCGTGGTCGTCGACATTT CTAAAGATGATCCTGAGGTTCAGTTCTCATGGTTCGTCGACGACGTGGAGGTACATACGGCCCAGACCCAGCCACGGGAGGAACAATTTAACTCCACTTTCCGGTCTGTGTCTGAATTGCCAATCATGCATCAGGACTGGCTGAACGGCAAGGAGTTCAAGTGTAGGGTGAACTCAGCTGCCTTCCCTGCGCCAATTGAGAAGACCATCTCTAAGACTAAGGGAAGACCCAAAGCGCCTCAGGTGTATACTATCCCCCCGCCCAAA GAGCAGATGGCCAAAGACAAAGTCTCTCTGACCTGCATGATCACCGACTTCTTTCCCGAAGACATAACAGTTGAGTGGCAATGGAACGGCCAGCCCGCCGAAAACTATAAAAACACACAGCCGATAATGAACACAATGGAAGCTACTTCGTCTATAGCAAGCTGAATGTACAGAAGTCTAACTGGGAAGCTGGGAACACCTTCACCTGCTCCGTGCTGCATGAAGGCCTTCACAATCACCATACAGAGAAGAGCTTGAGTCACTCCCC GGCAAA

IgG light chain DNA sequence (SEQ ID NO.2):

CAGGCAGTGGTTACTCAGGAGTCAGCATGACAACATCCCCTGGAGAGACTGTGACTCTTACCTGCCGGTCTAGCAGCGGGGCAATCACCACATCACACTATGCAAACTGGATTCAGGAGAAACCTGACCACCTGTTCACCGGACTTATCTCAGGGACAAATAATAGAGCCCCAGGGGTGCCCGCAAGGTTTAGTGGATCACTGATCGGCGACAAGGCCGCTCTCTGACTATCACCGGTGCTCAGACCGAGGATGAGGCCATTT ATATCTGTGCCCTCTGGTTTAGTAATCAGTTTATTTTTGGCTCTGGAACTAAGGTGACCGTCCTGGGGCAGCCGAAGTCAAGTCCAAGCGTGACGCTGTTTCCTCCGAGCAGCGAGGAGCTTGAGACAAACAAAGCCACACTCGTCTGCACCATCACCGATTTCTACCCGGGCGTGGTGACCGTCGATTGGAAAGTGGACGGAACCCCCGTGACTCAGGGGATGGAGACGACACAGCCTTCCAAGCAGTCCAACAAGTATA TGGCCAGTAGTTACCTTACTCTGACGGCCAGAGCTTGGGAGCGGCATAGTTCCTACTCTTGCCAGGTGACACATGAGGGACATACAGTGGAAAAAAGCCTGTCTCCCGCAGAATGTCTGTGA

Fab heavy chain DNA sequence (SEQ ID NO.3):

GAGGTGCAGCTGCAGCAGAGTGGAGCTGAACTGGCCAGACCTGGTGCCTCTGTCAAGTTGTCTTGCCGCACGTCCGGATACAGCTTTACAACCTACTGGATGCAGTGGGTTAGACAAAGACCAGGTCAAGGCCTGGAGTGGATTGCCGCTATATACCCCGGAGACGACGATGCAAGGTACACACAAAAATTTAAAGGCAAAGCTACCCTCACCGCAGACAGGAGCTCTTCCATTGTGTACCTCCAGCTCAACTCTCTGA CAAGTGAGGACAGCGCAGTGTATAGCTGCAGCAGGACGCAGTCTCTATTACACAATGGATTACTGGGGGCAGGGGTACTTCTGTGACTGTGAGCTCTGCGAAGACCACTCCTCCTTCCGTCTATCCACTTGCCCCAGGATCTGCC GCCCAGACAAATTCAATGGTTACCCTCGGGTGTCTGGTTAAAGGATACTTTCCGGAGCCTGTGACCGTGAGCTGGAACACAGGCTCATTGAGTAGCGGGGTTTCACACTTTCCCGGCG GTTCTGCAGAGTGACTTGTACACATTGAGCAGCAGCGTGACCGTTCCCTCCTCAACATGGCCATCTGAGACTGTGACGTGTAATGTGGCACACCCAGCATCCAGCACTAAAGTGGACAAAAAAATCGTGCCCCGAGATTGT

Example 2: Expression of antibody IgG and Fab in Expi293F cells

(1) Recovery of Expi293F cells

Take 30mL of Expi293F ^TM expression medium (Gibco), preheat it in a 37°C water bath, and then transfer it to a 125mL disposable cell culture bottle. Take one Expi293F cell out of the liquid nitrogen, immediately place it in a 37°C water bath, shake while melting, sterilize with alcohol, transfer it to the above 125mL cell culture flask with a pipette, and incubate at 37°C, 8% CO ₂ , 125 rpm Cell culture shaker culture.

(2) Transfection of recombinant plasmid

When the cell number reaches approximately 3×10 ⁶ cells/mL, centrifuge at 350g for 5 minutes and replace with fresh Expi293F ^TM expression medium for passage. After three passages, adjust the cell density to 2×10 ⁶ cells/mL and verify that the cell viability reaches more than 95% and prepare for transfection. Mix 30 μg of the plasmid constructed in Example 1 with 1.5 mL of Opti-MEMⅠ serum-free medium to obtain a mixture ①, in which the IgG and Fab antibody expression plasmids are respectively mixed with a heavy chain expression plasmid and a light chain expression plasmid in a mass ratio of 2:3 The total amount is 30μg. Mix 81 μL of cationic liposome transfection reagent with 1.5 mL of Opti-MEMⅠ serum-free medium, and incubate at room temperature for 5 minutes to obtain mixed solution ②. Mix the mixture ② into the mixture ① and let it stand at room temperature for 20 minutes to obtain the plasmid-liposome complex. Then add it dropwise to the cells to be transfected, shaking the culture bottle while dropping. The transfected cell culture was cultured in a culture shaker at 37°C, 125 rpm, 8% CO ₂ , and a relative humidity greater than 80%. 18-22 hours after transfection, mix 150 μL transfection enhancer I and 1.5 mL enhancer II, add cells, and shake the culture flask gently during the addition process. Place the culture flask back into the incubator for 7 days and collect the cell culture.

(3) Purification of IgG and Fab antibodies

Transfer the cell culture from step (2) to a 50 mL centrifuge tube, centrifuge at 4°C and 3200 g for 20 min, collect the supernatant, and filter with a sterile 0.22 μm filter membrane. Purify the supernatant with His-tagged protein purification kit. The specific steps are as follows:

①Put 2mL of His tag purification resin (Ni filler) into the empty affinity chromatography column;

②Add 10 times the column volume of ultrapure water to rinse the affinity column;

③ Add the filtered supernatant to the affinity column, collect the flow-through liquid, and cycle the sample 3 times;

④Add 3 times the column volume of non-denaturing washing solution to wash away impure proteins;

⑤ Add non-denaturing eluent to elute the target protein, collect the flow-through solution at 1 mL per tube, and elute a total of 5-6 mL. Temporarily store the collected protein eluate in an ice bath;

⑥Rinse the column with 3 times the column volume of ultrapure water, fill it with 10% ethanol-ultrapure water, seal both ends of the column tightly, and store at 4°C;

⑦Elute the target protein and stir and dialyze it in PBS at 4℃ for 48 hours. Collect and measure the concentration, aliquot and store at -20℃.

(4) SDS-PAGE verification of IgG and Fab antibody expression products

Take 10 μL of samples from each stage of the above purification process, add protein loading buffer, mix well, and heat at 95°C for 10 minutes to load the sample. Prepare a 10% concentration SDS-PAGE protein gel, load the sample, run the gel, end the electrophoresis, stain with Coomassie Brilliant Blue, and destain with 10% acetic acid-water. Clear protein bands are observed as shown in Figures 3 and 4, with full-length IgG. The molecular weight of the light chain and heavy chain of the antibody is around 25 and 50KDa; the molecular weight of the light chain and heavy chain of the Fab antibody is around 25 and 27KDa.

The results showed that using pTT5, pcDNA3.4, and pCEP4 as vectors respectively, the expression yields of full-length IgG antibodies were 27 mg/L, 7 mg/L, and 0.667 mg/L respectively.

Example 3: ELISA verification of estradiol IgG and Fab antibodies

Checkerboard titration method was used to optimize the concentration of estradiol-coated antigen E ₂ -BSA and antibody, and the OD value corresponding to the antigen and antibody concentration in the range of 1-1.5 (both 1 μg/mL) was used as the optimized concentration of E ₂ -BSA and IgG.

(1) Coat E ₂ -BSA in the wells of the enzyme plate at a concentration of 1 μg/mL, 100 μl/well, and keep overnight at 4°C;

(2) Discard the supernatant, wash, and block with 300 μL/well of 5% skimmed milk powder at 37°C for 2 hours;

(3) Discard the supernatant, wash 3-5 times with washing buffer (10mM PBST containing 0.5% v/v Tween-20, pH 7.4), and add 50 μL of _E2 standards of different concentrations (diluted in 10% methanol-PBS) Mix with 50 μL of the purified antibody in Example 2 (2 μg/mL, IgG antibody or Fab antibody), add it to the plate well, and incubate at 37°C for 1 hour;

(4) Discard the supernatant and wash 3-5 times with washing buffer. After washing, incubate with 100 μL/well anti-His-HRP enzyme-labeled secondary antibody and incubate at 37°C for 0.5 h;

(5) Discard the supernatant and wash 3-5 times with washing buffer. After washing, add 100 μL/well of TMB for color development reaction;

(6) Terminate the reaction with 50 μL/well of 2M sulfuric acid, and detect the OD450 nm absorbance value with a microplate reader.

Taking the logarithm of estradiol concentration as the abscissa and the inhibition rate B/B ₀ as the ordinate, construct an estradiol detection standard curve within the _E2 concentration range of 0.005ng/mL-100ng/mL, as shown in Figure 5 , the IC ₅₀ of the estradiol detection standard curve for IgG antibody and Fab antibody are 0.129ng/mL and 0.188ng/mL respectively. Therefore, the use of mammalian transient expression system to obtain estradiol antibody immune elements can be used for the construction of estradiol immunodetection methods.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (3)

1. A method for preparing an antibody to 17β-estradiol, characterized in that the antibody contains a heavy chain fragment and a light chain fragment; pTT5 is used as an expression vector to construct a plasmid expressing a heavy chain or a light chain respectively, and The plasmid expressing the heavy chain and the plasmid expressing the light chain are co-transfected into Expi293F cells at a mass ratio of 2:3, and the IgG antibody composed of the self-assembly of the heavy chain and the light chain is purified; The nucleotide sequence of the heavy chain fragment is shown in SEQ ID NO.1, and the nucleotide sequence of the light chain fragment is shown in SEQ ID NO.2. 2. The method according to claim 1, characterized in that a signal peptide is connected to the N-terminus of the heavy chain fragment and the light chain fragment. 3. The method according to claim 2, wherein the signal peptide is an Ig heavy chain signal peptide derived from mice, and the amino acid sequence is as shown in SEQ ID NO. 4.