CN114437213A

CN114437213A - Monoclonal antibody of resisting fluogesterone acetate and application thereof

Info

Publication number: CN114437213A
Application number: CN202210376454.3A
Authority: CN
Inventors: 魏单平; 吴迪; 赵荣茂; 王继圣; 李亚洲
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-05-06
Anticipated expiration: 2042-04-12
Also published as: CN114437213B

Abstract

The invention discloses a monoclonal antibody of resisting fluoprogesterone acetate and application thereof. The monoclonal antibody or antigen binding portion contains V_HAnd V is a heavy chain variable region_LLight chain variable region, said V_HAnd V_LBoth consist of an antigenic determinant complementary region and a framework region; the V is_HThe amino acid sequence of CDR1 of (1) is shown in positions 50-56 of SEQ ID No. 1; the V is_HThe amino acid sequence of CDR2 of (1) is shown in positions 76-92 of SEQ ID No. 1; what is needed isV is_HThe amino acid sequence of CDR3 is shown in position 125-134 of SEQ ID No. 1; the V is_LThe amino acid sequence of CDR1 of (1) is shown in positions 60-76 of SEQ ID No. 2; the V is_LThe amino acid sequence of CDR2 of (1) is shown in positions 92-98 of SEQ ID No. 2; the V is_LThe amino acid sequence of CDR3 is shown in position 131 and 138 of SEQ ID No. 2. The monoclonal antibody of the invention has the characteristics of strong specificity, high sensitivity and the like, and can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay.

Description

Monoclonal antibody of resisting fluogesterone acetate and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody for resisting fluogesterone acetate and application thereof.

Background

Fluoroprogesterone acetate (FGA for short, CAS number 2529-45-5) with molecular formula C₂₃H₃₁FO₅The steroid medicine has no smell, crystallization, melting point of 267.5 ℃, and is an artificially synthesized steroid medicine. The fluogesterone acetate is mainly used as a progestational drug clinically, is used for fertility regulation of animals, particularly sheep, has glucocorticoid drug activity, and has the functions of improving feed conversion rate and promoting growth of livestock and poultry. After the fluogesterone acetate enters the animal body, most of the fluogesterone acetate can be metabolized and discharged out of the body, and a small part of the fluogesterone acetate can be left in the tissues of the animal as the original medicine or metabolic residues. The hormone contained in the excrement causes environmental pollution, and the animal tissues are harmful to human beings after being eaten by the human beings.

The use of the fluoprogesterone acetate is strictly limited at home and abroad, and the European Union stipulates that the maximum residual quantity of the fluoprogesterone acetate in sheep's Yang and goat's milk is 1 mug/kg, and the maximum residual quantity of the fluoprogesterone acetate in muscles, fat, liver and kidney is 0.5 mug/kg. At present, the fluogesterone acetate is detected by adopting a High Performance Liquid Chromatography (HPLC), a liquid chromatography-mass spectrometry combined method (LCMS), a gas chromatography-mass spectrometry combined method (GC-MS) and the like. The quantitative analysis by the instrument method has lower detection limit, but the instrument operation is complex, the cost is high, and the requirement of rapid detection on site scale cannot be met.

Disclosure of Invention

Therefore, the invention provides a monoclonal antibody of resisting the fluoprogesterone acetate and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

the present embodiments provide monoclonal antibodies or antigen-binding portions thereof against fluogesterone acetate,the monoclonal antibody or antigen binding portion contains V_HAnd V is a heavy chain variable region_LLight chain variable region, said V_HAnd V_LBoth consist of an antigenic determinant complementary region and a framework region;

the V is_HThe amino acid sequence of CDR1 of (1) is shown in positions 50-56 of SEQ ID No. 1;

the V is_HThe amino acid sequence of CDR2 of (1) is shown in positions 76-92 of SEQ ID No. 1;

the V is_HThe amino acid sequence of CDR3 is shown in position 125-134 of SEQ ID No. 1;

the V is_LThe amino acid sequence of CDR1 of (1) is shown in positions 60-76 of SEQ ID No. 2;

the V is_LThe amino acid sequence of CDR2 of (1) is shown in positions 92-98 of SEQ ID No. 2;

the V is_LThe amino acid sequence of CDR3 is shown in position 131 and 138 of SEQ ID No. 2.

In one embodiment of the invention, the framework region is derived from a mouse.

In one embodiment of the present invention, said V_HThe amino acid sequence of (A) is shown as SEQ ID No. 1;

the V is_LThe amino acid sequence of (A) is shown in SEQ ID No. 2.

In one embodiment of the invention, the monoclonal antibody is a murine monoclonal antibody.

In one embodiment of the present invention, the antibody is any one of the following:

(a) v according to any one of the above_HAnd V as described in any of the above_LLinking the obtained single-chain antibody;

(b) a fusion antibody comprising the single chain antibody of (a);

(c) comprising a V as defined in any of the above_HAnd V as described in any of the above_LThe intact antibody of (a);

(d) monoclonal antibodies produced by hybridoma cell lines.

The embodiment of the present invention further provides a biomaterial related to the monoclonal antibody or the antigen binding portion thereof, wherein the biomaterial is any one of the following:

(1) a nucleic acid molecule encoding any of the monoclonal antibodies described above, or an antigen binding portion thereof;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1);

(4) a recombinant vector comprising the expression cassette of (2);

(5) a transgenic animal cell line comprising the nucleic acid molecule of (1);

(6) a microorganism containing the nucleic acid molecule of (1).

In one embodiment of the present invention, the nucleic acid molecule according to (1) above is a gene encoding any of the monoclonal antibodies described above or an antigen-binding portion thereof.

In one embodiment of the present invention, the gene is a DNA molecule as described in the following (A) or (B):

(A) the V is_HThe coding sequence of CDR1 is shown in position 148 and 168 of SEQ ID No.3,

the V is_HThe coding sequence of CDR2 is shown in position 226-276 of SEQ ID No.3,

the V is_HThe coding sequence of CDR3 is shown in position 373-402 of SEQ ID No.3,

the V is_LThe coding sequence of CDR1 is shown in position 178-228 of SEQ ID No.4,

the V is_LThe coding sequence of CDR2 is shown in position 274-294 of SEQ ID No.4,

the V is_LThe coding sequence of CDR3 of SEQ ID No.4 is shown in position 391-414;

(B) a DNA having 90% or more identity to the DNA molecule defined in (A) and encoding the monoclonal antibody or an antigen-binding portion thereof.

The embodiment of the invention also provides application of the monoclonal antibody or the antigen binding part thereof or the biological material in preparation of a reagent or a kit for detecting the fluogesterone acetate.

The invention has the following advantages:

the monoclonal antibody provided by the invention has higher affinity and detection sensitivity to the fluoprogesterone acetate, and lays a foundation for research and development and popularization of indirect competitive ELISA kits and colloidal gold test strips.

The test proves that: the monoclonal antibody of the invention has the characteristics of strong specificity, high sensitivity and the like, and can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a homology alignment diagram of a light chain gene sequence of a monoclonal antibody against fluogesterone acetate provided by the embodiment of the present invention;

FIG. 2 is a comparison chart of amino acid sequence homology of a monoclonal antibody against fluogesterone acetate provided by the embodiment of the present invention;

FIG. 3 is a homology alignment chart of the gene sequence of the heavy chain of monoclonal antibody against fluogesterone acetate provided by the embodiment of the invention;

FIG. 4 is a comparison chart of amino acid sequence homology of a heavy chain of a monoclonal antibody against fluogesterone acetate provided by the embodiment of the invention;

FIG. 5 shows the sensitivity and specificity of monoclonal antibody for detecting anti-fluogestone acetate by indirect competitive ELISA method according to the present inventionSexual RIDA SOFT four parameter method R²A graph;

FIG. 6 shows the sensitivity and specificity of the monoclonal antibody against fluogesterone acetate by indirect competitive ELISA method, RIDA SOFT four-parameter method IC₅₀A graph;

fig. 7 is a schematic structural diagram of a test strip for detecting fluogesterone acetate according to an embodiment of the present invention;

in the figure: 100-sample absorbent pad; 200-conjugate release pad; 300-nitrocellulose membrane; 310-a detection line; 320-quality control line; 400-absorbent pad; 500-bottom plate.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of monoclonal antibody against Fluoroprogesterone acetate

This example provides a method for preparing a monoclonal antibody against fluogesterone acetate, which comprises the following steps:

1. primary immunization

Emulsifying and emulsifying the fluogesterone acetate artificial antigen Freund complete adjuvant (1: 1), and injecting 6-week-old Balb/c mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd.) subcutaneously, wherein the immunization dose is 500 mu g/mouse; from the first immunization, the boosting immunization is carried out once every 4 weeks for 2 times, Freund's complete adjuvant is replaced by Freund's incomplete adjuvant, and the method and the dosage are the same as the first immunization; after 2 nd boosting immunization for one week, measuring the potency and inhibition in fundus venous blood sampling, when the potency is inhibited and reaches more than 1:10000, performing 1 impact immunization, namely injecting 0.5mL of immunogen solution into the abdominal cavity, taking splenocytes after three days, fusing with myeloma cells, and screening positive holes.

Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the fluoprogesterone acetate monoclonal antibody.

2. Preparation of monoclonal antibodies

Cell recovery: and taking out the cryo-preservation tube of the fluoprogesterone acetate monoclonal antibody hybridoma cell strain, immediately putting the cryo-preservation tube into a water bath at 37 ℃ for fast thawing, centrifuging to remove a frozen stock solution, and transferring the frozen stock solution into a culture bottle for culture.

Preparing ascites and purifying antibodies: injecting sterilized paraffin oil 0.8 mL/mouse (8 weeks old) into the abdominal cavity of Balb/c mouse by in vivo induction method, and injecting hybridoma cells 8 × 10 into the abdominal cavity 7 days later⁵Ascites were collected 10 days later.

Purifying by an octanoic acid-saturated ammonium sulfate method to obtain the monoclonal antibody of the fluogestone acetate.

Example 2 monoclonal antibody specificity and subclass identification against Fluoroprogesterone acetate

1. Flugesterone acetate monoclonal antibody sensitivity and specificity detection

The indirect competitive Elisa method is adopted to detect the sensitivity and specificity of the monoclonal antibody, and the results are shown in the following table 1. The abscissa of each concentration (0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/ml) of the fluoprogesterone acetate corresponds to the OD of each concentration of the fluoprogesterone acetate_450nmThe values are plotted on the ordinate, and the median Inhibitory Concentration (IC) is calculated using a RIDA SOFT four-parameter method to plot a standard curve₅₀) As shown in fig. 5 and 6.

The results show that the four-parameter method is used to fit a standard curve, curve R²=0.9999, using a Splinen fitting, IC₅₀=0.343ng/ml, the detection limit of the method is 0.1 ng/ml;

the subclass of the fluogesterone acetate monoclonal antibody is detected by using an IgG subclass detection kit (Sigma company in the United states), and the result shows that the fluogesterone acetate monoclonal antibody is IgG_1（κ）。

2. Monoclonal antibody light chain and heavy chain variable region gene clone for resisting fluogestone acetate

(1) Hybridoma cell culture and total RNA extraction

Hybridoma cells were cultured in RPMI 1640 complete medium at 37 ℃ with 5% carbon dioxide (Becton Dickinson) at 1X 10⁷. Total RNA of cells is extracted by a kit (purchased from Tiangen) for extracting total RNA of the cells.

(2) First Strand cDNA Synthesis

cDNA was synthesized by Takara inverted transcription kit (Japan).

(3) Gene amplification

Designing Lambda chain, Kappa chain, Heavy chain downstream primer and upstream universal primer.

Primer: AAGCAGTGGTATCAACGCAGA

R_κ:AACATTGATGTCTTTGGGGTAGAA

R_λ:AATCGTACACACCAGTGTGTGGG

R_H:AGGGATCCAGAGTTCCAGGT

PCR was carried out using the first strand of cDNA as a template, and the reaction system was 50. mu.l.

And (3) PCR reaction system: mu.l template, 1. mu.l dNTPs, 2.5. mu.l each of upstream and downstream primers (10. mu.M), 10. mu.l 5 XPCR buffer, ddH₂O30.5. mu.l, Taq enzyme (5. mu.l/. mu.l) 0.5. mu.l.

The PCR reaction conditions are as follows: 30s at 98 ℃; the temperature is lowered by 0.5 ℃ for each time at the temperature of 98 ℃ for 15s and at the temperature of 64 ℃ to 58 ℃ for 30s, and the circulation is carried out for 10 times; 30s at 72 ℃; 15 times of circulation at 98 ℃ for 15s, at 56 ℃ for 30s and at 72 ℃ for 30 s; 7min at 72 ℃.

(4) Cloning and screening of PCR amplification products

The PCR product was subjected to 1.5% agarose gel electrophoresis, the antibody Kappa chain, lambda chain and heavy chain fragments were recovered using a PCR product recovery kit (Beijing Tiangen), the fragments were inserted into pLB vector using pLB zero background quick cloning kit (Beijing Tiangen), transformed into DH5 alpha competent cells (ampicillin resistance), and screened for recombinant positive clone sequencing.

The PCR product was electrophoresed through 1.5% agarose gel, and the results showed that no band was found in the lambda strand, the heavy strand nucleotide sequence was shown in SEQ ID No.3, and the Kappa strand nucleotide sequence was shown in SEQ ID No. 4.

Wherein, V_HThe coding sequence of CDR1 such asV is shown at position 148 and 168 of SEQ ID No.3_HThe coding sequence of CDR2 is shown in position 226-276 of SEQ ID No.3, V_HThe coding sequence of CDR3 is shown in 373-402 th position of SEQ ID No.3, V_LThe coding sequence of CDR1 is shown in position 178-228 of SEQ ID No.4, V_LThe coding sequence of CDR2 is shown in position 274-294 of SEQ ID No.4, V_LThe coding sequence of CDR3 is shown in position 391-414 of SEQ ID No. 4.

(5) Variable region amino acid sequence and homology analysis

The results of comparison analysis in NCBI database showed that the monoclonal antibody light chain variable region gene has the highest homology with mouse immunoglobulin light chain variable region (Sequence ID: AF 321956.1), homology was 331/335, and percent homology was 99%, as shown in FIG. 1.

The amino acid Sequence of the light chain variable region of the monoclonal antibody against fluogesterone acetate has the highest homology with mouse mCG142167 (Sequence ID: EDK 98900.1), the homology is 120/122, and the homology percentage is 99%, as shown in FIG. 2.

The anti-fluoprogesterone acetate monoclonal antibody heavy chain variable region gene has the highest homology with mouse immunoglobulin heavy chain variable region (Sequence ID: AJ 851868.3), the homology is 296/326, and the homology is 91%, as shown in figure 3.

The amino acid Sequence of the heavy chain variable region of the monoclonal antibody against flurogestone acetate has the highest homology with the mouse immunoglobulin heavy chain (Sequence ID: AAO 21964.1), the homology is 112/124, and the homology percentage is 90%, as shown in FIG. 4. The results of homology analysis of the gene sequences and amino acid sequences encoding the variable regions of the light and heavy chains of the monoclonal antibody against fluogesterone acetate indicate that no sequences identical to those of the present invention are found. The light chain variable region and heavy chain variable region sequences were analyzed at https:// www.novopro.cn/tools/cdr.html to obtain the CDR regions.

The amino acid sequence of the heavy chain of the fluoprogesterone acetate monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of the Kappa chain is shown as SEQ ID No. 2.

Wherein, V_HThe amino acid sequence of CDR1 of (1) is shown in positions 50-56 of SEQ ID No. 1; v_HThe amino acid sequence of CDR2 of (1) is shown in positions 76-92 of SEQ ID No. 1; v_HThe amino acid sequence of CDR3 is shown in position 125-134 of SEQ ID No. 1.

V_LThe amino acid sequence of CDR1 of (1) is shown in positions 60-76 of SEQ ID No. 2; v_LThe amino acid sequence of CDR2 of (1) is shown in positions 92-98 of SEQ ID No. 2; v_LThe amino acid sequence of CDR3 is shown in position 131 and 138 of SEQ ID No. 2.

Example 3 preparation of Flugesterone acetate colloidal gold assay kit

As shown in fig. 7, the embodiment provides a fluogesterone acetate colloidal gold detection kit, which includes a detection test strip, and the detection test strip is composed of a bottom plate 500, a sample absorption pad 100, a conjugate release pad 200, a nitrocellulose membrane 300, a water absorption pad 400, and a card shell; wherein, the sample absorbing pad 100, the conjugate releasing pad 200, the nitrocellulose membrane 300 and the absorbent pad 400 are sequentially adhered on the PS base plate 500; the conjugate release pad 200 is covered with the sample absorbent pad 100 from the beginning with an area 1/4, the end of the conjugate release pad 200 is connected to the beginning of the nitrocellulose membrane 300, the end of the nitrocellulose membrane 300 is connected to the beginning of the absorbent pad 400, the beginning of the sample absorbent pad 100 is aligned with the beginning of the PS chassis 500, and the end of the absorbent pad 400 is aligned with the end of the PS chassis 500.

The nitrocellulose membrane 300 is provided with a detection line 310 and a quality control line 320, and the detection line T and the quality control line C are both strip-shaped strips which are vertical to the long phase of the test strip; detection line 310 is located on the side near the end of conjugate release pad 200; the control wire 320 is located on the side away from the end of the conjugate release pad 200. Cutting the test strip into small strips with the width of 4.05 mm by a machine, putting the small strips into a special plastic card shell, sealing the card shell by an aluminum foil bag, and storing the card shell for 12 months at the temperature of 2-30 ℃.

The preparation method of the fluogesterone acetate colloidal gold test strip mainly comprises the following steps:

1) preparing a nitrocellulose membrane with a detection line of a fluoprogesterone acetate artificial antigen and a quality control line coated with a goat anti-mouse antibody;

2) preparing a conjugate release pad sprayed with a colloidal gold-labeled fluoprogesterone acetate monoclonal antibody;

3) the sample absorbing pad, the conjugate releasing pad, the nitrocellulose membrane and the water absorbing pad are fixed on the bottom plate in sequence.

4) And packaging into a card shell.

Defining the detection limit of the test paper card: using goat milk completely not containing the fluoprogesterone acetate as a negative sample, preparing a sample 1 to be detected (fluoprogesterone acetate 0.1 ppb), a sample 2 to be detected (fluoprogesterone acetate 0.05 ppb) and a sample 3 to be detected (fluoprogesterone acetate 0 ppb). And sucking 100 muL of sample solution to be detected into the shell clamping hole by using a micropipettor, and judging the result after reacting for 10 minutes at room temperature, wherein the time is not more than 20 minutes.

And (4) judging a result:

negative (-): both line C and line T are developed, and the color development of line T is far stronger than that of line C, which indicates that the sample does not contain the fluoprogesterone acetate or is far lower than the detection limit.

Positive (+) position: c, developing color; the color development of the T line is the same as that of the C line, the color development of the T line is weaker than that of the C line or the T line does not develop, and the T line and the C line both indicate that the concentration of the fluoprogesterone acetate in the sample is equal to or higher than the detection limit.

And (4) invalidation: no C-line appears indicating an incorrect operating procedure or a test card has deteriorated and failed.

The results show that: the sample 1 to be tested (0.1 ppb of fluoprogesterone acetate) is positive, the sample 2 to be tested (0.05 ppb of fluoprogesterone acetate) is negative, and the sample 3 to be tested (0 ppb of fluoprogesterone acetate) is negative. The results show a test card detection limit of 0.1 ppb.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing Nabai Biotechnology Ltd

<120> monoclonal antibody of resisting fluoprogesterone acetate and application thereof

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Gly Ala Pro Gly Gly Gly Pro Leu Thr Met Gly Thr Ile Ala Thr Ala

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Leu Ala Gly Ala Thr Ala Thr Thr Pro Cys Ala Ala Gly Ala Gly Ser

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gggggttacg cagagtacat ggggaaggga gtgaccagtt agtcttaagg ccccattgag 60

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tcacagtctc catcctccct ggctgtgtca gtaggagaga aggtcactat gaactgcaaa 180

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cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcagtgtg 360

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Claims

1. A monoclonal antibody or antigen-binding portion thereof against flurogestone acetate, wherein said monoclonal antibody or antigen-binding portion comprises V_HAnd V is a heavy chain variable region_LLight chain variable region, said V_HAnd V_LBoth consist of an antigenic determinant complementary region and a framework region;

2. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the framework region is derived from a mouse.

3. The monoclonal antibody, or antigen binding portion thereof, of claim 1,

the V is_HThe amino acid sequence of (A) is shown as SEQ ID No. 1;

the V is_LThe amino acid sequence of (A) is shown in SEQ ID No. 2.

4. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the monoclonal antibody is a murine monoclonal antibody.

5. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the antibody is any one of:

(a) v according to any one of claims 1 to 4_HAnd V according to any one of claims 1 to 4_LLinking the obtained single-chain antibody;

(b) a fusion antibody comprising the single chain antibody of (a);

(c) comprising V according to any one of claims 1 to 4_HAnd V according to any one of claims 1 to 4_LThe intact antibody of (a);

(d) monoclonal antibodies produced by hybridoma cell lines.

6. A biological material associated with the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1-5, which is any one of:

(1) a nucleic acid molecule encoding the monoclonal antibody or antigen binding portion thereof of any one of claims 1-5;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1);

(4) a recombinant vector comprising the expression cassette of (2);

(5) a transgenic animal cell line comprising the nucleic acid molecule of (1);

(6) a microorganism containing the nucleic acid molecule of (1).

7. The biomaterial of claim 6, wherein the nucleic acid molecule of (1) is a gene encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-5.

8. The biomaterial according to claim 7, wherein the gene is a DNA molecule according to the following (A) or (B):

9. Use of a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-5, or a biological material according to any one of claims 6-8 in the manufacture of a reagent or kit for detecting fluoprogesterone acetate.