CN116041511A

CN116041511A - ACTH recombinant rabbit monoclonal antibody, preparation method and application thereof

Info

Publication number: CN116041511A
Application number: CN202310142820.3A
Authority: CN
Inventors: 董艳娜; 王桂芳; 柴丹丹; 韩阳瑞; 田晓平; 赵巧辉; 李桂林; 付光宇; 杨增利
Original assignee: Zhengzhou Immuno Biotech Co Ltd
Current assignee: Zhengzhou Immuno Biotech Co Ltd
Priority date: 2023-02-21
Filing date: 2023-02-21
Publication date: 2023-05-02

Abstract

The invention relates to the technical field of antibodies, in particular to an ACTH recombinant rabbit monoclonal antibody, a preparation method and application thereof. The ACTH recombinant rabbit monoclonal antibody is obtained by screening by utilizing phage display technology. The monoclonal antibody has strong specificity, high detection sensitivity and good affinity, and solves the problems of low content of adrenocorticotropic hormone in a sample and high detection difficulty. Through identification, repeated detection is carried out by using the monoclonal antibody, so that the stability is good and the accuracy is high.

Description

ACTH recombinant rabbit monoclonal antibody, preparation method and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an ACTH recombinant rabbit monoclonal antibody, a preparation method and application thereof.

Background

Adrenocorticotropic hormone (ACTH) is one of the important hormones secreted by the hypothalamic pituitary, and ACTH secretion is affected by feedback of the hypothalamic hormone Corticotropin Releasing Factor (CRF) and cortisol. In primary adrenocortical insufficiency, the typical manifestation is an elevated ACTH level, whereas low levels of ACTH are often found in adrenocortical insufficiency secondary to pituitary dysfunction. ACTH is valuable for differential diagnosis of hypoadrenal function and hypersecretion, and secondary hypertension. ACTH detection also helps identify the cause of cortisol hypersecretion in cushing's syndrome, which is characterized by a decrease in ACTH levels when cortisol hypersecretion is caused by adrenocortical lesions or hyperplasia, and an increase in ACTH levels if ACTH is generated by pituitary ectogenesis or is caused by ACTH hypersecretion.

Hypertension is a common disease worldwide, frequently occurring in spring and winter. Hypertension can cause lesions of heart, kidney, cerebral vessels and other parts, and is a main disease endangering human health. Adrenal hyperplastic hypertension refers to an adrenal secondary hypertension whose pathological basis is adrenal hyperplasia, and the diagnosis needs to be based on the increase of adrenal hormone secretion.

Therefore, the preparation of the ACTH monoclonal antibody with strong specificity, high sensitivity and good affinity has important significance for improving the diagnosis rate of primary hypertension.

Disclosure of Invention

In view of the above, the technical problem to be solved by the invention is to provide an ACTH recombinant rabbit monoclonal antibody, a preparation method and application thereof, and the invention provides the ACTH recombinant rabbit monoclonal antibody with strong specificity, high sensitivity and good affinity, and the preparation method and application thereof.

The ACTH recombinant rabbit monoclonal antibody provided by the invention comprises the following amino acid sequences:

(1) The CDR region of the heavy chain comprises at least one of the amino acid sequences shown as SEQ ID NO. 1, 2 or 3 and the CDR region of the light chain comprises at least one of the amino acid sequences shown as SEQ ID NO. 4, 5 or 6; or (b)

(2) An amino acid sequence which is identical or similar to the amino acid sequence shown in the formula (1) in function with the amino acid shown in the formula I through one or more of substitution, addition or deletion of amino acids; or (b)

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in (1) or (2).

In some implementations, the invention provides ACTH recombinant rabbit monoclonal antibodies,

the amino acid sequence of the CDR region of the heavy chain comprises at least one of the sequences shown as SEQ ID NO. 1, 2 or 3;

the amino acid sequence of the CDR region of the light chain comprises at least one of the sequences shown as SEQ ID NO. 4, 5 or 6.

In the invention, the ACTH recombinant rabbit monoclonal antibody comprises the following components:

i, at least one of the four FR regions of its heavy chain has an amino acid sequence as shown in SEQ ID NOs 7,8, 9 and 10; at least one of the four FR regions of its light chain has the amino acid sequence shown in SEQ ID NOs 11, 12, 13 and 14.

II, amino acid sequence which is one or more than one amino acid in the amino acid sequence shown in I through substitution, addition or deletion, but has the same or similar function with the amino acid shown in I; or (b)

III, an amino acid sequence having at least 80% homology with the amino acid sequence shown in I or II.

In the present invention, the sequence having at least 80% homology is an amino acid sequence obtained by substituting, adding or deleting one or more amino acids based on the original sequence, wherein the plurality is 2, 3, 4, 5, 6, 7,8, 9 or 10.

In some embodiments, the amino acid sequences of the three CDR regions of the heavy chain of the ACTH recombinant rabbit monoclonal antibody are shown in SEQ ID NOs 1, 2 and 3 in sequence;

in some embodiments, the amino acid sequences of the three CDRs of the light chain of the ACTH recombinant rabbit monoclonal antibody are shown in SEQ ID NOs 4, 5 and 6 in sequence.

In some embodiments, the amino acid sequences of the four FR regions of the heavy chain of said ACTH recombinant rabbit monoclonal antibody are shown in SEQ ID NO. 7,8, 9 and 10;

in some embodiments, the amino acid sequences of the four FR regions of the light chain of said ACTH recombinant rabbit monoclonal antibody are shown in SEQ ID NOs 11, 12, 13 and 14 in sequence.

In some embodiments, the heavy chain variable region of the ACTH recombinant rabbit monoclonal antibody comprises the CDR regions shown in SEQ ID NOs 1, 2, or 3 and the FR regions shown in SEQ ID NOs 7,8, 9, and 10.

In an embodiment of the present invention, the light chain variable region of the monoclonal antibody comprises the CDR region shown in SEQ ID NO. 4, 5 or 6 and the FR region shown in SEQ ID NO. 11, 12, 13 and 14.

In some embodiments, the heavy chain variable region of the ACTH recombinant rabbit monoclonal antibody has an amino acid sequence as set forth in SEQ ID No. 15; the light chain variable region has the amino acid sequence shown in SEQ ID NO. 16.

The ACTH recombinant rabbit monoclonal antibody provided by the invention further comprises a constant region, wherein the constant region of the heavy chain is of rabbit IgG subtype; the constant region of the light chain is kappa 1.

The ACTH recombinant rabbit monoclonal antibody is screened and obtained by utilizing phage display technology, and has the advantages of strong specificity, high sensitivity and good affinity. Solves the problems of low content of corticotropin in the sample and high detection difficulty. Through identification, repeated detection is carried out by using the monoclonal antibody, so that the stability is good and the accuracy is high.

The present invention provides a biomaterial comprising at least one of the following:

1) Nucleic acid encoding said ACTH recombinant rabbit monoclonal antibody;

2) An expression vector comprising the nucleic acid;

3) Transforming or transfecting a host cell of the expression vector;

4) The conjugate is prepared by coupling the ACTH recombinant rabbit monoclonal antibody with a solid medium or a semisolid medium;

5) Chemically or biologically labeled said ACTH recombinant rabbit monoclonal antibody;

6) And the conjugate is prepared by coupling the ACTH recombinant rabbit monoclonal antibody subjected to chemical labeling or biological labeling with a solid medium or a semisolid medium.

The nucleic acid sequence of the antibody is not limited, and all nucleic acids which can code the antibody are within the protection scope of the invention. In some embodiments, the nucleotide has a nucleotide sequence that is functionally identical or similar to the nucleotide sequences shown in SEQ ID NO. 17 and SEQ ID NO. 18, obtained by substitution, deletion, or addition of one or more nucleotides to the nucleotide sequences shown in SEQ ID NO. 17 and SEQ ID NO. 18. More specifically, the nucleic acid comprises: the nucleic acid encoding the heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO. 17; the nucleic acid encoding the light chain variable region has the nucleotide sequence shown as SEQ ID NO. 18.

The invention provides a preparation method of an ACTH recombinant rabbit monoclonal antibody, which is characterized by comprising the following steps: culturing said host cell to induce expression of said ACTH recombinant rabbit monoclonal antibody.

In some embodiments, the expression vector is RGFc-PCMV3 or RCL-PCMV3.

In some embodiments, the host cell is selected from the group consisting of E.coli, yeast, insect cells, and mammalian cells. Preferably, the host cell is a mammalian HEK293 cell.

The chemical label is an isotope, an immunotoxin and/or a chemical drug; the biomarker is biotin, avidin or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, jellyfish root toxin, PAP, ostosis, gelonin or retinervus Luffae fructus toxin

The solid or semi-solid medium refers to any support to which the recombinant antibodies, labeled recombinant antibodies, of the present invention can be attached, including but not limited to nitrocellulose membranes, polyvinylidene difluoride (PVDF) membranes, iPDMS chips, microwell plates, polystyrene plates, microparticles, microcarriers, gels, and the like.

The invention relates to an application of an ACTH recombinant rabbit monoclonal antibody, a biological material and/or an ACTH recombinant rabbit monoclonal antibody expressed by a host cell in preparation of a reagent or a kit for detecting diseases related to blood pressure.

In some specific embodiments, the index for detecting a hypertension-related disorder is detecting corticotropin levels.

Experiments show that the ACTH recombinant rabbit monoclonal antibody provided by the invention can be used as a detection antibody for detecting corticotropin, and has good sensitivity. The ACTH recombinant rabbit monoclonal antibody provided by the invention is suitable for immunological detection of corticotropin. In some embodiments, the corticotropin is detected using magnetic particle chemiluminescence.

The invention provides a detection reagent or a detection kit, which comprises the ACTH recombinant rabbit monoclonal antibody, the biological material and/or the ACTH recombinant rabbit monoclonal antibody expressed by the host cell.

The detection reagent or the detection kit also comprises acceptable auxiliary agents, buffer solutions, auxiliary materials or carriers.

In some embodiments, the kit is suitable for magnetic particle chemiluminescence of corticotropin.

The invention also provides a detection method of the hypertension related diseases, which comprises the step of detecting samples by adopting the detection reagent or the detection kit.

The invention utilizes phage display technology to screen and obtain the ACTH recombinant rabbit monoclonal antibody, and the heavy chain variable region of the ACTH recombinant rabbit monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 15; the light chain variable region has an amino acid sequence shown as SEQ ID NO. 16. The monoclonal antibody has strong specificity, high detection sensitivity and good affinity, and solves the problems of low content of adrenocorticotropic hormone in a sample and high detection difficulty. Through identification, repeated detection is carried out by using the monoclonal antibody, so that the stability is good and the accuracy is high.

Drawings

FIG. 1 is an agarose gel electrophoresis of extracted spleen RNA;

FIG. 2 shows agarose gel electrophoresis of VL gene PCR products;

FIG. 3 shows agarose gel electrophoresis of the VH gene PCR products;

FIG. 4 shows agarose gel electrophoresis of scFv gene PCR products;

FIG. 5 shows an agarose gel electrophoresis diagram of PCR identification of recombinant bacterial liquid of an antibody library;

FIG. 6 is a diagram of SDS-PAGE electrophoresis of recombinant antibody purification;

FIG. 7 shows the correlation analysis of the detection concentration of the antibody 1 and the antibody 2 with 25 clinical samples according to the present invention, wherein FIG. 7a shows the correlation analysis of the antibody 1 and FIG. 7b shows the correlation analysis of the antibody 2;

FIG. 8 is an analysis of the correlation of antibodies of the invention with reference manufacturer antibodies;

fig. 9 shows the deviation between the test result and the theoretical concentration.

Detailed Description

The invention provides an ACTH recombinant rabbit monoclonal antibody, a preparation method and application thereof, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The sequence shown in SEQ ID NO. 1 is GIDLSSYT;

the sequence shown in SEQ ID NO. 2 is ISTANRI;

the sequence shown in SEQ ID NO. 3 is ARSLTL;

the sequence shown in SEQ ID NO. 4 is QSVYNNN;

the sequence shown in SEQ ID NO. 5 is EAS;

the sequence shown in SEQ ID NO. 6 is LGIYDCKRADCNA;

the sequence shown in SEQ ID NO. 7 is QQQLEESGGRLVTPGTPLTLTCTAS;

the sequence shown in SEQ ID NO. 8 is MNWVRQAPGKGLEWIGL;

the sequence shown in SEQ ID NO. 9 is YYASWAKGRFTISKTSTTVDLKIISPTTEDT

ATYFC；

The sequence shown in SEQ ID NO. 10 is WGPGTLVTVSS;

the sequence shown in SEQ ID NO. 11 is ALVMTQTPPSVSAAVGSTVTINCQAS;

the sequence shown in SEQ ID NO. 12 is LAWYQQKPGQPPKLLIY;

the sequence shown in SEQ ID NO. 13 is KLASGVSSRFKGSGSGTQFTLTISDVQCD

DAATYYC；

The sequence shown in SEQ ID NO. 14 is FGGGTKLEIK;

the test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 phage platform screening preparation provided by the invention:

1. recombinant immunogens immunize animals:

new Zealand white rabbits were immunized 4 times with the prepared antigen ACTH-BSA. The immunization period is 30 days, the back subcutaneous multipoint injection is carried out, the immunization is carried out after the emulsification of 2mg of the immune antigen and the equal volume Freund complete adjuvant is fully carried out for the first time, and the immunization is carried out after the emulsification of 1mg of the immune antigen and the equal volume Freund incomplete adjuvant is fully carried out for the last three times. Blood is taken from the ear margin vein on day 10 after the third immunization, the mixture is kept stand at 37 ℃ for 1h and centrifuged at 6000r/min for 10min, and the supernatant (antiserum) is collected for ELISA detection of immune effect (see example 2).

2. Antisera titer assay

A detection plate was prepared, and RaIgGFc antibody was added to a coating buffer of 0.05mol/LCB (pH 9.6) at a coating concentration of 4. Mu.g/ml. The test serum is diluted from 1:200 in a doubling ratio, 50 μl/well, and meanwhile, an unimmunized rabbit serum control is arranged, and the test serum is incubated for 30min at 37 ℃; PBST was washed 5 times, patted dry, added with ACTH enzyme conjugate at working concentration, 50. Mu.l/well, incubated for 30min at 37 ℃. PBST was washed 5 times, dried by shaking, added with substrate A and developer B, reacted in 50. Mu.l/well for 5min in the absence of light, added with stop solution 50ul, and the signal value was measured. The test results are shown in Table 1. The final boost was performed after the titer reached 1/64K, animals were sacrificed three days later, spleen cells were extracted, total spleen RNA was routinely extracted by Trizol (FIG. 1) and cDNA was synthesized by reverse transcription.

TABLE 1 antiserum titre assay

Dilution factor	1#	2#	3#	4#	5#	6#	Negative control
									1/200	3.551	3.505	3.527	3.66	3.359	3.602	0.097
1/1k	4.5	4.028	3.903	4.5	3.692	3.806	0.062
								1/2k	1.638	3.173	2.627	3.021	2.364	3.63	0.048
1/4k	1.396	2.586	2.003	2.482	2.1	3.05	0.049
								1/8k	1.166	2.43	2.024	1.504	1.221	2.605	0.047
1/16k	0.537	1.679	1.115	0.775	0.708	1.704	0.052
								1/32k	0.355	1.345	0.873	0.529	0.427	1.314	0.046
1/64k	0.23	0.767	0.513	0.398	0.286	0.784	0.047

Example 2: scFv gene splicing and phage screening construction

1. scFv gene splicing:

PCR amplified the light chain variable region and heavy chain variable region of the antibody, respectively, and the PCR reaction procedure is shown in Table 2:

TABLE 2

The PCR products were recovered by 1% agarose gel as shown in FIGS. 2 and 3. The amplified light chain variable region and heavy chain variable region were spliced into scFv by overlap-PCR method, and the product was recovered by 1% agarose gel and stored at-20deg.C, as shown in FIG. 4.

2. Construction and screening of phage single-chain antibody library:

the phagemid vector pcomb3XSS and the ScFv fragment recovered by purification are subjected to enzyme digestion by SfiI to construct recombinant plasmids, the competent cells of the TG1 are electrically transformed by the recombinant plasmids to construct a rabbit immune single-chain antibody library (the recombinant rate bacterial liquid PCR identification of the antibody library is shown in figure 5), and a primary phage single-chain antibody library is prepared; the primary phage single-chain antibody library is enriched and screened for 3 rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; selecting 558 monoclonal strains, preparing monoclonal Phage supernatant, and identifying positive clones by using a Phage-ELISA method to obtain positive sequences.

Screening a plurality of variable region sequences through library establishment, wherein the diagnosis performance evaluation results of partial sequences and samples are shown in table 3:

antibody 1:

heavy chain variable region amino acid sequence:

QQQLEESGGRLVTPGTPLTLTCTASGIDLSSYTMNWVRQAPGKGLEWI GLISTANRIYYASWAKGRFTISKTSTTVDLKIISPTTEDTATYFCARSLTLWGP GTLVTVSS(SEQ ID NO:15)

heavy chain variable region nucleotide sequence:

CAGCAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCGGGGACACCCCTGACACTCACCTGCACAGCCTCTGGAATCGACCTCAGTAGCTATACTATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGACTCATTAGTACTGCTAATAGGATATACTACGCGAGCTGGGCGAAAGGCCGCTTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATCCTTGACTCTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA(SEQ IDNO:17)

CDR-H1 GIDLSSYT(SEQ ID NO:1)；

CDR-H2 ISTANRI(SEQ ID NO:2)；

CDR-H3 ARSLTL(SEQ ID NO:3)；

FR-H1 QQQLEESGGRLVTPGTPLTLTCTAS(SEQ ID NO:7)；

FR-H2 MNWVRQAPGKGLEWIGL(SEQ ID NO:8)；

FR-H3 YYASWAKGRFTISKTSTTVDLKIISPTTEDTATYFC(SEQ ID

NO:9)；

FR-H4 WGPGTLVTVSS(SEQ ID NO:10)；

the amino acid sequence of the light chain variable region:

ALVMTQTPPSVSAAVGSTVTINCQASQSVYNNNNLAWYQQKPGQPPK LLIYEASKLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGIYDCKRA DCNAFGGGTKLEIK(SEQ ID NO:16)

light chain variable region nucleotide sequence:

GCGCTTGTGATGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAAGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCGTCCAAACTGGCATCTGGGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACCCAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCATTTATGATTGTAAGCGTGCTGATTGTAATGCTTTCGGCGGAGGAACCAAGCTGGAGATCAAA(SEQ ID NO:18)

CDR-L1 QSVYNNNN(SEQ ID NO:4)；

CDR-L2 EAS(SEQ ID NO:5)；

CDR-L3 LGIYDCKRADCNA(SEQ ID NO:6)；

FR-L1 ALVMTQTPPSVSAAVGSTVTINCQAS(SEQ ID NO:11)；

FR-L2 LAWYQQKPGQPPKLLIY(SEQ ID NO:12)；

FR-L3 KLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYC(SEQ IDNO:13)；

FR-L4 FGGGTKLEIK(SEQ ID NO:14)；

antibody 2:

heavy chain variable region amino acid sequence:

QSVKESGGRLVTPGTPLTLTCTVSGIDLSRYSFHWVRQAPGKGLEYIGT ISNVGKTYFANWVKGRFTISKASTTVDLKLTSLTAPDTATYFCVRGFTLWG HGTLVTVSS(SEQ ID NO:19)

heavy chain variable region nucleotide sequence:

CAGTCGGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCGCTACAGCTTCCATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAATACATCGGAACCATTAGCAATGTTGGTAAGACATACTTCGCGAACTGGGTTAAAGGCCGATTCACCATCTCCAAAGCCTCGACCACGGTGGATCTGAAGCTGACCAGTCTAACAGCGCCAGACACGGCCACATATTTCTGTGTCAGAGGCTTTACCTTGTGGGGCCACGGCACCCTGGTCACCGTCTCCTCAGGCCAGGCCGG(SEQ ID NO:20)

CDR-H1 GIDLSRYS(SEQ ID NO:21)；

CDR-H2 ISNVGKT(SEQ ID NO:22)；

CDR-H3 VRGFTL(SEQ ID NO:23)；

FR-H1 QSVKESGGRLVTPGTPLTLTCTVS(SEQ ID NO:24)；

FR-H2 FHWVRQAPGKGLEYIGT(SEQ ID NO:25)；

FR-H3 YFANWVKGRFTISKASTTVDLKLTSLTAPDTATYFC(SEQ IDNO:26)；

FR-H4 WGHGTLVTVSS(SEQ ID NO:27)；

the amino acid sequence of the light chain variable region:

AIDMTQTPASVSAAVGGTVTIKCQSSQSVYNNWLSWYQQKPGQPPKV LIYEASKLASGVPSRFKASGSGTQFTLTISDVQCDDAATYYCLGSYDCKTA DCAAFGGGTKVEIK(SEQ ID NO:28)

light chain variable region nucleotide sequence:

GCCATCGATATGACCCAGACTCCAGCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAATAACTGGTTATCCTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGGTCCTTATCTACGAAGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAAAGCCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGTGACGTACAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTAGTTATGATTGTAAAACTGCTGATTGTGCGGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:29)

CDR-L1 QSVYNNW(SEQ ID NO:30)；

CDR-L2 EAS(SEQ ID NO:31)；

CDR-L3 LGSYDCKTADCAA(SEQ ID NO:32)；

FR-L1 AIDMTQTPASVSAAVGGTVTIKCQSS(SEQ ID NO:33)；

FR-L2 LSWYQQKPGQPPKVLIY(SEQ ID NO:34)；

FR-L3 KLASGVPSRFKASGSGTQFTLTISDVQCDDAATYYC(SEQ IDNO:35)；

FR-L4 FGGGTKVEIK(SEQ ID NO:36)；

TABLE 3 Table 3

From the results, the effect of antibody 1 was superior to that of antibody 2, and the subsequent experiments were performed.

Example 4: recombinant monoclonal antibody expression and antibody purification of the invention

1. Construction of stable transgenic cell lines

The heavy chain variable region and the light chain variable region of antibody 1 were amplified by PCR, respectively, to obtain antibody variable region genes. The heavy chain antibody gene and the light chain antibody gene are respectively recombined with RGFc-PCMV3 and RCL-PCMV3 vectors after KasI/BamHI double enzyme digestion in a homologous way to obtain expression vectors of rabbit total antibody sequences. After the recombined total-resistance plasmid is transferred into competent DH5 alpha cells, positive clone sequencing is selected and plasmid extraction is carried out. The extracted plasmids were transfected into HEK293 cells.

After 48h transfection, ELISA detection antibody is expressed, feeding material is added, and cell supernatant is collected after 7d transfection, thus obtaining target protein supernatant.

2. Identification of column equilibrium by SPA purification and SDS-PAGE of antibodies

Setting the flow rate to be 6.4mL/min, replacing the balance buffer solution to be 0.02mol/LPBSpH7.4, flushing the chromatographic column, setting the flow rate to be 3.8mL/min, carrying out sample loading and purification, taking a sample in a 10 mu L flow through tube, adding the sample into a 200 mu L G250 dye solution, putting the flow through tube into a 250mL conical flask to start collecting and penetrating when detecting bluing, and balancing: setting the flow rate to be 6.4mL/min, and flushing the chromatographic column again with 0.02mol/LPBSpH7.4 of balance buffer until no protein flows through; dissociation: 10 4mL centrifuge tubes are placed on a dissociation tube rack, 200 mu L of 1mol/L Tris (pH 8.5) is added into each tube, a constant flow pump is arranged to set the flow rate to be 6.4mL/min, dissociation buffer solution of 0.2mol/LGly+0.15mol/LNaClpH2.7 is used for dissociating target proteins, manual collection is started when 10 mu L of the equiflow tube is added into 200 mu L G250 dye liquor for detecting blue change, 4mL of each tube is collected, and collection is stopped when 10 mu L of the equiflow tube is added into 200 mu L G250 dye liquor for detecting colorless. SDS-PAGE detection of the collected proteins after combination is shown in FIG. 6. The purified antibody has high purity and high concentration.

Example 5: application of kit of ACTH recombinant rabbit monoclonal antibody prepared by the invention

The ACTH recombinant rabbit monoclonal antibody prepared by the invention is used as a magnetic bead sheep anti-rabbit complex binding antibody to be applied to a kit, and is matched with other components to measure the antigen in a sample. The specific performance evaluation is as follows:

1. preliminary evaluation of antibody-coated magnetic beads

Antibody 1 and antibody 2 obtained in the previous examples were applied to kit components, and 25 clinical samples within the detection range were randomly selected for detection. The correlation analysis of the detected concentration values with the hospital assigned clinical samples is performed, and the results are shown in fig. 7. Wherein, FIG. 7a is a correlation analysis of antibody 1, and FIG. 7b is a correlation analysis of antibody 2.

The results showed that antibody 2 had poor correlation and detected outliers and no subsequent evaluation was performed

2. Antibody 1 thermal stability acceleration test

The antibody-coated magnetic beads were subjected to a thermostable acceleration test at 37℃for 7d, and 5 clinical specimens were tested on-machine with the following results:

TABLE 4 evaluation results of antibody 1 stability

Performance index	2-8deg.C for 7 days	Stored at 37℃for 7 days
			Blank limit	0.04ng/ml	0.04ng/ml
Clinical 1	1.92ng/ml	1.99ng/ml
			Clinical 2	25.76ng/ml	25.88ng/ml
Clinical 3	46.53ng/ml	46.61ng/ml
			Clinical 4	68.65ng/ml	68.78ng/ml
Clinical 5	101.56ng/ml	102.36ng/ml

3. Accuracy measurement:

the detection is carried out by adopting 13 clinical samples with fixed values of a certain mainstream commercial kit in the market, the ACTH concentration in the samples is calculated back according to the system, and the correlation analysis is carried out with a reference manufacturer, and the result is shown in figure 8. According to the detection result, the obtained linear equation is as follows: y=8e-06x+2.0213, correlation coefficient R ² = 0.9829, which indicates that the ACTH recombinant rabbit monoclonal antibody provided by the invention has higher accuracy in detecting corticotropin clinical samples.

4. Recovery rate performance evaluation:

high value samples were selected 3 times according to 1:9 was added to 3 low value/matrix samples, respectively, to make a recycle sample, and the volume of the high value sample added was not more than 10% of the total volume. Each recovered sample was repeatedly tested 3 times to average and recovery was calculated. The recovery rates of the 3 recovered samples of the 1 st batch are 92.3%,94.4% and 96.7%, the recovery rates of the 3 recovered samples of the 2 nd batch are 103.6%,102.8% and 103.7%, the recovery rates of the 3 rd batch are 102.4%,103.0% and 103.8%, the deviation is less than 10%, and the recovery accuracy meets the requirements.

5. Linear analysis:

clinical high-value samples with ACTH concentration close to the expected linear range are respectively selected, 1 clinical low-value sample with concentration close to 0 or the lowest value which can be obtained clinically are mixed in different proportions to obtain 9 linear samples with different concentrations, and finally, the deviation between the test result and the theoretical concentration is calculated (figure 9). The results show that: the linear regression coefficient r is more than 0.99, and the linear sample deviation is less than 10%.

6. Precision evaluation:

the precision evaluation performed in this study was the precision in the batch. Three levels of samples with high, medium and low concentrations are carried out by using the same batch of reagent, each sample is repeatedly measured for 20 times in each batch of test, the average value and SD are obtained, and the variation coefficient CV% is calculated. The intra-batch coefficient of variation (Coefficient of Variation, CV) for the 3 samples was less than 5% and the results are shown in Table 5, indicating good reproducibility of the kit.

TABLE 5 precision results of ACTH rabbit monoclonal antibodies

7. Sensitivity performance evaluation:

selecting 5 clinical samples close to 0 value, repeating each sample for 3 times for 4 days to obtain 60 data, carrying out data inspection and result analysis according to an EP17 method, wherein LoB values of the clinical 0 value samples of 3 batches of kits are less than 0.15nmol/L, and the samples are detected in a blank limit, thus indicating that the sensitivity is higher

8. Affinity assay:

ACTH biotinylated recombinant rabbit monoclonal antibody 1 and antibody 2 were diluted in gradient for 6 concentrations, 2.08,4.17,8.33, 16.67, 33.33, 66.67nM, respectively, and binding and dissociation (binding for 60s, dissociation 300 s) experiments were performed on ACTH-immobilized sensors and different concentrations of biotinylated antibodies using Octet RED96E, and affinity measurements were performed, followed by data processing to obtain affinities, kd=2.76E-9 for antibody 1, kd=6.32E-8 for antibody 2. The ACTH biotinylation recombinant rabbit monoclonal antibody 1 provided by the invention has higher affinity.

From the results, the recombinant monoclonal antibody prepared by the invention reaches the level which is highly consistent with the detection results of mainstream factories in the market at present, and has the advantages of high precision, specificity and the like.

From the results, the recombinant monoclonal antibody prepared by the invention reaches the level which is highly consistent with the detection results of mainstream factories in the current market, has the advantages of high precision, and the like, and can be used as a detection antibody to be applied to a kit, and the detection antibody can meet the requirements of the kit according to the evaluation of various detection performances, thereby having important roles in later application and clinical diagnosis.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

ACTH recombinant rabbit monoclonal antibody, characterized in that

The amino acid sequence of the CDR region of the heavy chain comprises at least one of the sequences shown as SEQ ID NO. 1, 2 or 3;

the amino acid sequence of the CDR region of the light chain comprises at least one of the sequences shown as SEQ ID NO. 4, 5 or 6.
2. The ACTH recombinant rabbit monoclonal antibody of claim 1,

the amino acid sequences of the three CDR regions of the heavy chain are shown in SEQ ID NO. 1, 2 and 3 in sequence;

the amino acid sequences of the three CDRs of the light chain are shown in SEQ ID NOs 4, 5 and 6 in sequence.
3. The ACTH recombinant rabbit monoclonal antibody of claim 1 or 2,

the amino acid sequences of the four FR regions of the heavy chain are respectively shown as SEQ ID NOs 7,8, 9 and 10;

the amino acid sequences of the four FRs of the light chain are shown in SEQ ID NOs 11, 12, 13 and 14 respectively.
4. The ACTH recombinant rabbit monoclonal antibody according to claim 1 to 3,

the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 15;

the light chain variable region has an amino acid sequence shown as SEQ ID NO. 16.
5. The ACTH recombinant rabbit monoclonal antibody of claim 4, wherein the constant region of the heavy chain is of the rabbit IgG subtype; the constant region of the light chain is kappa 1.
6. A biomaterial comprising at least one of the following:

1) A nucleic acid encoding the ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5;

2) An expression vector comprising the nucleic acid;

3) Transforming or transfecting a host cell of the expression vector;

4) A conjugate prepared by coupling the ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5 with a solid medium or semi-solid medium;

5) The ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5, chemically-labeled or biomarker;

6) A conjugate prepared by coupling the ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5, chemically-labeled or biomarker, with a solid medium or semi-solid medium.
7. The biomaterial of claim 6, wherein the nucleic acid comprises:

the nucleic acid encoding the heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO. 17;

the nucleic acid encoding the light chain variable region has the nucleotide sequence shown as SEQ ID NO. 18.
8. The method for producing ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 5, comprising: culturing the host cell of claim 6, inducing expression of the ACTH recombinant rabbit monoclonal antibody.
9. Use of at least one of the following i-iii in the preparation of a reagent or kit for detecting a disease associated with hypertension:

the ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5;

ii) the biomaterial of claim 6 or 7;

iii, the ACTH recombinant rabbit monoclonal antibody prepared by the preparation method of claim 8.
10. A detection reagent or a detection kit, characterized by comprising at least one of the following (1) to (3):

(1) the ACTH recombinant rabbit monoclonal antibody of any one of claims 1-5;

(2) the biomaterial of claim 6 or 7;

(3) an ACTH recombinant rabbit monoclonal antibody produced by the method of claim 8.