CN116178536A - Mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody and application thereof - Google Patents

Abstract

The invention belongs to the field of antibodies, and particularly provides a mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody and application thereof, wherein the mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody is secreted by a hybridoma cell strain NC1#4 with a preservation number of CCTCC NO: C2022240. The invention firstly develops and successfully prepares the mouse anti-human IV type collagen alpha 5 chain NC1 section monoclonal antibody in China, the monoclonal antibody is the key of developing IV type collagen alpha 5 chain immunofluorescence or histochemical detection, and the monoclonal antibody has important significance for AS diagnosis and is beneficial to the determination of genetic mode. The pre-clinical verification experiment shows that the monoclonal antibody prepared by the invention is completely consistent with the immunofluorescence detection result of the commercial antibody, and the result is easier to read.

Description

Mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to a mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody, a preparation method and application thereof.

Background

Type IV collagen (type IV collagen) is an important framework protein constituting a basement membrane, and an α chain is a subunit constituting a type IV collagen molecule, one type IV collagen molecule is composed of 3 α chains. To date, 6 human type IV collagen alpha chains, respectively designated as alpha 1 to alpha 6 chains, have been found, and the coding genes thereof are COL4A1-6, respectively. The α5 chain can be divided into three domains according to amino acid sequence and function: 1. an amino terminal 7S region; 2. a collagen region comprising a "Gly-X-Y" repeat sequence; 3. carboxy-terminal non-collagenous region (NC 1). The NC1 region controls the association of type IV collagen monomers, the amino acid sequences of which are highly conserved among different species, but the amino acid sequences of different alpha chain NC1 regions are significantly different, which is the molecular basis for the preparation of monoclonal antibodies.

Alport Syndrome (AS), also known AS hereditary nephritis, eye-ear-kidney syndrome, is the most common hereditary glomerular disease, and is clinically manifested mainly AS hematuria, proteinuria, progressive renal failure with sensorineural deafness and ocular lesions. Atkin in 1988 first mapped AS-related genes to the X chromosome, and in 1990 Barker et al successfully cloned into the COL4A5 gene, a gene encoding the type IV collagen. Alpha.5 chain, and found the first COL4A5 gene mutation in X-concomitant inherited AS (XLAS) patients, followed by more and more COL4A5 gene mutations, while the COL4A3 and COL4A4 genes were related to autosomal inherited AS.

The immunofluorescence detection of the monoclonal antibody aiming at the NC1 segment of the IV type collagen alpha 5 chain has important diagnostic value on XLAS and autosomal recessive genetic AS (ARAS). Normally, the α5 (iv) chain is present in glomerular basement membrane, distal tubular basement membrane and bordetella capsule wall, immunofluorescence is positive, as a continuous line, whereas in XLAS male patients, the α5 chain immunofluorescence is mostly negative, and XLAS female patients immunofluorescence is mostly discontinuous positive. In addition, the IV type collagen alpha 5 chain is also found in the epidermal basal membrane of skin, and is similar to kidney, under normal conditions, alpha 5 chain immunofluorescence is positive and continuous line-shaped, while XLAS male immunofluorescence is mostly negative, XLAS female immunofluorescence is mostly discontinuous positive, so that the skin biopsy can replace kidney tissue to play a role in diagnosing diseases for patients with serious damage to kidney function and losing kidney puncture opportunity when part of diseases are found. For some ARAS patients, the researchers found that anti- α5 chain mab was deposited continuously on the distal tubular basement membrane and Bosch's capsule, but not on the glomerular basement membrane, and this specific immunofluorescence assay was found only in ARAS. Therefore, the alpha 5 chain immunofluorescence detection has important diagnostic value for XLAS and also has diagnostic value for partial ARAS patients, and is more beneficial to the determination of AS genetic modes.

Patent document CN110531088A discloses a type IV collagen detection kit comprising a reagent mixed with latex particles of a coated murine anti-human type IV collagen monoclonal antibody; patent document CN102010470B discloses an anti-type IV collagen monoclonal antibody. However, no mouse anti-human type IV collagen α5 chain NC1 fragment monoclonal antibodies as described herein are currently seen.

Disclosure of Invention

The invention aims to provide a monoclonal antibody which specifically binds to a human type IV collagen alpha 5 chain NC1 segment and is secreted by a hybridoma cell strain NC1#4 with a preservation number of CCTCC NO: C2022240.

The present patent also provides a monoclonal antibody comprising: an H chain variable region and an L chain variable region, wherein (a) the H chain variable region comprises three complementarity determining regions, a CDR1 sequence represented by SEQ ID NO:17, a CDR2 sequence represented by SEQ ID NO:19 and a CDR3 sequence represented by SEQ ID NO:21, and (b) the L chain variable region comprises three complementarity determining regions, a CDR1 sequence represented by SEQ ID NO:37, a CDR2 sequence represented by SEQ ID NO:39 and a CDR3 sequence represented by SEQ ID NO: 41.

Preferably, the monoclonal antibody recognizes an epitope comprising the amino acid sequences at positions 251-475 represented by SEQ ID NO. 1.

Preferably, the monoclonal antibody is humanized.

The patent also provides application of the monoclonal antibody in preparing a kit for detecting human IV type collagen.

Preferably, the kit is used for immunofluorescence detection of human type IV collagen α5 chain.

Preferably, the kit is for diagnosing Alport syndrome.

The patent also provides a hybridoma cell strain, which is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2022240.

The invention has the beneficial effects that: the monoclonal antibody of the NC1 segment of the anti-human IV type collagen alpha 5 chain of the mouse is developed and successfully prepared at home, is the key of developing the immunofluorescence or the histochemical detection of the IV type collagen alpha 5 chain, has important significance for AS diagnosis and is beneficial to the determination of genetic modes. At present, only Japan has the monoclonal antibody product, and the price is high. The pre-clinical verification experiment shows that the monoclonal antibody prepared by the method is completely consistent with the antibody immunofluorescence detection result of Japanese company, and the result is easier to read.

Drawings

Fig. 1: SDS-PAGE and Western Blot analysis of CO4A 5-Ecoli.

Fig. 2: and (5) cloning immunofluorescence detection of the renal tissue cell supernatant. Supernatant 4,11,19 immunofluorescence is positive, positive part supernatants 4 and 19 are the same as control antibodies, but the fluorescence intensity of the 19 supernatant is obviously weak; the 11# supernatant fluorescent clearly positive sites were similar to the control antibodies, but there were also nonspecific weak positives of other tubular basement membranes except the distal tubular basement membrane. A: cosmosobio antibodies; b: supernatant 2; c: supernatant 3; d: supernatant 4; e: supernatant 5; f, supernatant 6; g: supernatant 8; h: supernatant 9; i: a supernatant 10; j: supernatant 11; k: a supernatant 12; l: a supernatant 13; m: a supernatant 14; n: a supernatant 15; o: supernatant 17; p: a supernatant 18; q: supernatant 19; r: supernatant 21. (400 x)

Fig. 3: and cloning immunofluorescence detection of skin tissue cell supernatant. a: cosmobio antibody (200×); b: supernatant #4 (200 x); c:11L# supernatant (200X); d:19# supernatant (200 x).

Fig. 4: immunofluorescence detection of kidney tissue with anti- α5 (IV) monoclonal antibody. A and B: cosmobrio antibodies (A: 100x; B:400 x); c, D: monoclonal antibody 4 (C: 100x; D:400 x); e, F: monoclonal antibody 5 (E: 100x; F: 400); g, H:11# mab (G: 100x; H:400 x).

Fig. 5: immunofluorescence detection of skin tissue with anti- α5 (IV) monoclonal antibody. a: cosmobio antibody (200×); b:4# mab (200 x); c: mab # 5 (200 x); d:11# mab (200 x).

Fig. 6: and 4# monoclonal antibody kidney tissue immunofluorescence detection. A and B: immunofluorescence of Cosmobi antibody in kidney tissue of control non-Alport syndrome patients (A: 100x; B:400 x); c, D: immunofluorescence of renal tissue in control non-Alport syndrome patients with monoclonal antibody 4 (C: 100x; D:400 x); e, F: cosmobi antibodies showed positive discontinuities in renal tissue immunofluorescence detection results in female patients with X concomitant inherited Alport syndrome (E; 100X; F: 400X); g, H: the 4# monoclonal antibody shows positive discontinuity (G: 100X; H: 400X) in the result of immunofluorescence detection of kidney tissue of female patients with X-concomitant genetic Alport syndrome.

Fig. 7: immunofluorescence detection of skin tissue #4 monoclonal antibody. a: immunofluorescence of Cosmobio antibody against skin tissue of non-Alport syndrome patient (200 x); b: immunofluorescence detection result of kidney tissue of the patient with the non-Alport syndrome of the control model 4 monoclonal antibody (200 x); c: the Cosmobio antibody shows negative (200X) in the result of immunofluorescence detection of skin tissue of male patients with X concomitant genetic Alport syndrome; d: the result of the immunofluorescence detection of the skin tissue of the male patient with the X-concomitant genetic Alport syndrome by the monoclonal antibody 4 shows negative (200X).

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1

1. Antigen preparation: gene cloning, protein expression purification

The ordered gene human CO4A5 is subcloned on pGS-21a plasmid vector with His tag and GST tag simultaneously, E.coli is transformed for expression identification, experimental results show that the protein is expressed in inclusion bodies, the inclusion bodies are dissolved by 8M urea PBS, the subsequent affinity purification of the human CO4A5 by nickel column is carried out, the protein is dissolved in 8M urea PBS, the concentration is 1.05mg/ml,6ml, and the purity is 90%.

Cloning strategy: ATG-His tag-GST tag-KpnI-TEV protease site-CO 4A 5-Ecoli-Stop codon-HindIII, the aa sequence of which is shown as SEQ ID NO:1, wherein, the 5 th to 10 th bits are His tag, the 14 th to 233 th bits are GST tag, the 244 th to 250 th bits are TEV protease site, and the 251 th to 475 th bits are CO4A5-Ecoli, namely CO4A5 NC1 section. The coding sequence of the CO4A5 NC1 segment is shown as 28-702bp of SEQ ID NO. 2.

Prokaryotic expression and affinity purified protein detection. The results are shown in FIG. 1.

2. Immunization of mice

5 balb/c mice (5 subcutaneous injections) were each immunized (11 mice were actually immunized and 9 mice survived) with periodic adjuvant (complete and incomplete adjuvant), and after three immunizations, small serum samples were collected for Elisa testing as shown in the following table.

Elisa data analysis

The CO4A5 protein has GST and HIS labels, the protein immunizes 11 mice, and the number of the mice is C-1# to C-9# is 9;

2. coating with CO4A5 protein, GST protein and His protein respectively, starting from the four-way blood of 9 mice at a ratio of 1:100, starting from the positive control of GST and His monoclonal antibody at a ratio of 1ug/ml, and carrying out gradient dilution at a ratio of 1:3;

co4a5 protein: three mice (1 #,4#,6 #) were null and the other 6 mice were titered: 9# > 3# =5 # > 2# =7 # > 8#;

gst protein assay, three mice: 3#,7#,9# react with GST protein, 7# has poor specificity;

his protein assay, confirming that # 2, # 3 mice did not react with His protein.

(II) conclusion

1. Mouse serum has weak cross reaction with GST and His, and mainly reacts with CO4A5 protein;

2. 6 mice were selected for cell fusion according to titers preferentially at 9# > 3# =5 # > 2# =7 # > 8 #.

3. Cell fusion screening and subcloning

Time	Content	Remarks
			Day
1	Mouse terminal reinforcement	80 μg mouse without adjuvant and by intraperitoneal injection
			Day 3	Cell fusion	PEG method
Day 7-10	Fusion primary screen	Coating conjugate ELISA
			Day 9	Fusion rechecking	Coating conjugate ELISA, competition reaction and ELISA imitation method
Day 19	First subcloning	Primary screening hybridoma monoclonal
			Day 29	Second subcloning
Day 39	Third subcloning

Cell fusion was performed in 6 mice, and cell supernatants were screened by Elisa to obtain 21 positive clones, elisa values:

the Immunofluorescence (IF) assay was performed on 21 clones, and the experimental procedure was as follows:

1. taking frozen kidney tissue slices (0.2-0.3 um), and air-drying in air;

2. acetone was fixed for 10 min, and washed 3 times with PBS for 3 min each;

3. denaturing agent (0.1M glycine, 6M urochorda, pH 3.5) was added dropwise and incubated in air for 10 min;

4. flushing with double distilled water for 3 times, each time for 3 minutes;

5. dripping diluted I antibody (obtained supernatant of 21 cells, stock solution, blank control, equal volume PBS) as positive control, stock solution, and incubating at 37deg.C for 30 min;

pbs rinse 3 times for 3 minutes each;

7. diluted FITC labeled goat anti-mouse F (ab) is added dropwise ₂ IgG secondary antibody (Sigma),incubation at 37 ℃ for 30 minutes;

pbs rinse 3 times for 3 minutes each; PBS-glycerol seal, microscopy.

9. Results: immunofluorescence positives were detected for a total of 3 clones: 4#,11#,19#, wherein the positive fluorescent sites of the 4# and 19# supernatants are identical to the control antibody, but the 19# fluorescent intensity is very weak, the 11# supernatant fluorescent light is obvious, the positive sites are similar to the control antibody, but the tubular basement membrane except the distal tubular basement membrane has weak immunofluorescence positive, and a nonspecific signal exists. (see FIG. 2)

The detected positive clone is further subjected to immunofluorescence detection on skin tissues, the experimental steps are the same as those described above, and the result is the same as kidney tissues. (see FIG. 3)

4. Cell line freezing and antibody mass production

Cell clones screened by immunofluorescence are established, frozen and stored, and a small amount of antibody is produced.

Time	Content	Remarks
			Day
1	96-well cells were transformed into 24-well cells	Cell expansion culture and identification
			Day 14	24-hole cell T25 bottle
Day 28	T25 bottle cell to T75 bottle
			Day 38	Cell cryopreservation	Cell cryopreservation
Day
	42	Cell injection mice	Production of ascites
Day 56				Collecting ascites
	Day 58	Ascites Protein A purification	Ascites purification

Finally, three monoclonal antibodies, namely 4# antibody, 5# antibody, 11# antibody, 4# and 11# corresponding to the original 4# and 11# cell clones, and 5# antibody corresponding to the 19# cell clone were obtained, and 5mg antibody was produced respectively. The 4# cell clone was preserved in China center for type culture collection (university of Chinese Wuhan, 430072) at 2022, 09 and 06, with a preservation number of CCTCC NO: c2022240, culture designation hybridoma cell line NC1#4. The sequence information is as follows:

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5. 3 antibodies immunofluorescence detection, further verification

1. Taking frozen kidney and skin tissue slice (thickness 0.2-0.3 um), and air drying in air;

2. acetone was fixed for 10 min, and washed 3 times with PBS for 3 min each;

3. denaturant (0.1M glycine, 6M urea, pH 3.5) is added dropwise, and the mixture is incubated in air for 10 minutes;

4. flushing with double distilled water for 3 times, each time for 3 minutes;

5. dripping diluted I antibody (the dilution of the obtained monoclonal antibodies of different clones is 1:100, and the anti-alpha 2/alpha 5 antibody is a positive control (Cosmobrio, japan, stock solution), dripping an equal volume of PBS (phosphate buffer solution) in a blank control, and incubating for 30 minutes at 37 ℃;

pbs rinse 3 times for 3 minutes each;

7. diluted FITC labeled goat anti-mouse F (ab) is added dropwise ₂ IgG secondary antibody (Sigma), 37 ℃, incubated for 30 min;

pbs rinse 3 times for 3 minutes each; PBS-glycerol seal, microscopy.

9. Results:

skin tissue: a total of 12 skin samples were tested, of which 1 was COL4A5 mutant and 2 COL4A4 mutant, and the remainder was kidney disease patients with non-IV collagen alpha chain mutation;

kidney tissue: a total of 15 specimens were tested, 1 of which was a COL4A5 gene mutation and 1 COL4A4 gene mutation patient, and the other was a non-Alport syndrome kidney patient or surgically resected kidney tissue;

fluorescence results: the 4# antibody has best effect, is consistent with the detection result of the Cosmobrio antibody, is matched with the Cosmobrio antibody under the condition of 100 times dilution no matter the patients of Alport syndrome and non Alport syndrome, and the 5# antibody has very weak fluorescence intensity even though the detection result is consistent with the Cosmobrio antibody, and the 11# antibody has a non-specific signal even though the original concentration is the same, so that the finally screened 4# monoclonal antibody meets the final requirement. (see FIGS. 4,5,6, 7)

The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

Claims (8)

1. The monoclonal antibody is characterized in that the monoclonal antibody specifically binds to a human IV type collagen alpha 5 chain NC1 segment and is secreted by a hybridoma cell strain NC1#4 with a preservation number of CCTCC NO: C2022240.

2. A monoclonal antibody, comprising: an H chain variable region and an L chain variable region, wherein (a) the H chain variable region comprises three complementarity determining regions, a CDR1 sequence represented by SEQ ID NO:17, a CDR2 sequence represented by SEQ ID NO:19 and a CDR3 sequence represented by SEQ ID NO:21, and (b) the L chain variable region comprises three complementarity determining regions, a CDR1 sequence represented by SEQ ID NO:37, a CDR2 sequence represented by SEQ ID NO:39 and a CDR3 sequence represented by SEQ ID NO: 41.

3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody recognizes an epitope comprising the amino acid sequence at positions 251-475 represented by SEQ ID No. 1.

4. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody is humanized.

5. Use of a monoclonal antibody according to claim 1 or 2 for the preparation of a kit for detecting human type IV collagen.

6. The use according to claim 5, wherein the kit is for immunofluorescence detection of human type IV collagen α5 chain.

7. The use according to claim 5, wherein the kit is for diagnosing Alport syndrome.

8. A hybridoma cell strain is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2022240.

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