CN116621986A

CN116621986A - anti-GAPDH monoclonal antibody and application thereof

Info

Publication number: CN116621986A
Application number: CN202310603687.7A
Authority: CN
Inventors: 吴川; 漆彦婷; 潘炜松; 陈刚; 徐超
Original assignee: Hunan Nuohe New Biotechnology Co ltd
Current assignee: Hunan Nuohe New Biotechnology Co ltd
Priority date: 2023-05-26
Filing date: 2023-05-26
Publication date: 2023-08-22

Abstract

The invention discloses a monoclonal antibody against GAPDH and application thereof, comprising a heavy chain of a heavy chain CDR1 or a variant thereof, a heavy chain of a heavy chain CDR2 or a variant thereof, a heavy chain of a heavy chain CDR3 or a variant thereof, and a heavy chain of a light chain CDR1 or a variant thereof, a heavy chain of a light chain CDR2 or a variant thereof, and a light chain of a light chain CDR3 or a variant thereof; the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 1; the CDR2 of the heavy chain is shown as SEQ ID NO. 2; the CDR3 of the heavy chain is shown as SEQ ID NO. 3; the CDR1 of the light chain is shown as SEQ ID NO. 4; the CDR2 of the light chain is shown as SEQ ID NO. 5; the light chain CDR3 is shown in SEQ ID NO. 6. The titer of the monoclonal antibody against GAPDH can reach 8.75X10 ⁶ The affinity reaches 6.317E-10, the antibody activity is high, and the application potential is great.

Description

anti-GAPDH monoclonal antibody and application thereof

Technical Field

The invention belongs to the field of molecular immunology, and in particular relates to an anti-GAPDH monoclonal antibody and application thereof.

Background

At present, the detection method for carrying out expression quantification on genes is commonly used for real-time quantitative PCR, RNA blotting, ribonuclease protection analysis, gene chips and the like; the detection method for quantification at the protein level includes western blotting, co-immunoprecipitation and the like. However, these detection methods all need to correct the target gene and the target protein by using the reference protein, so as to obtain a true and reliable result, for example, the target protein content is divided by the reference protein content, and the obtained value is the corrected target protein relative content, which can be used for correcting the protein level, and is beneficial to comparing the target protein expression amounts under different conditions or in different cell tissues. The antibody corresponding to the reference protein is called reference antibody, namely the reference antibody (Internal Control Antibody) is used for detecting the reference protein, so that experimental errors in the protein quantification process can be corrected, and whether the steps of transferring films, developing colors and the like are normal or not can be verified.

The currently common internal references include GAPDH, beta-actin and beta-tubulin, the mitochondrial internal reference COX IV and the nuclear internal reference Histone H3 and PCNA, etc. Wherein GAPDH is an abbreviation of Glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase), and the GAPDH gene also belongs to a commonly used reference gene, such as in eukaryotes, GAPDH is used as the reference gene for quantitatively determining the expression level of a target gene; in prokaryotes, however, E.coli GAPDH was used as a reference gene for fluorescent quantitative PCR detection. The existing research also considers that GAPDH can be used as an internal reference protein common to eukaryotes and prokaryotes for Western-blot analysis.

At present, GAPDH antibody is used as one of the most widely applied reference antibodies, is often used for Western blot and other experiments, and can be used for researching physiological and biochemical changes of various agricultural products. However, most of the currently commercialized GAPDH antibodies are derived from mammals, and they are of a small variety and expensive. Therefore, more anti-GAPDH monoclonal antibodies with good affinity and high expression are searched, which is helpful for further understanding and mastering the physiological and biochemical change rules of various agricultural products and developing the agricultural products which are more beneficial to human health.

Disclosure of Invention

Aiming at the defects or improvement demands of the prior art, the invention provides an anti-GAPDH monoclonal antibody and application thereof, and aims to stimulate mice by taking N-terminal short peptide of human GAPDH as antigen, find a GAPDH antibody with high titer in the bodies, clone and sequence the variable region genes of the antibody to obtain the amino acid sequences of the heavy chain and the light chain of the antibody, and prepare the anti-GAPDH monoclonal antibody based on the amino acid sequences, thereby solving the technical problems of few types and high price of the existing GAPDH antibody.

To achieve the above object, according to one aspect of the present invention, there is provided an anti-GAPDH monoclonal antibody comprising a heavy chain of a heavy chain CDR1 or variant thereof, a heavy chain of a heavy chain CDR2 or variant thereof, and a heavy chain of a heavy chain CDR3 or variant thereof;

and a light chain of light chain CDR1 or variant thereof, a light chain of light chain CDR2 or variant thereof, and a light chain of light chain CDR3 or variant thereof;

the heavy chain CDR1 comprises or consists of an amino acid sequence N-Y-G-M-N shown in SEQ ID NO. 1; the heavy chain CDR2 comprises or consists of an amino acid sequence W-I-G-T-N-T-G-E-P-T-Y-G-E-E-F-K-G shown in SEQ ID NO. 2; the heavy chain CDR3 comprises or consists of an amino acid sequence D-G-Y-Y-D-F-D-Y shown in SEQ ID NO. 3;

the light chain CDR1 comprises or consists of an amino acid sequence K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence L-V-S-K-L-D-S shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of a sequence W-Q-G-T-H-F-K-T shown in SEQ ID NO. 6.

Preferably, the heavy chain of said variant of said monoclonal antibody against GAPDH comprises or consists of the amino acid sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID No. 7,8,9, 10, or an amino acid sequence having one or more amino acid mutations compared to the sequence shown in SEQ ID No. 7,8,9, 10.

Preferably, the anti-GAPDH monoclonal antibody, the light chain of said variant thereof, the light chain variable region of which comprises or consists of the amino acid sequence shown in SEQ ID NOs 11, 12, 13, 14, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID NOs 11, 12, 13, 14, or an amino acid sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NOs 11, 12, 13, 14.

Preferably, the heavy chain of said variant of said monoclonal antibody against GAPDH comprises the amino acid sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having a mutation of 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids compared to the sequence shown in SEQ ID No. 7,8,9, 10, said mutation being a conservative mutation, including a substitution, insertion or deletion of an amino acid.

Preferably, the anti-GAPDH monoclonal antibody, the light chain of said variant thereof, the light chain variable region of which comprises the amino acid sequence shown in SEQ ID No. 11, 12, 13, 14, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 11, 12, 13, 14, or a sequence having a 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutation, said amino acid mutation being a conservative mutation, including a substitution, insertion or deletion.

Preferably, the heavy chain amino acid sequence of the anti-GAPDH monoclonal antibody is shown as SEQ ID NO. 15, and the light chain amino acid sequence of the anti-GAPDH monoclonal antibody is shown as SEQ ID NO. 16.

According to another aspect of the invention there is also provided a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against GAPDH according to the invention, said nucleic acid sequence comprising a gene, a cDNA molecule, an mRNA molecule and fragment oligonucleotides thereof.

According to another aspect of the present invention there is provided a biological material comprising a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against GAPDH according to the invention, said biological material comprising an expression vector, an expression cassette, a host cell, an engineered bacterium or a hybridoma cell line.

According to another aspect of the invention there is also provided the use of a monoclonal antibody against GAPDH according to the invention for the detection of the reference protein GAPDH.

According to another aspect of the invention there is also provided a test kit comprising a monoclonal antibody against GAPDH according to the invention.

In general, compared with the prior art, the above technical solutions contemplated by the present invention can achieve the following beneficial effects due to the high potency of GAPDH antibodies discovered by the present invention:

the invention obtains the high-titer anti-GAPDH antibody in the mouse antigen immune experiment, obtains the heavy chain and light chain amino acid sequences of the antibody through cloning and sequencing, and prepares the anti-GAPDH monoclonal antibody by adopting a recombination technology. The titer of the monoclonal antibody against GAPDH provided by the invention can reach 8.75X10 ⁶ The purified anti-GAPDH monoclonal antibody has good affinity, high activity and wide market prospect application.

Drawings

FIG. 1 is a heavy and light chain amino acid sequence of a monoclonal antibody against GAPDH;

FIG. 2 is a comparison of the affinity of a monoclonal antibody against GAPDH provided by the invention to a commercially available GAPDH antibody for antigen.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Terminology and definition:

the following terms used in the present invention have the following meanings unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art.

Naturally occurring amino acids include alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-naturally occurring amino acids include, but are not limited to, alpha-amino adipic acid, aminobutyric acid, citrulline, homocdown, homoleucine, homoarginine, homoleucine, and the like.

Peptides, polypeptides, proteins are not strictly distinguished in the present invention and may in some cases be used interchangeably and generally refer to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic. The polypeptide may also comprise non-amino acid components such as carbohydrate groups, metal ions or carboxylic acid esters. The non-amino acid component may be added by the cell expressing the polypeptide and may vary with cell type. A polypeptide is defined in the present invention with respect to its amino acid backbone structure or the nucleic acid encoding it. Such as the addition of carbohydrate groups, is not generally specified, but may be present. All polypeptide sequences are written according to commonly accepted practices, with the α -N-terminal amino acid residue on the left and the α -C-terminal amino acid residue on the right. When used in the present invention, the term N-terminus refers to the free α -amino group of an amino acid in a polypeptide and the term C-terminus refers to the free α -carboxylic acid end of an amino acid in a polypeptide. A polypeptide ending with a group at the N-terminus refers to a polypeptide that carries a group on the alpha-amino nitrogen of the N-terminal amino acid residue. An amino acid ending with a group at the N-terminus refers to an amino acid bearing a group on the alpha-amino nitrogen.

The invention stimulates mice through a synthetic N-terminal short peptide (GAPDH-N) antigen of human GAPDH, and a high-potency GAPDH antibody is found in the mice. Specifically, GAPDH-N antigen is utilized to immunize a BALB/C mouse for multiple times, after the immunization is enhanced to four needles, tail blood is collected for titer detection, hybridoma cell strains are prepared, positive cells of secreted antibodies are screened, stable anti-GAPDH antibody hybridoma cell strains are obtained together, ascites is prepared, monoclonal antibodies are purified and obtained, a hybridoma cell 4C7 with the highest secreted antibody titer is screened through antibody titer measurement, and experimental results show that the titer of ascites antibodies secreted by the hybridoma cell strain 4C7 can reach 8.75x10 ⁶ Is significantly higher than other 4 hybridoma cell strains; the amino acid sequences of the heavy chain and the light chain of the antibody are obtained through gene cloning and sequencing of the variable region of the antibody, and the complementarity determining regions of the heavy chain are analyzed: CDR1: N-Y-G-M-N; CDR2: W-I-G-T-N-T-G-E-P-T-Y-G-E-E-F-K-G; CDR3: D-G-Y-Y-D-F-D-Y; complementarity determining regions of the light chain: CDR1: K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N; CDR2: L-V-S-K-L-D-S; CDR3: W-Q-G-T-H-F-K-T; and preparing a recombinant antibody according to the amino acid sequences of the heavy chain and the light chain of the antibody, namely the preferred anti-GAPDH monoclonal antibody.

The present invention provides a monoclonal antibody against GAPDH comprising a heavy chain of a heavy chain CDR1 or variant thereof, a heavy chain of a heavy chain CDR2 or variant thereof, a heavy chain of a heavy chain CDR3 or variant thereof, and a heavy chain of a light chain CDR1 or variant thereof, a heavy chain of a light chain CDR2 or variant thereof, a light chain CDR3 or variant thereof;

the light chain CDR1 comprises or consists of an amino acid sequence K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N shown in SEQ ID NO. 4; the light chain CDR2 comprises or consists of an amino acid sequence L-V-S-K-L-D-S shown in SEQ ID NO. 5; the light chain CDR3 comprises or consists of the sequence W-Q-G-T-H-F-K-T shown in SEQ ID NO. 6.

A heavy chain of the variant comprising or consisting of:

in some embodiments the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 7,8,9, 10, or a sequence having at least 80%,85%,90% identity to the sequence set forth in SEQ ID NO. 7,8,9, 10; preferably at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity;

or an amino acid sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 7,8,9, 10, preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations; the amino acid mutation is preferably a conservative mutation, more preferably a substitution, insertion or deletion.

A light chain of the variant comprising or consisting of:

in some embodiments the light chain variable region comprises the amino acid sequence set forth in SEQ ID NOs 11, 12, 13, 14, or a sequence having at least 80%,85%,90% identity, preferably at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity to the sequence set forth in SEQ ID NOs 11, 12, 13, 14;

or an amino acid sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 11, 12, 13, 14, preferably 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acid mutations; the amino acid mutation is preferably a conservative mutation, more preferably a substitution, insertion or deletion.

In some embodiments, the invention also provides a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against GAPDH according to the invention. In the present invention, a nucleic acid sequence comprises a variant (e.g., substitution of degenerate codons) of its conservative substitution and a complementary sequence. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof, e.g., oligonucleotides.

In some embodiments, the invention also provides a biological material comprising a nucleic acid sequence encoding a monoclonal antibody or antibody fragment thereof against GAPDH of the invention, including an expression vector, an expression cassette, a host cell, an engineered bacterium, or a hybridoma cell line.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" refers to a coding sequence being linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in a suitable host cell, and include promoters, enhancers and other expression control elements.

In addition, the invention also provides application of the anti-GAPDH monoclonal antibody in detecting the reference protein GAPDH.

The invention also provides a detection kit comprising the anti-GAPDH monoclonal antibody of the invention.

The following are examples:

EXAMPLE 1 monoclonal antibodies against GAPDH

The heavy chain and light chain amino acid sequences of the anti-GAPDH monoclonal antibody are shown in figure 1, and are specifically as follows:

the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the heavy chain CDR1 has an amino acid sequence N-Y-G-M-N shown in SEQ ID NO. 1; the heavy chain CDR2 has an amino acid sequence W-I-G-T-N-T-G-E-P-T-Y-G-E-E-F-K-G as shown in SEQ ID NO. 2; heavy chain CDR3, the amino acid sequence shown as SEQ ID NO 3 is D-G-Y-Y-D-F-D-Y;

the light chain comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein the light chain CDR1 has an amino acid sequence K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N as shown in SEQ ID NO. 4; light chain CDR2, the amino acid sequence L-V-S-K-L-D-S shown in SEQ ID NO. 5; light chain CDR3, sequence W-Q-G-T-H-F-K-T as shown in SEQ ID NO: 6.

EXAMPLE 2 monoclonal antibodies against GAPDH

The heavy chain of the anti-GAPDH monoclonal antibody comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the heavy chain CDR1 comprises an amino acid sequence N-Y-G-M-N shown as SEQ ID NO. 1; a heavy chain CDR2 comprising the amino acid sequence W-I-G-T-N-T-G-E-P-T-Y-G-E-E-F-K-G as shown in SEQ ID NO. 2; a heavy chain CDR3 comprising the amino acid sequence D-G-Y-Y-D-F-D-Y as shown in SEQ ID NO. 3; the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO. 7,8,9 and 10;

the light chain comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein the light chain CDR1 comprises an amino acid sequence shown as SEQ ID NO. 4, K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N; light chain CDR2 comprising the amino acid sequence L-V-S-K-L-D-S as shown in SEQ ID NO. 5; a light chain CDR3 comprising the sequence W-Q-G-T-H-F-K-T as shown in SEQ ID NO. 6; the light chain variable region comprises the amino acid sequences shown in SEQ ID NO. 11, 12, 13 and 14.

EXAMPLE 3 screening of monoclonal antibodies against GAPDH

1. Antigen immunization

The synthesized N-terminal short peptide of human GAPDH (hereinafter referred to simply as GAPDH-N) was mixed with Freund's complete adjuvant to give an oily emulsion. The emulsion is subcutaneously injected into a BALB/c mouse at a dosage of 0.15mL, the immunity is enhanced in the abdominal cavity (equal amount of antigen is mixed with Freund's incomplete adjuvant) after 14 days of the first immunization, and after the immunity is enhanced to four needles, tail blood is collected for potency detection, and the potency meets the fusion requirement.

3 days before fusion, the same dose of antigen is used for intraperitoneal injection to boost immunity, and the immunization method is the same.

The amino acid sequence of GAPDH-N is as follows:

wherein freund's complete adjuvant: appearance: amber cell suspension; the components are as follows: 85% of paraffin oil, 15% of mannitol monooleate and 1mg/mL of mycobacterium tuberculosis (Mycobacterium smegmatis).

2. Preparation of hybridoma cell lines

(1) Preparation of feeder cells

BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mice were sacrificed by pulling the neck, 75% alcohol was soaked whole body, in a super clean bench, the abdominal skin was cut off with scissors under aseptic operation, the peritoneum was exposed, 5mL of RPMI1640 basal medium was injected with a syringe, washing was repeated, the washing liquid was recovered, 1000rpm, centrifugation was carried out for 5 minutes, precipitation was left, the culture broth (in the complete culture broth of RPMI1640 containing HAT) was screened with RPMI1640, and the cell concentration was adjusted to 1X 10 ⁵ mu.L/well of 96-well plate, 150. Mu.L/well, 37℃and 5% CO were added per mL ₂ Culturing overnight.

(2) Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI1640 basal culture solution, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifuging, discarding supernatant, re-suspending RPMI1640 basic culture solution, repeating for three times to obtain immune spleen cells, and counting.

(3) Preparation of myeloma cells

After 8-azaguanine screening, the mouse myeloma cells Sp2/0 are cultured to the logarithmic phase, two large bottles are taken to prepare cell suspension, the cell suspension is centrifuged, the supernatant is discarded, the cell suspension is resuspended by using RPMI1640 basic culture solution, and myeloma cells are obtained by repeating the steps for three times if needed, and the number of the myeloma cells is counted.

(4) Cell fusion and HAT selection hybridomas

Myeloma cells and immune splenocytes were mixed at a ratio of 1:10, washed 1 time with RPMI1640 basal medium in a 50mL plastic centrifuge tube, and centrifuged at 1200rpm for 10 minutes. The supernatant was discarded, the cells were mixed well, 1mL of 50% PEG 1500 was slowly added for fusion, and after 1 minute of fusion, 15mL of RPMI1640 basal medium was added to terminate the cell fusion. 1000rpm, and centrifuging for 5-10 minutes. The supernatant was discarded, and the culture broth was gently resuspended in 50mL of RPMI1640, halved in 10 96-well plates, 50. Mu.L/well, 37℃and 5% CO ₂ Culturing. Culturing was carried out until the sixth day, and the HAT medium (the complete medium of RPMI1640 containing HAT) was changed twice.

(5) Hybridoma cell screening

Antigen GAPDH-N was diluted to a final concentration of 1. Mu.g/mL with 0.05M carbonate buffer, pH 9.6. 0.1mL per well, 96 well polystyrene plates were added at 37℃for 2 hours or at 4℃overnight. The following day, 0.02M pH 7.2PBS,0.15mL/well containing 10% calf serum or 1% skim milk powder was blocked at 37 ℃ for 2 hours for detection. On the seventh day after fusion, 0.1mL of cell supernatant was taken out in the 96-well assay plate, washed with water for 30 minutes at 37℃for six times, then 2000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG was added, and after 30 minutes at 37℃and the same wash, 100. Mu.L of a phosphate buffer containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH 5.0 citric acid phosphate buffer solution, 15 minutes at 37℃was added, 50. Mu.L of a diluted sulfuric acid solution was added per well, and the absorption value at 450nm was measured. RPMI1640 complete culture solution was used as a negative control, and positive cells were obtained at a ratio of the measured value to the control value of ∈ 2.0.

The positive cell holes of the secreted antibody are cloned by a limiting dilution method on a 96-well culture plate with 1 cell/well, the positive holes are screened and cloned three times continuously according to the upper method, after the expansion culture, the positive cells are frozen in a culture solution containing 10% DMSO, and the cell density is 10 ⁶ individual/mL; five stable anti-GAPDH antibody hybridoma cell lines were obtained altogether.

3. Preparation of monoclonal antibodies

Selecting BALB/c mice with 6-8 weeks of robustness, and injecting 0.5mL of pristane into the abdominal cavity of each mouse; intraperitoneal injection 1X 10 after 10 days ⁶ And a hybridoma cell. Ascites can be generated 7-10 days after inoculating cells, the health condition and the symptoms of the ascites of animals are closely observed, the mice are killed before death as much as possible, the ascites is sucked into a test tube by a dropper, and generally 5-10 mL of ascites can be obtained by one mouse. Collecting ascites, centrifuging to obtain supernatant, and storing in a refrigerator at-20deg.C. The ascites supernatant was diluted with 3 volumes of PBS and filtered through filter paper. The resulting filtrate was applied to a protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL/min. The non-protein G adsorbed material was then washed with PBS at a flow rate of 1mL/min until the absorbance at OD280nm reached baseline. The antibody was eluted with a 0.1M glycine eluent (pH 2.5) and recovered. The recovered solution was neutralized with 0.1M Tris (pH 8.8),the antibody concentration was adjusted to a suitable concentration by ultrafiltration and sub-packaged for frozen storage at-20 ℃.

4. Potency determination

The titers of the 5 hybridoma cells and the secreted ascites antibodies were detected by an indirect ELISA method, and the antibody titers are shown in Table 1 below.

TABLE 1 determination of different antibody titers

anti-GAPDH antibody cell lines	Hybridoma cell culture supernatant titers	Ascites antibody titre
			4E8	5.24×10 ³	6.13×10 ⁴
6E4	3.23×10 ⁴	4.59×10 ⁵
			5B11	4.27×10 ³	5.34×10 ⁵
4C7	6.47×10 ⁶	8.75×10 ⁶
			5G9	3.49×10 ³	6.32×10 ⁵

The specific experimental steps are as follows:

(1) Coating: antigen GAPDH-N was diluted to 1. Mu.g/mL, 100. Mu.L/Kong Jiazhi ELISA plate, 37℃for 2 hours or 4℃overnight;

(2) Washing the plate with a plate washing machine for 5 times, injecting 350 mu L of washing liquid into each hole, and staying for 20 seconds; finally, beating to dryness;

(3) Washing off the coating liquid with washing liquid, sealing with sealing liquid, placing 150 μl of each hole at 37deg.C for 1.5-2 hr;

(4) The plate washer washes the plate 5 times, and each time, each hole is filled with 350 mu L of washing liquid, and the plate stays for 20 seconds; finally, beating to dryness;

(5) Adding a sample: respectively adding cell culture supernatant diluted into different gradients and ascites into an ELISA plate coated with antigen, reacting at 37 ℃ for 1 hour (simultaneously making a negative control hole and a positive control hole) at 100 mu L/hole;

(6) The plate washer washes the plate 5 times, and each time, each hole is filled with 350 mu L of washing liquid, and the plate stays for 20 seconds; finally, beating to dryness;

(7) Adding horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (diluted 6000 times with blocking solution), and reacting at 37 ℃ for 1 hour at 100 mu L/hole;

(8) The plate washer washes the plate 5 times, and each time, each hole is filled with 350 mu L of washing liquid, and the plate stays for 20 seconds; finally, beating to dryness;

(9) Adding a color development liquid TMB: the preparation is ready to use, 100 mu L/hole and is reacted for 30 minutes at 37 ℃ in a dark place;

(10) Terminating the reaction: 2M sulfuric acid, 50. Mu.L/well, was added to each reaction well;

(11) Microplate reader reading: 450nm,630nm wavelength.

Finally, the strain 4C7 with the highest titer is determined and screened.

EXAMPLE 4 monoclonal antibodies against GAPDH

1. Antibody variable region gene cloning and sequencing

Total RNA was extracted from hybridoma cell line 4C7 secreting anti-GAPDH antibody of example 2 using SMART ER ^TM RACE cDNA Amplification Kit kit and SMARTER II A Oligonucleotides and 5' -CDS primer in the kit are used for first-strand cDNA synthesis, and the obtained first-strand cDNA product is used as a PCR amplification template. The light chain genes were amplified with Universal Primer AMix (UPM), nested Universal Primer A (NUP) and mIgG CKR primers and the heavy chain genes were amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIgG CHR primers. Wherein the primer pair of the light chain amplifies a target band of about 0.7Kb, and the primer pair of the heavy chain amplifies a target band of about 1.5 Kb.

Purifying and recovering by agarose gel electrophoresis, adding A reaction of the product by rTaq DNA polymerase, inserting into pMD-18T vector, transforming into DH5 alpha competent cells, taking 4 clones of heavy chain and light chain gene clone respectively after bacterial colony growth, and sequencing by the company of the family Praeparatae.

The antibody obtained by analyzing the hybridoma cell strain 4C7 has the following light chain and heavy chain sequences; wherein the complementarity determining region of the heavy chain:

CDR-H1：N-Y-G-M-N；

CDR-H2：W-I-G-T-N-T-G-E-P-T-Y-G-E-E-F-K-G；

CDR-H3：D-G-Y-Y-D-F-D-Y。

complementarity determining regions of the light chain:

CDR-L1：K-S-S-Q-S-L-L-D-S-G-G-K-T-Y-L-N；

CDR-L2：L-V-S-K-L-D-S；

CDR-L3：W-Q-G-T-H-F-K-T。

2. preparation of recombinant antibodies for affinity analysis and Activity characterization

Enzyme-free indirect method was used as data in the same manner as for activity identification, and the coating was used as four gradients of 1. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.125. Mu.g/ml; antibodies were loaded from a 2-fold gradient dilution of 1000ng/ml to 0.97656 ng/ml. The OD values corresponding to the different antibody concentrations at the coating-free concentration were obtained.

Under the same coating concentration, the antibody concentration is plotted with the antibody concentration as an abscissa and the OD value as an ordinate, and the antibody concentration at the maximum OD value of 50% is calculated according to a fitting equation; the reciprocal of the affinity constant was calculated according to the following formula:

K＝(n-1)/(2*(n*Ab`-Ab))；

wherein Ab and Ab ' respectively represent the antibody concentration at 50% of the maximum OD value at the corresponding coating concentration (Ag, ag '), n=ag/Ag ';

every two coating concentrations can be combined to calculate a K value, and finally six K values can be obtained, the average value is obtained, and the reciprocal is obtained as an affinity constant KD, as shown in table 2.

TABLE 2 affinity constant KD determination results

Sample name	KD
		4C7	6.317E-10
6E4	7.934E-8
		5B11	8.446E-9
4E8	3.386E-8
		5G9	4.165E-9

The antibody affinity refers to the binding force between an antibody and an epitope or an antigenic determinant, and is essentially a non-covalent acting force, comprising attractive force, hydrogen bond, hydrophobic acting force and the like between amino acids. The affinity of an antibody can be expressed by an affinity constant KD, wherein the higher the affinity constant KD, the greater the ability of the antibody to bind to a hapten.

As can be seen from Table 2, the affinity of the purified anti-GAPDH antibody 4C7 monoclonal antibody reaches 6.317E-10, and the affinity is obviously better than that of the antibodies secreted by the other 4 hybridoma cells.

Activity assay

The antigen GAPDH-N was diluted to 1. Mu.g/mL with 50mM carbonate buffer coating solution and coated on microwell plates at 100. Mu.L per well overnight at 4 ℃; the next day, the washing liquid is washed for 2 times by PBST, and is patted dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted anti-GAPDH monoclonal antibody 4C7, performing 5-fold specific dilution from 1000ng/ml, loading, 100 μl/well, and 37 ℃ for 30min (part of supernatant 1 h); washing with PBST washing solution for 5 times, and drying; adding horseradish peroxidase-labeled goat anti-mouse IgG (immunoglobulin G) into each hole at 100 mu L and 37 ℃ for 30min; washing with PBST washing solution for 5 times, and drying; urea peroxide (50 μl/well) was added, and tetramethylbenzidine (50 μl/well) was added for 10min; adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 mu L/hole; OD values were read on the microplate reader at 450nm (reference 630 nm) as shown in Table 3.

TABLE 3 characterization of purified anti-GAPDH antibody Activity

Sample concentration ng/ml	1000	250	50	10	1	0.5	0
								OD450	4.165	3.863	0.926	0.264	0.103	0.094	0.042

The obtained anti-GAPDH antibody was compared with a commercially available product (control) and the resultant HeLa cell lysate was assayed for GAPDH by the protein assay, wherein the primary antibody (1 mg/mL) was diluted 1/10000 and the secondary antibody was ProteinFind Goat Anti-Mouse IgG HRP conjugate (HS 201-01), and the results are shown in FIG. 2.

As shown in FIG. 2, the anti-GAPDH antibody provided by the invention can still detect target protein GAPDH when the HeLa cell lysate is diluted by 25 times, and the target protein is not detected by the commercially available GAPDH antibody under the same dilution, which indicates that compared with the existing commercially available GAPDH antibody, the GAPDH antibody provided by the invention has better affinity to antigen.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A monoclonal antibody directed against GAPDH comprising a heavy chain CDR1 or a variant thereof, a heavy chain CDR2 or a variant thereof, and a heavy chain CDR3 or a variant thereof;

2. The monoclonal antibody against GAPDH of claim 1, wherein the heavy chain variable region of said variant comprises or consists of the amino acid sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID No. 7,8,9, 10, or an amino acid sequence having one or more amino acid mutations compared to the sequence shown in SEQ ID No. 7,8,9, 10.

3. The monoclonal antibody against GAPDH of claim 2, wherein the variant light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NOs 11, 12, 13, 14, or a sequence having at least 80%,85%,90% identity to the sequence shown in SEQ ID NOs 11, 12, 13, 14, or an amino acid sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NOs 11, 12, 13, 14.

4. The monoclonal antibody against GAPDH of claim 2, wherein the heavy chain variable region of said variant comprises the amino acid sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 7,8,9, 10, or a sequence having a mutation of 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids compared to the sequence shown in SEQ ID No. 7,8,9, 10; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion of an amino acid.

5. The monoclonal antibody against GAPDH of claim 3, wherein the variant light chain variable region comprises the amino acid sequence shown in SEQ ID No. 11, 12, 13, 14, or a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the sequence shown in SEQ ID No. 11, 92%,93%,94%,95%,96%,97%,98%,99% or 100% or a sequence having a mutation of 1, 2, 3, 4, 5, 6, 7,8,9 or 10 amino acids compared to the amino acid sequence shown in SEQ ID No. 11, 12, 13, 14; the amino acid mutation is a conservative mutation, including substitution, insertion or deletion of an amino acid.

6. The anti-GAPDH monoclonal antibody of any one of claims 1 to 5, wherein the heavy chain amino acid sequence of said antibody is as set forth in SEQ ID No. 15 and the light chain amino acid sequence is as set forth in SEQ ID No. 16.

7. A nucleic acid sequence encoding the anti-GAPDH monoclonal antibody or antibody fragment thereof of any one of claims 1 to 6, comprising a gene, a cDNA molecule, an mRNA molecule, and fragment oligonucleotides thereof.

8. A biological material comprising a nucleic acid sequence encoding the anti-GAPDH monoclonal antibody or antibody fragment thereof of any one of claims 1 to 6, comprising an expression vector, an expression cassette, a host cell, an engineered bacterium, or a hybridoma cell line.

9. Use of a monoclonal antibody against GAPDH as claimed in any of claims 1 to 6 for the detection of the internal reference protein GAPDH.

10. A test kit comprising the anti-GAPDH monoclonal antibody of any one of claims 1 to 6.