CN113249445B

CN113249445B - Nucleic acid-antibody dual cancer detection kit

Info

Publication number: CN113249445B
Application number: CN202110587789.5A
Authority: CN
Inventors: 马红妙; 张玲
Original assignee: Hangzhou Kangbaiyu Medical Laboratory Co ltd
Current assignee: Hangzhou kangbaiyu medical laboratory Co.,Ltd.
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2022-01-04
Anticipated expiration: 2041-05-28
Also published as: CN113249445A

Abstract

The invention relates to a nucleic acid-antibody dual cancer detection kit. The invention obtains the specific RPA primer and probe aiming at CXCL1 detection by analyzing the CXCL1 gene sequence; meanwhile, monoclonal antibodies with good effects are screened and obtained aiming at the CXCL1 protein, and the monoclonal antibodies are used for preparing an immunodetection kit. The two detection methods are combined for use, so that the detection accuracy can be further improved, the cost is low, and the method is suitable for large-scale popularization and use.

Description

Nucleic acid-antibody dual cancer detection kit

Technical Field

The invention relates to the field of biological medicine, in particular to a nucleic acid-antibody dual cancer detection kit.

Background

Bladder cancer is the most common malignancy in the urinary system and urothelial cancer is the most common type of pathology worldwide. In male malignant tumors, the incidence rate of bladder cancer is 4 th, the mortality rate is 9 th, and the incidence rate ratio of male and female is 3: 1. Over 33 million patients with bladder tumors are newly diagnosed each year, and over 13 million die of bladder cancer. More than 2/3 patients are diagnosed with non-muscle invasive bladder urothelial cancer, but are highly recurrent post-operatively and 10% to 30% of recurrent patients will progress to muscle invasive bladder urothelial cancer, with about 30% of patients diagnosed with distant organ metastasis. Therefore, the method for quickly and accurately detecting the bladder cancer is established, and has great significance for clinical diagnosis and timely and effective treatment of bladder cancer patients.

CXCL1 (Chemokine (C-X-C motif) ligand 1), Chemokine CXC motif ligand 1, is a cytokine belonging to the CXC Chemokine family, which was also previously known as GRO1 (Human growth-regulated oncogene 1, growth-regulating oncogene 1), KC (Keratinocyte chemoattractant, Keratinocyte chemotaxis), NAP-3 (neutrophiling protein 3, Neutrophil activating protein 3) and MSGA-alpha (Melanoma growth stimulating hormone alpha). In humans, the protein is encoded by the cxcl1 gene and is located on human chromosome 4. CXCL1 is expressed in macrophages, neutrophils and epithelial cells and exhibits neutrophil chemotactic activity, CXCR2 is a chemokine receptor for CXCL and exhibits chemotactic effects upon binding to CXCL 1. Research shows that CXCL1 is highly expressed in bladder cancer, and in the case of poor differentiation, late clinical stage and recurrence, the expression degree is obviously higher than that in the case of good differentiation, early clinical stage and no recurrence, so CXCL1 can be used as a urothelial cell tumor marker for diagnosis, early intervention and treatment of bladder cancer. "W02007026895" discloses the use of CXCL1 protein as a tumor marker for detecting urothelial cancer, and W02010/050554 discloses CXCL1 antibody and a detection kit therefor, but there is still a great need for a new CXCL1 detection kit. Therefore, developing a detection method which is simple to prepare, low in cost, convenient to use and free of a high-precision instrument is an important aspect of the current research.

Disclosure of Invention

Based on the above findings, the primary object of the present invention is to provide a novel bladder cancer detection kit. The invention overcomes the defects of the prior art and provides the detection kit which has the advantages of simple preparation, low cost, convenient use and no need of a high-precision instrument. The kit detects the bladder cancer patient by a nucleic acid-antibody dual detection method, and further improves the detection accuracy.

The invention provides the following technical scheme:

the invention provides a nucleic acid detection reagent aiming at CXCL1, which comprises a detection primer pair and a probe, wherein the sequence of an upstream primer is shown as SEQ ID NO. 2, the sequence of a downstream primer is shown as SEQ ID NO. 3, and the sequence of the probe is shown as SEQ ID NO. 4.

In some embodiments, the probe of the invention is modified with dSpacer at a position 36bp apart from the 5 ' end in the probe, thymine (dT) at positions 34bp and 39bp apart from the 5 ' end on both sides of the dSpacer molecule is replaced with a fluorophore FAM and a quencher BHQ1, respectively, and is modified at the 3 ' end of the probe with a blocking group C3 Spacer.

A monoclonal antibody that specifically binds to CXCL1, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, the amino acid sequences of the heavy chain CDR1 region, CDR2 region and CDR3 regions are set forth in SEQ ID NOs 7, 8 and 9, respectively; the light chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the amino acid sequences of the light chain CDR1 region, the CDR2 region and the CDR3 region are shown in SEQ ID NOs 10, 11 and 12, respectively.

In another aspect, the invention provides a monoclonal antibody that specifically binds CXCL1, comprising a heavy chain variable region comprising the amino acid sequence SEQ ID No. 5 and a light chain variable region comprising the amino acid sequence SEQ ID No. 6.

In some embodiments, the anti-CXCL 1 antibodies of the invention comprise or consist of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region sequence, a heavy chain variable region sequence, or a CDR sequence as described above, and each light chain comprises a light chain constant region sequence, a light chain variable region sequence, or a CDR sequence as described above. The antibody of the invention may be a full length antibody comprising a constant region, the full length antibody light chain constant region further comprising murine kappa, lambda chain sequences. The full-length antibody heavy chain constant region further comprises murine IgG1, IgG2a, IgG2b, IgG3, IgA or IgM sequences.

In some embodiments, an anti-CXCL 1 antibody of the invention is a Fab fragment, Fab 'fragment, F (ab')2 fragment, Fv fragment, diabody, linear antibody, single chain antibody molecule, or multispecific antibody formed from an anti-CXCL 1 antibody or antibody fragment described above.

In some embodiments, the anti-CXCL 1 antibody of the invention is used for the preparation of a diagnostic kit for bladder cancer.

In some embodiments, the nucleic acid detection reagent for CXCL1 described in the present invention is used for preparing a bladder cancer diagnostic kit. In some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody;

in some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA antibody, wherein the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region which comprises a CDR1 shown in SEQ ID NO. 7, a CDR2 shown in SEQ ID NO. 8 and a CDR3 shown in SEQ ID NO. 9, and a light chain variable region which comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 shown in SEQ ID NO. 11 and a CDR3 shown in SEQ ID NO. 12.

In some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA antibody, wherein the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO. 5, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 6.

Use of a kit for detecting bladder cancer by in vitro determination of the expression of CXCL1 protein, or a gene encoding the protein, in a biological sample from a subject.

In some embodiments, the biological sample of the invention is blood, plasma, serum, urine, bladder tissue or cells.

Advantageous effects

The invention prepares CXCL1 nucleic acid detection reagent by specific RPA primer and probe aiming at CXCL 1; meanwhile, a monoclonal antibody with a good effect is screened and obtained aiming at the CXCL1 antigen, the monoclonal antibody is prepared into a kit for detecting the CXCL1 protein, and the two detection methods are combined for use in the diagnosis of patients with bladder cancer, so that the detection accuracy can be further improved, the cost is low, and the method is suitable for large-scale popularization and use.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

Figure 1 RPA method detects CXCL1 mRNA expression in cells.

Fig. 2 CL228 specifically binds CXCL 1.

Figure 3 ELISA method detects CXCL1 protein.

Figure 4 the ELISA method detects CXCL protein in urine.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 design of RPA-specific detection primers

The gene sequence of CXCL1 is compared with the homologous sequence thereof, and a more specific region is selected as a target region for primer detection, and the sequence of the region is shown as SEQ ID NO. 1.

According to the rule of RPA primer design and the optimization of the primer, a specific primer sequence is obtained, and the sequence is shown as follows:

upstream primer (SEQ ID NO: 2):

CGAGTGGCACTGCTGCTCCTGCTCCTGGTAGCCG

downstream primer (SEQ ID NO: 3):

Biotin- CTATGGGGGATGCAGGATTGAGGCAAGCTTTCC

probe sequence (SEQ ID NO: 4):

FAM-GAATTCACCCCAAGAACATCCAAAGTGTGAACGTGAAGTCCCCCGGACCC, dSpacer is adopted to modify the position 36bp apart from the 5 ' end in the probe, thymine (dT) 34bp and 39bp apart from the 5 ' end on both sides of dSpacer molecule is replaced by a fluorescent group FAM and a quenching group BHQ1 respectively, and the 3 ' end of the probe is modified by a blocking group C3 Spacer.

Example 2 detection of CXCL1 Gene expression by RPA method

Separately culturing the bladder cancer cell strain T24 and normal bladder epithelial cell SV-HUC-1. Total RNA from cells was extracted using Trizol reagent (Thermo Co.) and following the instructions. The extracted RNA was used as a template for the RPA test, and the primers designed in example 1 were used for the RPA test

Amplifying, setting ultrapure water as a negative control, setting the reaction duration (namely the amplification cycle number) at 40 ℃ of the reaction to be 25min respectively, and setting an RPA reaction system to be 50 mu l, wherein 2 mu l of forward and reverse primers (10 mu M), 2 mu l of reverse primers (10 mu M), 0.6 mu l of probe, 25 mu l of buffer solution containing recombinase, DNA polymerase, single-strand binding protein, endonuclease IV and reverse transcriptase, 1 mu l of template and 17.9 mu l of lddH2O, fully oscillating, uniformly mixing and instantaneously separating, finally adding 2.5 mu l of 280mM magnesium acetate (MgOAc), and placing the reaction tube in a real-time fluorescence PCR instrument for constant temperature reaction at 40 ℃ for corresponding duration; as shown in fig. 1, the CXCL1 gene was highly expressed in the bladder cancer cell line T24, and CXCL1 expression could be detected in the human normal bladder epithelial cells SV-HUC-1, but the mRNA level of CXCL1 in the bladder cancer cell line was significantly higher than that of the human normal bladder epithelial cells.

Example 3 anti-CXCL 1 antibody preparation

After 100 μ g of CXCL1 antigen and equal volume of Complete Freund's Adjuvant (CFA) are fully and uniformly mixed to form a water-like oil pocket, a BALB/C mouse is primarily immunized through intraperitoneal injection. And after 3 weeks, 100 microgram of CXCL1 and Incomplete Freund's Adjuvant (IFA) are mixed uniformly in equal volume and emulsified, and the booster immunization is carried out by back multi-point injection. Thereafter, the immunization was performed every 2 weeks, and tail blood was collected 7 days after each immunization, and the antibody titer was measured by ELISA. When the antibody titer is proper, 100 mug of CXCL1 antigen and Incomplete Freund's Adjuvant (IFA) are mixed uniformly in equal volume and emulsified, and back multi-point injection is adopted for the last immunization. Spleens were harvested on day four and subjected to cell fusion. Spleen cells of immunized Balb/c mice were fused with a myeloma Sp2/0 cell line, and the fused cells were diluted to an appropriate concentration in Iscove's medium (0.1 mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) containing 10% serum, and then cultured in a 96-well plate. After 10 days, cell supernatants were removed and primary cultures showing a positive reaction with CXCL1 protein in the supernatants were examined by high throughput ELISA. And diluting the hybridoma cells in the holes for subcloning, and screening by an ELISA method to finally obtain a positive hybridoma cell strain which is named as CL-228. After expansion culture, the hybridoma cells were cryopreserved.

EXAMPLE 4 purification of monoclonal antibodies

BALB/c mice were injected intraperitoneally with 0.5 ml/mouse, 1 week before hybridoma inoculation. After 1 week, each mouse was inoculated intraperitoneally at about 1X10⁶(ii) individual hybridoma cells; and after 7-10 days, collecting ascites. Centrifuging ascites at 10000 Xg for 30min, removing precipitate, salting out with 50% ammonium sulfate, coarse extracting, dissolving with PBS, and dialyzing with flowing water for 5 hr; dialyzing and equilibrating with 0.1mol/L phosphate buffer (pH 8.0) overnight; and (3) loading, eluting the hybrid protein by using 0.1mol/L phosphate buffer solution (pH 8.0), eluting by using citrate eluents with different pH values, collecting elution peaks in a segmented mode, and concentrating to obtain the purified anti-CXCL 1 antibody CL-228.

Example 5 anti-CXCL 1 antibody subtype identification

The positive mouse monoclonal cell line selected by indirect ELISA was subjected to subclass measurement using a subclass measuring reagent (Sigma). The microplate provided in the kit had been pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-CXCL 1 antibody sample purified in example 4 was added to the sample wells at 50 μ l per well without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. The results show that the monoclonal antibody of the present invention is a murine monoclonal antibody of the IgG2a type, designated CL-228.

EXAMPLE 6 monoclonal antibody sequencing

The CL-228 hybridoma cell cryopreservation tube is taken out from liquid nitrogen, is rapidly thawed at 37 ℃, is centrifuged at 1000rpm for 5min to remove the cryopreservation liquid, is placed in a 100mm pore plate, is cultured until the culture liquid accounts for about 80 percent of the culture plate, is added with 1ml of Trizol reagent (Thermo company), and extracts the total RNA of the hybridoma cell according to the instruction.

Mu.g of the above total RNA was taken, DECP water was added thereto to make the volume 11. mu.l, 1.0. mu.l of oligo (dT) (10. mu.M) was added thereto, 1. mu.l of dNTPs (10mM) was added thereto, the mixture was mixed well, incubated at 65 ℃ for 5 minutes and then placed on ice for 1 minute, followed by addition of 4. mu.l of RT buffer (5X), 1.0. mu.l of DTT (100mM), 1. mu.l of Ribonucleae Inhibitor and 1. mu.l of reverse transcriptase (takara Co., Ltd.), and reacted at 50 ℃ for 10 minutes. The reaction was terminated by incubation at 80 ℃ for 10 minutes, and the obtained cDNA was stored at-20 ℃. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. The primer sequences were designed according to the literature (Bodo Brocks. Specifes-Cross active scFv Against the turbine Stroma Marker "fibrous Activation Protein" Selected by phase Display From an amplified FAP)^-/-Knock-Out Mouse).

Sequencing results show that the amino acid sequences of the heavy chain and light chain variable regions of the anti-CXCL 1 antibody CL-228 are respectively shown in SEQ ID NO:5 and SEQ ID NO:6 is shown in the specification; the amino acid sequences of 3 CDRs in the heavy chain variable region of the antibody are respectively shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9; the amino acid sequences of 3 CDRs in the light chain variable region are shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively.

Example 7 identification of antibody specificity

The monoclonal antibody purified in example 4 was cross-reacted with ELISA plates coated with CXCL2, CXCL5 and CXCL8, which are homologous proteins of CXCL1, respectively, by an indirect ELISA method, and the specificity was analyzed. The specific experimental process is that CXCL1, CXCL2, CXCL5 and CXCL8 are respectively coated, the concentration is 0.1mg/ml, the antibodies obtained in the embodiment 3 are respectively added into four different coating antigens, and the degree of cross reaction is detected by indirect ELISA. As shown in fig. 2, anti-CXCL 1 monoclonal antibody CL-228 reacted only with CXCL1 and did not cross-react with CXCL2, CXCL5 and CXCL 8.

Example 8 anti-CXCL 1 antibody affinity assay

An antibody capture Antibody (AHC) is coated on the surface of a CM5 chip by means of amino coupling, chip activation buffer solution N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), AHC and ethanolamine for blocking are prepared according to the specifications of an amino coupling kit and an anti-capture kit, a coating program in a Biacore3000 system is selected, and AHC amino is coupled on the surface of a CM5 chip. The anti-CXCL 1 antibody CL-228 obtained in example 4 was captured on the chip surface. The antibody was diluted to 1. mu.g/mL with HBS-EP + buffer, set to a flow rate of 5. mu.l/min, and coated to a response value of 300 RU. CXCL1 antigen was applied in 7 different concentration gradients and serially diluted 2-fold in HBS-EP buffer from 40nM to 0 nM. The CXCL1 antigen flow rate was set at 30. mu.l/min and the binding time was set at 3 min. The flow rate of HBS-EP + buffer was set at 30. mu.l/min, and the dissociation time was set at 10 min. Use of 3M MgCl₂As a regeneration buffer, the chip was regenerated according to the regeneration procedure. In addition, affinity assays were performed following the same procedure as for the control antibody IgG1-14 (see CN103408663B patent page 20, line 4). Calculation of binding Rate (K) by Simultaneous fitting of binding and dissociation sensorgrams_a) And dissociation Rate (K)_d). Equilibrium dissociation constant (K)_d) Using dissociation rate (K)_d) Rate of binding (K)_a) And (4) calculating. The results are shown in table one: the antibody of the invention has high affinity, and the affinity K_DThe value reaches 6.84x10^-9M。

Watch 1

Antibodies	K_a（1/Ms）	K_d（1/s）	K_D(M)
				CL-228	3.67x10⁴	2.51 x10^-4	6.84x10^-9
IgG1-14	5.77x10⁴	4.14x10^-3	7.18x10^-8

Example 9 detection of CXCL1 by ELISA

The anti-CXCL 1 monoclonal antibody CL-228 and the control antibody IgG1-14 obtained in example 4 are diluted to 2 mu g/ml, and are respectively added into an ELISA plate hole with each hole being 100 mu l, the ELISA plate is placed in an environment at 4 ℃ to be coated overnight, 100 mu l of blocking solution is respectively added into each hole, the ELISA plate hole is placed in an incubator at 37 ℃ for 30 minutes, the blocking solution is removed after the ELISA plate is taken out from the incubator. The CXCL1 protein was diluted with artificial serum to concentrations of 200, 100, 50, 25, 10, 5, 1pg/ml, respectively. Mu.l of the suspension was added to each well and incubated at 37 ℃ for 1 hour. The supernatant was removed, followed by PBST washing. The CXCL1 antigen is used to immunize rabbit to obtain polyclonal antibody of CXCL1, which is labeled with horseradish peroxidase to obtain enzyme labeled antibody. This was added to wells 100. mu.l per well and incubated at 37 ℃ for 30 minutes in an incubator. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing for 3 times. Mu.l TMB was added to each well and incubated for 15-30min at room temperature in the dark. The chromogenic reaction was stopped by adding 50. mu.l of stop solution to each well. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. The results are shown in fig. 3, in which the CL-228 signal intensity of the anti-CXCL 1 monoclonal antibody of the present invention is higher than that of the control antibody IgG1-14, and the human CXCL1 has higher detection ability.

Example 10 detection of CXCL1 in urine

The anti-CXCL 1 monoclonal antibody CL-228 and the control antibody IgG1-14 obtained in example 4 are diluted to 2 mu g/ml, and are respectively added into an ELISA plate hole with each hole being 100 mu l, the ELISA plate is placed in an environment at 4 ℃ to be coated overnight, 100 mu l of blocking solution is respectively added into each hole, the ELISA plate hole is placed in an incubator at 37 ℃ for 30 minutes, the blocking solution is removed after the ELISA plate is taken out from the incubator. The CXCL1 protein is diluted with human urine and the concentrations after dilution are 600, 300, 150, 75, 30, 15 and 10pg/ml respectively. Mu.l of the suspension was added to each well and incubated at 37 ℃ for 1 hour. The supernatant was removed, followed by PBST washing. The CXCL1 antigen is used to immunize rabbit to obtain polyclonal antibody of CXCL1, which is labeled with horseradish peroxidase to obtain enzyme labeled antibody. This was added to wells 100. mu.l per well and incubated at 37 ℃ for 30 minutes in an incubator. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing for 3 times. Mu.l TMB was added to each well and incubated for 15-30min at room temperature in the dark. The chromogenic reaction was stopped by adding 50. mu.l of stop solution to each well. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. The results are shown in fig. 4, the anti-CXCL 1 monoclonal antibody CL-228 of the invention can also detect CXCL1 protein in urine, and the effect is better than that of the control antibody IgG 1-14.

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Claims

1. A nucleic acid-antibody dual bladder cancer detection kit, which comprises an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit containing monoclonal antibody for specifically detecting CXCL 1; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA antibody, wherein the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region, sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, and sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12.

2. A nucleic acid-antibody dual bladder cancer detection kit, which comprises an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit containing monoclonal antibody for specifically detecting CXCL 1; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA antibody, wherein the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 5 and SEQ ID NO. 6 respectively.