CN118443952A - Melanoma glycoprotein B content detection kit and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a melanoma glycoprotein B content detection kit and a preparation method thereof, wherein the kit comprises a standard substance, an immune magnetic bead coated by a melanoma glycoprotein B monoclonal antibody I, an enzyme-labeled melanoma glycoprotein B monoclonal antibody II, a blocking agent, a chemiluminescent substrate and an auxiliary reagent; the blocking agent is an APS-S100a8/a9 combined blocking agent. The preparation method comprises the following steps: s1, preparing immunomagnetic beads; selecting magnetic nano particles as a carrier, and coating by adopting a mouse anti-human PD-L1 monoclonal antibody; s2, preparing an enzyme-labeled antibody: goat anti-mouse PD-1 monoclonal antibodies were labeled with alkaline phosphatase. The invention can effectively eliminate interference factors by adding the blocking agent and combining with an advanced magnetic particle chemical immunoassay method, has simple operation and high sensitivity, and greatly improves the detection accuracy of the magnetic particle chemical luminescence immunoassay method.

Description

Melanoma glycoprotein B content detection kit and preparation method thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a melanoma glycoprotein B content detection kit and a preparation method thereof.

Background

Neurodegenerative diseases are a group of heterogeneous diseases characterized by progressive neuronal loss and decreased brain function. With aging population, the incidence rate of neurodegenerative diseases is increased year by year, and great burden is brought to families and society. However, effective prevention methods are rare. GPNMB (Glycoprotein Non-METASTATIC MELANOMA PROTEIN B) is a membrane protein and has attracted considerable attention in the fields of oncology and immunology. Recent studies have shown that there is a relationship between GPNMB and Parkinson's Disease (PD).

PD is a neurodegenerative disease of the nervous system and is mainly characterized by symptoms such as dyskinesia, muscle stiffness, tremors, and the like. Although the exact cause of PD remains unclear, it has been determined that inflammation, oxidative stress, and degenerative damage to neurons play an important role in their development. GPNMB plays an important role in the immunomodulation and inflammation process. Recent studies have found that the expression level of GPNMB is significantly increased in brain tissue of PD patients. This finding suggests that GPNMB may be involved in the pathogenesis of PD.

Further studies revealed the potential role of GPNMB in PD. Overexpression of GPNMB is associated with degenerative damage to neurons and inflammatory responses. It may have an impact on the development of PD by modulating neuronal survival, improving oxidative stress and reducing inflammatory responses.

Melanoma is a malignant tumor, usually associated with abnormal proliferation of melanocytes. Melanoma has many antigen types, mainly including MAGE, NY-ESO-9, gp72, etc., and therefore, in order to eliminate interference and ensure accuracy of detection results, a suitable detection method is required to be selected when GPNMB content test is performed.

Colloidal gold immunochromatography is favored because of simple and rapid operation, but has lower sensitivity and low accuracy for detecting substances with low concentration; the enzyme-linked immunosorbent assay (ELISA) has long detection time, is easy to interfere, has limited sensitivity, has high requirement on operators, has inaccurate quantification and low detection flux; the magnetic particle chemiluminescence immunoassay method is a rapid, sensitive, high in anti-interference capability and high in repeatability, and can detect substances with extremely low concentration, but the detection result is interfered by multiple factors, and an undetermined or false positive or false negative result can appear, so that the problem of interference is very important to be solved.

Disclosure of Invention

The invention aims to solve the technical problem of providing a melanoma glycoprotein B content detection kit and a preparation method thereof, wherein interference factors can be effectively eliminated by adding a blocking agent and combining with an advanced magnetic particle chemical immunoassay method, the operation is simple, the sensitivity is high, and the detection accuracy of the magnetic particle chemical luminescence immunoassay method is greatly improved.

In order to solve the technical problems, the invention adopts the following technical scheme:

The melanoma glycoprotein B content detection kit is characterized in that: the kit comprises a standard substance, immune magnetic beads coated by a melanoma glycoprotein B monoclonal antibody I, an enzyme-labeled melanoma glycoprotein B monoclonal antibody II, a blocking agent, a chemiluminescent substrate and an auxiliary reagent; the blocking agent is an APS-S100a8/a9 combined blocking agent.

Preferably, the melanoma glycoprotein B monoclonal antibody I is a mouse anti-human PD-L1 monoclonal antibody; the melanoma glycoprotein B monoclonal antibody II is a goat anti-mouse PD-1 monoclonal antibody.

Preferably, the biological enzyme used for the enzyme labeling is alkaline phosphatase.

Preferably, the chemiluminescent substrate is an enzymatic chemiluminescent substrate solution which is AMPPD and analogues thereof or luminol and derivatives thereof.

Preferably, the auxiliary reagent comprises the following reagents:

antibody dilution buffer: PBS +0.5% bsa +0.05% tween20;

washing buffer: PBS +0.05% tween20;

blocking buffer: pbs+2% bsa or pbs+1% hsa;

Termination agent: h ₂SO₄.

The method for preparing the melanoma glycoprotein B content detection kit according to any one of the above, comprising the following steps:

S1, preparing immunomagnetic beads; selecting magnetic nano particles as a carrier, coating by adopting a mouse anti-human PD-L1 monoclonal antibody, and diluting by adopting an antibody dilution buffer solution to obtain a magnetic separation reagent;

s2, preparing an enzyme-labeled antibody: goat anti-mouse PD-1 monoclonal antibodies were labeled with alkaline phosphatase.

Preferably, the specific method for preparing the magnetic separation reagent in step S1 is as follows:

s11, placing the magnetic nano particles on a magnetic rack after re-suspending, magnetically separating for 1min, and removing supernatant;

S12, adding an antibody dilution buffer solution to fully resuspend the magnetic beads, then placing the magnetic beads on a magnetic rack for magnetic separation for 1min, and removing supernatant, and repeating the steps three times;

S13, adding an antibody dilution buffer solution, fully suspending magnetic beads, adding an APS-S100a8/a9 combined blocker and EDC, uniformly mixing at room temperature in a dark place for reaction for 30min, and removing supernatant after magnetic separation on a magnetic frame for 1 min;

s14, adding a washing Buffer solution for washing once, placing the mixture on a magnetic rack for magnetic separation for 1min, removing supernatant, adding a Binding Buffer, adding GPNMB, uniformly mixing, and uniformly mixing at room temperature in a dark place for reaction for 3h;

S15, adding a washing buffer solution to wash the magnetic beads for 3 times, removing the supernatant for the last time, adding glycine, uniformly mixing, and uniformly mixing at room temperature in a dark place for reaction overnight;

S16, adding a washing buffer solution, washing for three times, adding a blocking buffer solution, and adding a joint blocker, an Abeta 1-40 antibody and an Abeta 1-42 antibody; mixing, and storing at 4deg.C.

Preferably, the mouse anti-human PD-L1 monoclonal antibody adopts a nano antibody.

The invention has the beneficial effects that:

1. The method has the characteristics of high specificity, time saving, safety, no toxicity and the like, can obviously improve the detection rate, and provides a reliable and efficient sample pretreatment method for pathogen detection.

2. Compared with the prior art, the invention adopts the magnetic nano particles as the carrier to prepare the immunomagnetic beads, has the advantages of narrow particle size distribution range, low magnetic targeting property, stable crystal form and single component, and the enrichment rate is up to more than 90 percent, so that the test result of the kit is more accurate and stable.

3. According to the invention, an APS-S100a8/a9 combined blocker is adopted, and researches show that astragalus polysaccharide can sensitize the blocking effect of the blocker, and can effectively remove interference antigens of melanoma in a sample in the detection process.

4. The invention adopts the blocking agent and the advanced magnetic particle chemical immunoassay method to effectively eliminate interference factors, has simple operation and high sensitivity, and greatly improves the detection accuracy of the magnetic particle chemical luminescence immunoassay method.

Detailed Description

The invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention, in order to facilitate the understanding of those skilled in the art.

Example 1

1. Preparation of nanobody of mouse anti-human PD-L1 monoclonal antibody:

1.1 immunization of mice: healthy adult mice are selected, recombinant protein PD-L1 and Freund's adjuvant are uniformly mixed according to the proportion of 1:1, and 6-7 mug/Kg are immunized by adopting a back subcutaneous multipoint injection mode, and the immunization is performed four times, wherein the immunization interval is 2 weeks. The mouse peripheral blood was then collected and used to construct phage display libraries.

1.2 Isolation of murine lymphocytes: lymphocytes are analyzed from the collected camel source anticoagulated whole blood according to routine procedures in the technical field, 1mL of RNA separating reagent is added to every 2.5X10 living cells, 1mL is taken for RNA extraction, and the rest is preserved at-80 ℃.

1.3 Total RNA extraction: total RNA was extracted according to procedures conventional in the art and adjusted to a concentration of 1. Mu.g/. Mu.L with RNase-free water.

1.4 Reverse transcription synthesis of cDNA:

Reverse transcription of cDNA was performed using the RNA obtained in step 1.3 as a template according to the instructions of the reverse transcription kit (transcripor FIRST STAND CDNA SYNTHESIS KIT of Roche Co.).

1.5 Antibody variable region Gene amplification: the cDNA obtained by reverse transcription was used as a template for PCR reaction. Amplification was performed in two rounds, and the primer sequences for the first round of PCR were as follows:

CALL001：GTCCTGGCTGCTCTTCTACAAGG

CALL002：GGTACGTGCTGTTGAACTGTTCC

The PCR reaction conditions and procedures were: 95 ℃ for 5min;95℃for 30s,57℃for 30s,72℃for 30s,30cycles;72 ℃ for 7min

The band of about 700bp was recovered by using agarose gel recovery kit gel, and finally the nucleic acid concentration was adjusted to 5 ng/. Mu.l with water.

The primer sequences for the second round of PCR were as follows:

VHH-Back：GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH-For：CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

the PCR reaction conditions and procedures were: 95 ℃ for 5min;95℃for 30s,55℃for 30s,72℃for 30s,15cycles;72 ℃ for 7min

The PCR product was purified using a PCR product recovery kit.

1.6 Vector construction

PMES 4.5. Mu.g of the digested vector and 450ng of the digested secondary PCR product were subjected to PstI and BstEII double digestion, respectively, 15. Mu. L T4 DNA ligase was added to the secondary PCR product, buffer and water were added to a total volume of 150. Mu.L, and the ligation was performed overnight at 16℃and the ligation product was recovered. The PCR product recovery kit was used for product recovery, eluting with 20. Mu.L water.

1.7 Electric conversion and storage Capacity determination

10. Mu.L of the purified ligation product was added to a pre-chilled electrocuvette containing 50. Mu.L of E.coli TG1 competent cells, and electrotransformation was performed using an electrotransformation apparatus (ECM 630 electrotransformation apparatus of BTX, USA), the electrocuvette was removed, and the transformant was resuscitated and cultured. Clones were randomly picked and colony PCR identified. The stock volume was estimated from the PCR positive rate (stock volume = clone number x dilution x [ positive rate ] PCR identification x 10).

The primer sequences were as follows:

MP57：TTATGCTTCCGGCTCGTATG

GIII：CCACAGACAGCCCTCATAG

1.8 phage amplification

Resuscitated bacterial solution was inoculated into YT-AG medium and cultured at 37℃at 200rpm until the culture OD600 = 0.5. 1010ml of the bacterial liquid was taken out and added to 4X 10VCSM13 and the mixture was allowed to stand at 37℃for 30min for infection. Centrifuging at 4000rpm at normal temperature for 10min, and removing the supernatant. The cells were resuspended in 2 XYT-AK (ampicillin and kanamycin) medium and incubated overnight at 37℃at 200 rpm. The supernatant was centrifuged in a 40ml tube, 10ml of PEG/NaCl (20%/2.5M) solution was added and thoroughly mixed, the supernatant was discarded by centrifugation, the pellet was washed with 1ml of ice PBS and centrifuged, 250. Mu.l of pre-chilled PEG/NaCl was taken, thoroughly mixed and washed for resuspension.

Phage titer was determined: TG1 was grown to od600=0.4, phages were diluted in gradient with LB medium, phage TG1 cultures diluted in doubling ratios were mixed, plaque formation in the plates was observed the next day, diluted gradient plates with plaque numbers between 30 and 300 were counted and phage titer (pfu) was calculated according to the following formula.

Phage titer (pfu/ml) =dilution x number of plaques x 100

1.9 Nanobody screening

Positive clones were screened by ELISA with recombinant PD-L1 antigen. ELISA plates were coated with recombinant PD-L1 antigen, blocked with 5% BSA, and washed with PBST. Mu.l phage supernatant was added to each well and left at 37℃for 1h. The supernatant was discarded, and HRP-labeled secondary antibody against M13 was added and left at 37 ℃ for 1h. The supernatant was discarded, TMB solution was added, incubated at room temperature for 5min, 2M sulfuric acid stop solution was added to each well, and the wells were read with an ELISA reader at 450 nm.

Clones positive for phage ELSIA were selected and plasmids were extracted. Sequencing results were analyzed using Vector NTI software, and the antibody light and heavy chain genes were analyzed by registering IMGT (https:// www.imgt.org/IMGT_ vquest) to determine the framework regions (framework regions, FR) and complementarity determining regions (complementarity determining regions, CDR) of the variable regions.

2. Preparation of anti-PD-L1 nanobody

2.1 Nanobody original strain TG1 amplification and nanobody recombinant plasmid transformation E.coli BL21 (DE 3) original strain TG1 glycerol bacteria containing nanobody nucleic acid were transformed according to 1:1000 ratio was inoculated into 5mL fresh LB-A medium, cultured at 37℃and 200rpm overnight. The following day, plasmids were extracted using PLASMID MINI KIT (OMEGA) according to the instructions. After verification, 1. Mu.l of the above plasmid was transformed into 100. Mu.l of competent cells, gently mixed, placed on ice for 30min, heat-shocked in a water bath at 42℃for 90s, and cooled in an ice bath for 3min. 600 mu lLB of culture medium was added to the centrifuge tube and cultured with shaking at 37℃for 60min. 100 μl of the supernatant was spread on LB-A plates with ase:Sub>A triangular spreader and incubated overnight at 37deg.C with inversion.

2.2 Induction of expression of nanobodies

The above monoclonal colonies were picked up in LB-A medium and cultured overnight at 37℃with shaking. The following day, the bacterial liquid is taken according to the following formula 1:100 ml of fresh LB-A medium was added at ase:Sub>A ratio of 100, and the culture was performed at 37℃for 3 hours with shaking until the bacterial liquid OD600 = 0.8 or so, and 1mM IPTG was added at ase:Sub>A final concentration and induced overnight at 30 ℃. On the third day, 8000rpm, centrifugation is performed for 10min to collect the cells, and 1.5mL of precooled TES buffer is added to resuspend the pellet. After 2min of ice bath, the cycle was repeated 6 times with gentle shaking for 30 s. 3.0mlTES/4 (TES was diluted 4-fold with water) was added, and after gentle shaking for 30s, the ice bath was allowed to stand for 2min, and the shaking and standing steps were repeated 6 times as well. Centrifuging at 9000rpm and 4deg.C for 10min, and collecting supernatant to obtain periplasm extract.

2.3 Purification and identification of nanobodies

After the IMAC Sepharose (GE company) was resuspended, 2ml was added to the gravity column and allowed to stand for 30min to allow the Sepharose to naturally settle to the bottom of the gravity column and the preservation buffer was drained. 2 column volumes of nickel sulfate solution (0.1M) were added and the nickel sulfate solution was tapped at a flow rate of about 8 s/drop; adding 10 times of column volume of balance buffer to balance and wash sepharose, and keeping the flow rate unchanged; diluting a sample with a balancing buffer solution for 2 times, adding the diluted sample into a gravity column, regulating the flow rate to 6 s/drop, and collecting penetrating fluid; adding a washing buffer solution with a volume of 10 times of the column volume to wash sepharose, maintaining the flow rate unchanged, and collecting washing solution; adding an elution buffer solution with the volume of 3 times of the column, maintaining the flow rate at 6 s/drop, and collecting the elution solution containing the target protein; finally, sequentially adding 10 times of column volume of balance buffer, 10 times of column volume of pure water and 10 times of column volume of 20% ethanol to wash sepharose, and finally reserving 4ml of 20% ethanol for preserving chromatographic columns. And (3) performing SDS-PAGE detection on the collected samples to obtain the purified nano antibody.

Example 2

Preparation of immunomagnetic beads:

magnetic nanoparticles are selected as carriers and coated with a mouse anti-human PD-L1 monoclonal antibody.

Polylysine modified iron oxide magnetic nanoparticle (PLL@Fe2O3) product No. Mag1100 of Corp technology, beijing, among magnetic nanoparticle selections.

Assembling a kit:

standard substance, melanoma glycoprotein B monoclonal antibody I coated immunomagnetic bead, enzyme-labeled melanoma glycoprotein B monoclonal antibody II, blocker, chemiluminescent substrate and auxiliary reagent.

Wherein the blocking agent is APS-S100a8/a9 combined blocking agent, and is formed by combining astragalus polysaccharide and S100a8/a9 through peptide chain modification; the melanoma glycoprotein B monoclonal antibody I is a mouse anti-human PD-L1 monoclonal antibody; the melanoma glycoprotein B monoclonal antibody II is a goat anti-mouse PD-1 monoclonal antibody; the biological enzyme adopted by the enzyme labeling is alkaline phosphatase; the chemiluminescent substrate is an enzymatic chemiluminescent substrate solution, and is AMPPD and analogues thereof or luminol and derivatives thereof.

Example 3

Use of the above kit

3.1 Preparation of magnetic separation reagents

3.1.1 After re-suspending the magnetic nanoparticles, 1ml was taken in a 15ml centrifuge tube, placed on a magnetic rack for magnetic separation for 1min, and the supernatant was carefully removed.

3.1.2, Adding 10ml of antibody dilution buffer (PBS+0.5% BSA+0.05% tween 20) to fully resuspend the beads, placing on a magnetic rack for magnetic separation for 1min, carefully removing the supernatant, and repeating the steps three times.

3.1.3, Adding 10ml of antibody dilution buffer (PBS+0.5% BSA+0.05% tween 20) to fully suspend the magnetic beads, adding 1mlAPS-S100a8/a9 combined blocker and 1ml EDC (dichloroethane), uniformly mixing at room temperature in a dark place for reaction for 30min, placing on a magnetic frame for magnetic separation for 1min, and carefully removing the supernatant.

3.1.4, 10Ml of washing buffer (PBS+0.05% tween 20) is added for washing once, the mixture is placed on a magnetic frame for magnetic separation for 1min, the supernatant is carefully removed, 10ml Binding Buffer mg of GPNMB is added, and the mixture is uniformly mixed and reacted for 3h at room temperature in a dark place.

3.1.5, 10Ml of washing buffer (PBS+0.05% tween 20) is added to wash the magnetic beads for 3 times, 5ml of glycine is added to the mixture after the supernatant is removed for the last time, and the mixture is uniformly mixed at room temperature and in a dark place for reaction overnight.

3.1.6, 10ML of wash buffer (PBS+0.05% tween 20) was added for three washes, 10mL of blocking buffer (PBS+2% BSA or PBS+1% HSA) was added and the combination blocker and 0.03ng/mL Abeta 1-40 antibody and 0.05ng/mL Abeta 1-42 antibody were added at 10 mg/L. Mixing, and storing at 4deg.C.

3.2 Preparation of enzyme-labeled antibodies

3.2.1 AP (alkaline phosphatase) was prepared as an AP solution at a concentration of 1.0-5.0mg/mL with TRIS buffer.

3.2.2, Respectively activating the goat anti-mouse PD-1 monoclonal antibody and the AP solution, and mixing the activated AP solution and the goat anti-mouse PD-1 monoclonal antibody according to a molar ratio of 1: (1-3) and carrying out coupling reaction under the action of 1M magnesium chloride solution to obtain a coupling product.

3.2.3 Adding the conjugate into a separation and purification instrument, using Tris buffer with pH of 7.5 as a mobile phase and a flow rate of 1ml/min, and collecting the separated product for 25-45min to obtain the AP-labeled anti-GPNMB.

Example 4 (addition of blocking agent)

The accurate detection effect of the blocking agent kit is verified by collecting 96 normal human samples and blood samples of 46 patients subjected to PET-CT examination and clinical parkinsonism, and the detection is judged by taking the reference range of normal human as 0-125pg/mL and the reference range of the normal human as more than 125pg/mL as positive. The test is carried out by using the kit, and the results are shown in the following table, wherein the table is a statistical table of the detection results of the kit, PET-CT examination and clinical coincidence rate.

TABLE 1 comparison of the test results of the kit with the results of the PET-CT examination

The sensitivity of the kit (adding blocking agent) is far higher than that of the traditional ELISA kit in the sensitivity comparison experiment of the kit (adding blocking agent) and the traditional ELISA kit. The test results are shown in the following table

TABLE 2 sensitivity of the kit

Concentration pg/mL	0	100	500	2500	5000	10000
							Luminescence value	9561	144447	719871	3401173	6998744	12947783

Table 3 zero emission values

TABLE 4 sensitivity of traditional ELISA kits

TABLE 5 zero absorbance

The serum sample and the plasma sample are detected by the kit (adding blocker) and the traditional ELISA reagent respectively, and the comparison data are as follows:

TABLE 6 comparison of detection results of the present kit and conventional ELISA reagents

Example 5

The accurate detection effect of the kit without adding the blocking agent is verified by collecting 96 normal human samples and blood samples of 46 patients subjected to PET-CT examination and clinical manifestation of Parkinson, and the detection is judged by taking the reference range of normal human as 0-125pg/mL and the reference range of the normal human as positive with the reference range of more than 125 pg/mL. The test was performed with a kit (without blocking agent) and the results are shown in the following table, which is a statistical table of the detection results of the kit of the present invention and the clinical compliance rate of PET-CT examination.

TABLE 7 comparison of detection results of kit (without blocker) with PET-CT examination results

The serum sample and the plasma sample are detected by the kit (without blocking agent) and the traditional ELISA reagent, and the comparison data are as follows:

Table 8 comparison of the test results of the kit (without blocking agent) and the conventional ELISA reagent

Example 6

The detection accuracy effect of the astragalus polysaccharide kit is verified by collecting 96 normal human samples and blood samples of 46 patients subjected to PET-CT examination and clinical parkinsonism, and the detection accuracy effect is judged by taking the reference range of normal human as 0-125pg/mL and the reference range of the normal human as more than 125pg/mL as positive. The test was performed with a kit (without astragalus polysaccharide), and the results are shown in the following table, which is a statistical table of the detection results of the kit (without astragalus polysaccharide) and the clinical compliance rate of PET-CT examination.

Table 9 comparison of the detection results of the kit (without Astragalus polysaccharides added) with the PET-CT examination results

The serum sample and the plasma sample are detected by a kit (without adding astragalus polysaccharide) and a traditional ELISA reagent respectively, and the comparison data are as follows:

TABLE 9 comparison of the test results of the present kit and the conventional ELISA reagents

The test result shows that the kit can detect samples with lower content, and the test sensitivity is far higher than that of the prior art.

The kit added with the blocking agent has obviously improved clinical compliance and sensitivity.

In particular, the kit adopting the combination blocker added with astragalus polysaccharide has better performance in terms of clinical compliance rate and sensitivity.

All technical features in the present embodiment can be modified in appearance according to actual needs.

The foregoing embodiments are preferred embodiments of the present invention, and in addition, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.

Claims (8)

1. The melanoma glycoprotein B content detection kit is characterized in that: the kit comprises a standard substance, immune magnetic beads coated by a melanoma glycoprotein B monoclonal antibody I, an enzyme-labeled melanoma glycoprotein B monoclonal antibody II, a blocking agent, a chemiluminescent substrate and an auxiliary reagent;

the blocking agent is an APS-S100a8/a9 combined blocking agent.

2. The melanoma glycoprotein B content detection kit according to claim 1, wherein: the melanoma glycoprotein B monoclonal antibody I is a mouse anti-human PD-L1 monoclonal antibody; the melanoma glycoprotein B monoclonal antibody II is a goat anti-mouse PD-1 monoclonal antibody.

3. The melanoma glycoprotein B content detection kit according to claim 1, wherein: the biological enzyme adopted by the enzyme labeling is alkaline phosphatase.

4. The melanoma glycoprotein B content detection kit according to claim 1, wherein: the chemiluminescent substrate is an enzymatic chemiluminescent substrate solution, and is AMPPD and analogues thereof or luminol and derivatives thereof.

5. The melanoma glycoprotein B content detection kit according to claim 1, wherein: the auxiliary reagent comprises the following reagents:

antibody dilution buffer: PBS +0.5% bsa +0.05% tween20;

washing buffer: PBS +0.05% tween20;

blocking buffer: pbs+2% bsa or pbs+1% hsa;

Termination agent: h ₂SO₄.

6. The method for preparing the melanoma glycoprotein B content detection kit according to any one of claims 1 to 5, characterized in that: the method comprises the following steps:

S1, preparing a magnetic separation reagent; selecting magnetic nano particles as a carrier, coating by adopting a mouse anti-human PD-L1 monoclonal antibody, and then diluting by adopting a buffer solution to obtain a magnetic separation reagent;

s2, preparing an enzyme-labeled antibody: goat anti-mouse PD-1 monoclonal antibodies were labeled with alkaline phosphatase.

7. The method for preparing the melanoma glycoprotein B content detection kit according to claim 6, wherein the method comprises the steps of: the specific method for preparing the magnetic separation reagent in the step S1 is as follows:

s11, placing the magnetic nano particles on a magnetic rack after re-suspending, magnetically separating for 1min, and removing supernatant;

S12, adding an antibody dilution buffer solution to fully resuspend the magnetic beads, then placing the magnetic beads on a magnetic rack for magnetic separation for 1min, and removing supernatant, and repeating the steps three times;

S13, adding an antibody dilution buffer solution, fully suspending magnetic beads, adding an APS-S100a8/a9 combined blocker and EDC, uniformly mixing at room temperature in a dark place for reaction for 30min, and removing supernatant after magnetic separation on a magnetic frame for 1 min;

s14, adding a washing Buffer solution for washing once, placing the mixture on a magnetic rack for magnetic separation for 1min, removing supernatant, adding a Binding Buffer, adding GPNMB, uniformly mixing, and uniformly mixing at room temperature in a dark place for reaction for 3h;

S15, adding a washing buffer solution to wash the magnetic beads for 3 times, removing the supernatant for the last time, adding glycine, uniformly mixing, and uniformly mixing at room temperature in a dark place for reaction overnight;

S16, adding a washing buffer solution, washing for three times, adding a blocking buffer solution, and adding a joint blocker, an Abeta 1-40 antibody and an Abeta 1-42 antibody; mixing, and storing at 4deg.C.

8. The method for preparing the melanoma glycoprotein B content detection kit according to claim 6, wherein the method comprises the steps of: the mouse anti-human PD-L1 monoclonal antibody adopts a nano antibody.