CN108570104B

CN108570104B - Recombinant adiponectin antigen, antibody and adiponectin nano latex enhanced immunoturbidimetry kit

Info

Publication number: CN108570104B
Application number: CN201810211206.7A
Authority: CN
Inventors: 徐爱民; 周鹏程; 钟铃; 赖华
Original assignee: Guangdong Yingnuo Biotechnology Co ltd
Current assignee: Inno Biotechnology Shenzhen Co ltd
Priority date: 2018-03-14
Filing date: 2018-03-14
Publication date: 2021-11-12
Anticipated expiration: 2038-03-14
Also published as: CN108570104A

Abstract

The invention discloses a recombinant adiponectin antigen, which is prepared by the following steps: a. culturing the cell strain of the high-expression recombinant human full-length adiponectin protein in a high-glucose DMEM culture medium under the conditions of oxygen introduction of 0.25-0.75L/min, carbon dioxide content of 3-6%, bovine serum content of 5-10% and rotating speed of 100-200 rpm to enable the cell strain to reach 1-3 multiplied by 10⁷The number of cells of (a); b. replacing the culture medium with serum-free medium, adding 0.5% vitamin C, and maintaining the cell strain at 1-3 × 10⁷The number of cells in (2) continuously expressing adiponectin for 4 to 10 days. The invention also discloses the adiponectin antibody, the adiponectin antibody nano latex particle and a kit containing the antigen and antibody nano latex particle. The kit adopts a nano latex enhanced immunoturbidimetry method to detect adiponectin, has high accuracy and real and reliable data, and has good prediction effect on type 2 diabetes and early stage diabetes.

Description

Recombinant adiponectin antigen, antibody and adiponectin nano latex enhanced immunoturbidimetry kit

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant adiponectin antigen, antibody and adiponectin nano latex enhanced immunoturbidimetry kit and application thereof.

Background

With the progress of society, the change of age and structure of population and the change of living habits, the incidence of Diabetes rapidly increases, and has become a global epidemic (American Diabetes Association, Diabetes care 36Suppl 1: S67-74 (2013)). According to the data of the international diabetes association, there are currently over 3 hundred and 8 million diabetics worldwide, and 46% of them are not discovered until after serious diabetic complications occur. Statistically, the global annual expenditure on Diabetes health care is more than 5480 billion dollars (International Diabetes Federation, IDF Diabetes Atlas,6th edition/2013update (2013)). According to recent epidemiological investigations, the prevalence of diabetes in our country has risen from 1.4% in 1994 to 11.6% in 2010, with about 1.139 million diabetic patients present, and over 4 million 9 million people in pre-diabetes (Xu et al, JAMA 310:948-959 (2013)). The direct medical cost related to diabetes mellitus in China accounts for 7.6% of the total national sanitation cost, and the diabetes mellitus becomes the first killer threatening the health of the nation. It is expected that in the next 10 years, the medical costs of China for chronic diseases such as diabetes, cardiovascular disease and stroke will be up to $ 5580 million, and the number of deaths from these chronic diseases will exceed 8000 ten thousand.

Diabetes is also the culprit of many diseases. With the development of diabetes, patients often develop a series of complications including cardiovascular diseases, neuropathy, retinopathy and nephropathy, and in severe cases, myocardial infarction, stroke, gangrene and renal failure can occur. Once the diabetes is diagnosed, the diabetes cannot be reversed, no medicine for curing the diabetes and the complications thereof exists at present, the existing medicine can only control blood sugar, delay the occurrence of the complications and relieve the symptoms, and patients need to take the medicine for a lifetime. In contrast, approximately 30% of patients in the pre-diabetic stage progress to Diabetes in about ten years (Ferranini E.et al. Lancet Diabetes Endocrinol 2:667-675 (2014)). The key point of diabetes prevention and treatment lies in early discovery and prediction of diabetes and pre-diabetic people.

Adiponectin (Adiponectin) is a protein hormone secreted mainly from adipocytes, and is abundantly present in the blood circulation, playing an important role in regulating insulin sensitivity and glucose metabolism. In metabolic diseases, adiponectin has anti-diabetic, anti-atherosclerotic and anti-inflammatory effects. A number of epidemiological investigations have shown that adiponectin levels in serum/plasma are closely related to insulin resistance, pre-diabetes and type 2 diabetes, and that adiponectin levels are significantly reduced in these diseases. The association of the adiponectin level in serum/plasma with diabetes and the early stage of diabetes can provide valuable information for diseases, and the tracking follow-up and intervention treatment of people with high diabetes incidence are promoted, so that the early discovery, early intervention and early treatment of people with high diabetes risk are realized, and the incidence of diabetes is reduced.

However, clinical spread of adiponectin detection also faces a series of difficulties or obstacles. Firstly, the clinical popularization is limited by the current method for detecting adiponectin. The method for detecting adiponectin mainly comprises the following steps: Enzyme-Linked immunoassay (Enzyme-Linked ImmunoSorbent Assay, ELISA, adiponectin detection kit, CN 105548173 a), Radioimmunoassay (ra, a method for measuring adiponectin concentration, CN103076452A), chemiluminescence (chemiluminiscence immunoassay, CLIA, adiponectin chemiluminescence immunoassay kit, a preparation method and application thereof, CN 106199011 a), and the like, the Enzyme-Linked immunoassay has high sensitivity, but is cumbersome to operate and difficult to realize full-automatic detection; the radioimmunoassay method is harmful to operators and pollutes the environment, and the chemiluminescence method also has the defects of complex operation, high cost, low instrument popularization rate and the like. These disadvantages limit the popularization and application of the existing adiponectin detection method, making it difficult to be widely applied in scientific research and clinic.

The Latex nanoparticle enhanced immunoassay (Latex enhanced immunoassay) is a relatively rapid, accurate and stable liquid-phase immunoturbidimetric detection method which appears in recent years, such as CN103777023B and CN 202066863U. The basic principle is that antigen and antibody are specifically combined to generate an immunoprecipitation reaction to cause liquid phase turbidity change so as to detect the concentration of the antigen. Within a certain range, the higher the concentration of antigen, the faster the immunoprecipitation reaction, and the greater the change in absorbance in the liquid phase. According to the technology, the nano latex microspheres are coupled with the specific antibody, so that the volume of an immunoprecipitate formed by the antigen antibody is increased, a detection signal is amplified, and the detection sensitivity is obviously improved.

However, since natural adiponectin in blood circulation exists in various forms such as monomers, trimers, hexamers and polymers, and also has high glycosylation, the current laboratory detection methods and the existing adiponectin detection methods in the market are limited by the antibodies used in the detection methods, and only can recognize one or more adiponectin forms in blood circulation, and it is difficult to recognize glycosylated adiponectin. The complexity of the native adiponectin form results in a large difference in circulating adiponectin levels measured by different detection methods, and even with the same detection method, there is a difference in adiponectin levels measured due to the different adiponectin-specific antibodies used. Therefore, the reference range of the normal value of the adiponectin is difficult to unify, and the clinical popularization of adiponectin detection is limited. In addition, natural adiponectin can be used only for the production of a small amount of antibodies in the laboratory and cannot be produced on a large scale due to the limitation of the concentration of natural adiponectin in the blood circulation (about 6mg/L), and thus recombinant adiponectin protein is used for the mass production of antibodies in industrial applications.

The recombinant protein for immunizing animals to produce adiponectin specific polyclonal antibody/monoclonal antibody is mainly produced by the following protein expression systems: prokaryotic cell expression system, yeast expression system, insect cell expression system and mammal cell expression system, the higher the expression system is, the more comprehensive and complex the posttranslational modification can be completed, and the more the expressed recombinant protein is close to the natural protein in human blood circulation. At present, adiponectin recombinant protein is mainly produced through a prokaryotic expression system, and because the prokaryotic expression system is too simple and cannot complete complex post-translational modification, the expressed protein may have differences from natural protein in tertiary structure, and the system can only express adiponectin monomers and cannot form quaternary structures such as tripolymers and polymers. These recombinant antigens are structurally very different from adiponectin in human blood circulation, and the use of such recombinant antigens to produce adiponectin-specific antibodies results in insufficient specificity and sensitivity of the antibodies, thereby affecting the sensitivity and specificity of downstream reagents. The recombinant adiponectin protein produced by a mammalian cell expression system, particularly by adopting human embryonic kidney cells HEK293, not only has a three-stage structure completely consistent with the natural protein in the human body, but also can form four-stage structures such as tripolymers, hexamers and polymers besides monomers, and has high structural consistency with the natural adiponectin in the human blood circulation. The antibody, especially polyclonal antibody, generated by immunizing animals with the recombinant adiponectin can identify all forms of adiponectin in human blood circulation; meanwhile, the recombinant adiponectin is used as a calibrator of a detection reagent, so that the level of adiponectin in human blood circulation can be reflected most accurately, and the recombinant adiponectin is an ideal choice for determining a reference range of a normal value of adiponectin. However, the prior art of producing recombinant adiponectin internationally through mammalian systems has low yield and high cost (the yield is about 2mg/L, patent: US 7,365,170B2), and the popularization and application of the recombinant antigen in adiponectin detection are limited.

More importantly, most of the studies on adiponectin such as the above patents do not carry out detailed clinical verification on the produced reagents, do not describe the normal level and reference range of adiponectin, fail to specify the range of adiponectin abnormal values and the significance of the abnormal values, and further do not determine the function of adiponectin prediction on type 2 diabetes and diabetes prophase through clinical verification, thereby further limiting the clinical popularization and application of adiponectin detection.

Disclosure of Invention

In order to solve the problems in the prior art, the present invention aims to provide a recombinant adiponectin antigen, an antibody prepared from the recombinant adiponectin antigen, an adiponectin antibody nano latex particle prepared from the antibody, a kit containing the adiponectin antibody nano latex particle, and applications of the kit in predicting pre-diabetes and type 2 diabetes.

The above-mentioned pre-diabetes means an intermediate zone between normal and diabetes, and includes fasting blood glucose abnormality (fasting blood glucose value is between 6.1 and 6.9 mmol/l) and glucose tolerance abnormality (blood glucose value is 7.8 to 11.0mmol/l after receiving oral glucose tolerance test for two hours). When a diabetic is diagnosed with type 2 diabetes, the disease often reaches a middle or even late stage and misses a highly effective treatment period. While the pre-stage of type 2 diabetes is the golden period for treating diabetes and even cardiovascular and cerebrovascular diseases.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a recombinant adiponectin antigen prepared by the steps comprising:

a. culturing cell strain with high expression recombinant human full-length adiponectin protein in culture medium, and culturing carbon dioxide in environmentThe concentration is 3-6%, the culture speed is 100-200 rpm, the culture medium is a high-sugar DMEM culture medium containing 5-10% by mass of bovine serum, and the oxygen introduction amount in the culture is 0.25-0.75L/min, so that the cell strain reaches 1-3 x 10⁷The number of cells of (a);

b. replacing all the culture medium with a serum-free culture medium added with 0.5% by mass of vitamin C to maintain the cell strain at 1-3 × 10⁷Cell number of (4), continuous expression.

Preferably, the method further comprises the step of replacing half volume of the culture medium every 2-3 days, wherein the culture medium is a serum-free culture medium added with 0.5% of vitamin C by mass percent.

More preferably, the recombinant human full-length adiponectin protein adopts an expression vector skeleton of pRSET A; the oxygen introduction amount is 0.5L/min, the concentration of the carbon dioxide is 5%, the mass percentage of the bovine serum is 10%, the rotating speed is 120rpm, and the continuous expression time is 4-10 days, preferably 7 days; and/or the high expression is an expression amount of 10mg/ml or more, preferably 20mg/ml or more, more preferably 40mg/ml or more.

The recombinant adiponectin prepared by the method is in a polymer, trimer and monomer coexisting state, is highly consistent with adiponectin in human blood circulation in structure, and is high in yield and purity, and the adiponectin antibody prepared by the recombinant adiponectin antibody is better in effect than the adiponectin antibody prepared by adiponectin antigen (US 7,365,170B2) produced by mammalian cells in the prior art, so that the recombinant adiponectin is beneficial to subsequent application.

In order to solve the above technical problems, one of the technical solutions of the present invention is: an adiponectin antibody prepared by using the recombinant adiponectin antigen; preferably a polyclonal antibody; more preferably, the polyclonal antibody is a goat or rabbit polyclonal antibody.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a method for producing an adiponectin antibody, which can be a method for producing an existing antibody, particularly a polyclonal antibody, comprising the steps of:

a. emulsifying the recombinant adiponectin antigen by equivalent Freund complete adjuvant, and injecting goats or rabbits at multiple points subcutaneously on the back;

b. emulsifying the recombinant adiponectin antigen by Freund incomplete adjuvant every 1-2 weeks, and repeatedly injecting for 2-4 times by the same method;

c. extracting blood, and purifying to obtain serum.

Preferably, the rabbit is a New Zealand white rabbit.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a method for preparing adiponectin antibody nano latex particles comprises the following steps: mixing latex particle solution with the adiponectin antibody to carry out crosslinking reaction; preferably, the latex particles are polystyrene microspheres (commonly called functional polystyrene microspheres) formed by polymerizing styrene and acrylic acid, and carboxyl groups exist on the surfaces of the latex particles; preferably polystyrene microspheres (commonly known as functional polystyrene microspheres) from Bangslab; more preferably, the average particle size of the latex particles is in the range of 120 to 200 nm.

The crosslinking agent used for the crosslinking may be selected from EDC, N-hydroxysuccinimide, N-hydroxythiosuccinimide, carboximide, hydrazide, potassium isocyanate or a combination thereof, preferably the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the final concentration of the adiponectin antibody is 0.5-5 mg/ml; preferably, the final concentration of the adiponectin antibody is 3 mg/ml; the final concentration of the cross-linking agent is 200 mM; and/or, the crosslinking is carried out at room temperature for 4-12 hours. The final concentration refers to the final concentration in the crosslinking reaction system.

In order to solve the above technical problems, one of the technical solutions of the present invention is: an adiponectin antibody nano latex particle obtained by the preparation method.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a kit for detecting adiponectin, the kit comprising said adiponectin antibody nanoemulsion particles.

Preferably, the kit is a kit for a nano latex enhanced immunoturbidimetry, and further comprises the recombinant adiponectin antigen as a calibrator, an R1 reagent and an R2 reagent, wherein the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution, and the R2 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative, a buffer solution and adiponectin antibody nano latex particles.

More preferably, the R1 reagent contains the following components: 0.5-2% of sodium chloride, 5-30 g of PEG8000, 0.05-1% of sodium azide and 100-300 mmol of MOPS buffer solution, wherein the pH value is 7.0-8.0; the R2 reagent contains the following components: 1-3 mg/mL adiponectin antibody nano latex particles, 1-2% of sodium chloride, 1-2% of BSA, 0.5-1% of triton X-100, 0.05-0.5% of sodium azide and 100-300 mmol of MOPS buffer solution, wherein the pH is 7.4-8.0, and the% is in mass percentage;

preferably, the R1 reagent contains the following components: the sodium chloride is 1%, the PEG8000 is 15g, the sodium azide is 0.09%, and the MOPS buffer solution is 250mmol, and the pH value is 7.4; the R2 contains the following components: 1mg/mL of latex particles coated with anti-human adiponectin polyclonal antibody, 1% of sodium chloride, 1% of BSA, 0.5% of triton X-100, 0.09% of sodium azide, pH 8.0, and 250mmol of MOPS buffer, the% being mass%.

In order to solve the above technical problems, one of the technical solutions of the present invention is: the method for detecting the concentration of the adiponectin, which is not used for diagnosis, is characterized in that the method is a nano latex enhanced immunoturbidimetry method using the kit.

In order to solve the above technical problems, one of the technical solutions of the present invention is: the adiponectin antibody, the adiponectin nano latex particle or the kit is applied to preparation of a reagent for predicting pre-diabetes or type 2 diabetes.

Preferably, the prediction standard of the prediabetes stage is that the content of adiponectin is 4-6.5 mg/L; the prediction standard of the type 2 diabetes is that the content of adiponectin is less than 4 mg/L.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: 1. the accuracy is high: the specific antibody can identify various forms of adiponectin in human circulation and the adiponectin after hyperglycosylation, can accurately reflect the real adiponectin level, and has high specificity and sensitivity; 2. the adiponectin level is measured truly and reliably: the adiponectin produced by the mammalian expression system is used as a calibrator and is highly consistent with adiponectin in human circulation, so that the deviation between a detection result and a real level, which is generated by using an adiponectin monomer as the calibrator, is avoided. 3. The immunoprecipitation reaction is simple and direct, the time from the beginning of the reaction to the equilibrium detection point is only 5 minutes, and the operation steps of incubation, cleaning and the like required by ELISA, CLIA and other methods are saved; 4. the precision is high: the biochemical analyzer is used for automatic detection, the steps are simple, the interference caused by manual operation and complicated steps is avoided, and the repeatability is good. 5. The detection result can accurately reflect the real level of the adiponectin in the human circulation, and has good prediction effect on the early-stage diabetes and the type 2 diabetes.

Drawings

FIG. 1 shows the expression of adiponectin antigen by mammalian cells of the present invention;

FIG. 2 is a graph of the calibrator response of adiponectin reagents prepared with different concentrations of antibody;

FIG. 3 is a standard curve for the kit;

FIG. 4 is a graph showing the linear range of the kit, wherein 4A is the linearity at an adiponectin concentration of 0.8-8 mg/L, and 4B is the linearity at an adiponectin concentration of 8-80 mg/L;

FIG. 5 is a graph of serum adiponectin levels in a population

FIG. 6 is a diagnosis of type 2 diabetes and pre-diabetes using the kit of the present invention.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

EXAMPLE 1 expression of adiponectin antigen

1) Recombinant adiponectin antigens expressed by mammalian cells of the prior art

Reference reports (US 7,365,170B 2; Xu A, et al. Testosterone selective processes for the high molecular weight of the inhibition of adiponectin by inhibition of cleavage from adipocytes. J Biol chem.2005May 6; 280(18): 18073-80). First, using an upstream primer: 5'-GCCCG CGGAT CCATG CTGTT GCTGG GAGCT GTTC-3' and the downstream primer: 5'-GGCCG CGAAT TCTCA CTTGT CATCG TCGTC CTTGT AGTCG TTGGT GTCAT GGTAG AGAAG-3' the gene encoding adiponectin was amplified from a human adipocyte DNA library (Clontech, USA). After treatment with restriction enzyme BamHI/EcoRI, the human adiponectin gene was inserted into an expression vector pRSET A which is stronger than the vector pcDNA3.1 promoter reported in the literature, and a DNA sequence expressing a FLAG tag was inserted at the carboxyl terminal of the adiponectin gene to construct an expression vector pR-Ad-F of the full-length adiponectin fusion protein. The expression vector was transfected into mammalian HEK293 cells, and stable cell lines expressing high levels of human adiponectin were selected using geneticin G418 (available from Life Technology) at a concentration of 1mg/ml and stored in liquid nitrogen. Serum-free conditioned medium, DMEM, was collected from the cells and the FLAG-labeled recombinant adiponectin antigen was affinity gel purified using an anti-FLAG M2 monoclonal antibody.

2) The recombinant adiponectin antigen expressed by the mammalian cell

Because the recombinant adiponectin antigen expressed by the method of 1) has low yield and high cost, the genetic mycin G418 is used for screening cell strains for 2 weeks to kill cells which can not express adiponectin each time the cells are thawed on the premise of 1); then passed through a constant temperature bio-fermenter (brand: Sartorius stedim biotech,

B) culturing cell strain, and optimizing oxygen introduction amount (0.5L/min), carbon dioxide amount (5%), bovine serum amount (5-10%), rotation speed (120rpm), and culture medium formula (high-sugar DMEM culture medium purchased from Thermo) to make cell strain reach 1 × 10 in fermentation tank⁷The number of cells of (a), the fermentation temperature is 37 ℃; then, the culture medium was changed to a serum-free nutrient formula (SFM medium, purchased from Thermo, and 0.5% vitamin C was added), so as to promote the secretion of adiponectin of the cell line. Maintaining the cell line at above 1X 10⁷The cell number level of (2) secreted adiponectin for one week, thereby greatly improving the yield of adiponectin (40 mg/L). Finally, adiponectin fusion protein was affinity purified from the medium by high pressure liquid chromatography (purchased from Bio-Rad) using a FLAG affinity chromatography column (purchased from Sigma). FIG. 1A shows purified recombinant adiponectin antigen of the present invention, in which lane 1 shows protein marker, 2 shows adiponectin after complete reduction treatment as a monomer, 3 shows adiponectin after semi-reduction treatment as a trimer and a monomer, and 4 shows adiponectin without reduction treatment as a multimer, trimer and monomer coexisting. In the above results, "reduction" means: the disulfide bond connecting the adiponectin monomers and the monomers to form the polymer and the trimer is opened to become the adiponectin monomers again. Thus, it was confirmed that adiponectin produced by the method described above coexists in multimers, trimers and monomers, whereas adiponectin produced by the conventional general techniques is mostly in monomers. FIG. 1B is SDS-PAGE of adiponectin antigen prepared by the prior art, which is mainly represented by a monomer (33kDa position) and has insufficient purity, and even after affinity purification and other processes, a great deal of small-molecule hetero-protein still exists, which affects the quality of the antibody produced subsequently. In addition, the expression level of adiponectin in the prior art is less than 5mg/L, and the invention leads the expression level of adiponectin to reach 40-50mg/L through process improvement. Thereby reducing the production cost, reducing the batch difference of the recombinant adiponectin and improving the consistency of the subsequent antibody production and reagents.

EXAMPLE 2 preparation of adiponectin antibodies

Respectively taking 300 mu g of natural adiponectin antigen (CN 103777023B, purchased from Bio-Rad company, abbreviated as antigen A, and abbreviated as antibody A) disclosed by the prior art, recombinant adiponectin antigen (US 7,365,170B2, abbreviated as antigen B, and abbreviated as antibody B) produced by the prior art, and mammalian cells expressing adiponectin antigens of the invention, emulsifying the antigens by equivalent Freund's complete adjuvant (purchased from Sigma), injecting 2.2-2.5 kg of New Zealand white rabbits which are bred in a Specific Pathogen Free (SPF) animal room by back subcutaneous multipoint injection, emulsifying the antigens by Freund's incomplete adjuvant (Sigma) every two weeks, and performing booster immunization 3 times by the same method repeatedly, and immunizing for 4 times in total. Then 100ml of blood was collected from the vein of rabbits of different treatment groups, and centrifuged to obtain serum. Passing 10ml of each rabbit serum through a Protein G (Thermo, reagents or consumables not specifically described below are purchased from Thermo) immunoaffinity chromatography column, repeatedly washing the column with 1000ml of TBS (20mM Tris, pH7.4,500mM NaCl, 0.05% Tween-20) buffer, discarding the effluent, eluting the bound antibody with 10ml of 100mM glycine/HCl (pH2.5) buffer after washing, collecting the antibody in a dialysis bag, placing the dialysis bag in 1000ml of TBS (20mM Tris, pH7.4,500mM NaCl, 0.05% Tween-20) buffer, dialyzing overnight at 4 ℃ in a refrigerator, and repeating the dialysis steps 3 times to obtain three purified anti-human adiponectin IgG polyclonal antibodies: antibody a, antibody B and antibodies of the invention.

EXAMPLE 3 preparation of latex particles coated with polyclonal anti-human adiponectin antibody

The 3 antibodies obtained in example 2, antibody a, antibody B and the antibody of the present invention, were coupled to the surface of latex particles, purchased from Bangslab, which had been treated to have carboxyl chemical groups attached thereto, by chemical bonding. And the antibody prepared by using the adiponectin antigen expressed by the mammalian cells is used for optimizing the concentration of the antibody for preparing the latex particles.

The method comprises the following specific steps:

1)100mg of the latex particles containing carboxyl groups were dissolved in 5ml of MOPS buffer (50mM, pH 6.4) to obtain a latex particle solution.

2) Dissolving the adiponectin specific antibody to be crosslinked into 5ml of MOPS buffer solution (50mM, pH 6.4) to obtain an antibody solution with the concentration of 1-5 mg/ml; the optimized antibody concentration is 3 mg/ml.

3) And (3) fully mixing the latex particle solution and the antibody solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) with the final concentration of 20mM, dissolving in the mixed solution, and carrying out crosslinking reaction for 2-4 h at room temperature.

4) The crosslinking reaction was terminated by adding 1ml of 1M Tris pH10 to the final concentration and incubating for 10 minutes.

5) The antibody-crosslinked latex particles were collected in dialysis bags (Thermo) and placed in 1000ml MOPS (50mM, pH 8.0) buffer and dialyzed overnight at 4 ℃ in a refrigerator, and the chromatography step was repeated 3 times.

6) The antibody-crosslinked latex particles were diluted with MOPS (50mM, pH 8.0) to a final concentration of 1 mg/ml.

The above steps were used to obtain 3 latex particles coated with the antibody: antibody latex particles a, antibody latex particles B, and antibody latex particles of the present invention.

TABLE 1 Signal values obtained by preparing reagents with different concentrations of the antibodies of the invention

As shown in table 1: the reagent prepared by the adiponectin antibody with the concentration of 1mg/ml to 5mg/ml can obtain higher signal values and detection upper limits when detecting adiponectin calibrators with different concentrations, wherein the effect is the best when the antibody with the concentration of 3mg/ml is used. The antibody concentration is lower than 1mg/ml, such as 0.5mg/ml, and the measured signal value is obviously reduced; whereas when the antibody concentration is higher than 5mg/ml, for example, 6mg/ml, the upper limit of detection is affected, and a banding effect with a signal value lower than 20mg/L occurs at a concentration of 40 mg/L.

EXAMPLE 4 preparation of the kit and Performance testing

1) Preparation of R1 and R2 reagents

The R1 reagent is prepared from the following components in percentage by weight: the concentration of sodium chloride can be 0.5-2%, 1% in the invention, the dosage of PEG8000 can be 5-30 g, 15g in the invention; the concentration of sodium azide may be 0.05 to 1%, in the present invention, 0.09%, the pH may be 7.0 to 8.0, in the present invention, 7.4, and the concentration of MOPS buffer may be 100 to 300mmol, in the present invention, 250 mmol.

The R2 reagent is prepared from the following components in percentage by weight: example 3 latex particles coated with anti-human adiponectin polyclonal antibody were prepared at 0.5-1.25mg/mL, 1mg/mL in the present invention, 1% sodium chloride, 1% BSA, 0.5% Triton X-100, 0.05-0.5% sodium azide, 0.09% in the present invention, pH was 7.4-8.0, 8.0 in the present invention, MOPS buffer was 100-300 mmol, 250mmol in the present invention.

2) Performance testing of the kit

The kit is used for determining the dominant wavelength by using a Hitachi 7100 full-automatic biochemical analyzer: 630nm, secondary wavelength: 800nm, reaction direction: and (4) rising.

The dosage of the reagent is as follows: sample 1.5 μ 1; 150 μ 1 of R1 reagent; r2 reagent 50. mu.1.

Measurement method (two-point end-point method): adding 150 mu 1R1 reagent into 1.5 mu l of sample, reacting at 37 ℃ for 3-5 minutes, adding 50 mu 1R2 reagent into R2, mixing uniformly for 10-30 seconds, and determining absorbance A₁And the absorbance A was measured after another 5 minutes₂Calculating the signal value, i.e. the absorbance difference Δ a ═ a₂-A₁。

3) Performance comparison of kit produced by antibodies prepared from antigens of different sources

The signal values for the kits prepared with the different antibody latex particles are shown in table 2.

TABLE 2 kit for preparation of different antibody latex particles (unit: absorbance. times.10000)

The performance comparison of the enhanced immunoturbidimetric kit prepared from the antibody latex particles from different sources is carried out, and the result shows that the performance of the kit prepared from the antibody latex particles is obviously superior to that of the kits prepared from other 2 antibody latex particles under the same preparation process and preparation parameters, and the average signal value of the calibrator with different concentrations is 4.65 times that of the kit prepared from the antibody latex particles A and 2.16 times that of the kit prepared from the antibody latex particles B, so that the performance of the reagent is obviously improved, and the sensitivity and the precision are improved.

Example 5 detailed Performance testing of kits prepared according to the invention

5.1 standard curve preparation:

by using the calibrator of the present invention, 6 calibrator solutions with different concentrations, such as 40mg/L, 20mg/L, 10mg/L, 5mg/L, 2mg/L and 1mg/L, and a blank control, were prepared with physiological saline, and a standard curve of the recombinant adiponectin calibrator of the present invention was obtained according to the above procedure, as shown in fig. 3. Each point on the curve in figure 3 represents a quantity of calibrator, where the x-axis represents the concentration of adiponectin and the y-axis represents the difference in absorbance. The standard curve equation is: -1.4434x²+215.08x-38.69, correlation coefficient r is 0.9999.

5.2 quality control result:

the reagent of the invention is adopted to test two quality control products with known concentration for 3 times respectively in a calibrated Hitachi 7100 full-automatic biochemical analyzer, the concentration value of the adiponectin is recorded and measured, and the average value is obtained by calculation, and the result is shown in Table 3.

TABLE 3 control of adiponectin levels

The measured water average of the quality control adiponectin is in the concentration range of the quality control product, which indicates that the reagent loading position, the instrument parameter setting and the calibration meet the standard.

5.3 in-batch precision:

4 portions of serum were measured according to the kit of the present invention, and the same serum sample was tested 20 times, and the measurement mean and the batch precision were calculated as shown in Table 4. The results show that the in-batch precision is 1.1% -3.96%.

TABLE 4 in-batch precision

5.4 Linear Range:

80mg/L of the recombinant adiponectin high-concentration sample and 8mg/L of the recombinant adiponectin low-concentration sample of the present invention prepared in example 1 near the upper limit of the linear range were diluted with physiological saline at 9/10,8/10,7/10,6/10,5/10,4/10,3/10,2/10,1/10 to prepare 10 calibrator solutions of different concentrations, respectively. Each concentration was measured by the method described in example 4, and the measured concentration value and the theoretical concentration were subjected to linear regression analysis, and the regression equation was calculated as: y is 1.0109x-0.1865, and the correlation coefficient r is 0.9956, which shows that the kit has good correlation in the linear range of 0.8-40 mg/L, and the hook effect does not occur when the sample concentration reaches 80 mg/L. As shown in FIG. 4 and Table 5, FIG. 4A shows the linear range of the recombinant adiponectin concentration of 0.8-8 mg/L, and FIG. 4B shows the linear range of the adiponectin concentration of 8-80 mg/L.

TABLE 5 adiponectin Linear Range

5.5 assay sensitivity:

the analytical sensitivity refers to the lowest measured concentration which can be detected by a detection method, also called the detection limit or the minimum detected concentration, and the experimental method comprises the steps of repeatedly measuring a blank sample for 20 times in one operation, calculating the Mean value Mean and the standard deviation SD of the 20-time result, and reporting the detection limit (Mean +2SD) of the method by adding two times of the standard deviation to the blank Mean value. The analysis sensitivity of the invention is 0.3mg/L, and the specific content is shown in Table 6.

TABLE 6 sensitivity analysis

5.6 hemoglobin interference assay:

the interferent selection formulations and test results are shown in table 7. The result shows that the reagent kit and the computer parameter 150 mu 1R1 reagent are used, 1.5 mu l of the sample and 50 mu 1R2 have good anti-interference capability, and the result is not obviously influenced when the concentration of the heme reaches 500 mg/dL.

TABLE 7 anti-interference Capacity of antibody reagents of the invention

Example 6 detection data for diabetic samples

Prospective data analysis: samples of 1476 healthy persons were collected and the adiponectin levels in the serum were measured using a calibrated Hitachi 7100 full-automatic biochemical analyzer according to the methods described in examples 4 and 5. The adiponectin level distribution is detected to be between 0.55 and 35.43mg/L (see figure 5).

Type 2 diabetes and pre-diabetic prediction: blood samples were collected and stored in 1649 normal populations (age distribution 30 to 81 years) and were taken annually at a follow-up visit of 5 years and tested for adiponectin levels in their sera. Analysis revealed that 522 cases developed pre-diabetes and 88 cases developed pre-diabetes within 5 years (see fig. 6), and that baseline adiponectin levels were significantly lower in patients who progressed to diabetes than in healthy and pre-diabetes, and significantly lower in patients who progressed to pre-diabetes than in healthy. Through statistical analysis of SPSS software, the judgment standard of the invention is summarized as follows: more than 6.5mg/L is a healthy group, 4-6.5 mg/L is a high risk group in the early stage of diabetes, and less than 4mg/L is a high risk group in diabetes. The experimental result of the invention shows that the adiponectin level can be used as an index for predicting type 2 diabetes and early stage diabetes.

Claims

1. A recombinant adiponectin antigen prepared by the steps of:

a. culturing a mammalian cell strain highly expressing recombinant human full-length adiponectin in a culture medium, wherein the concentration of carbon dioxide in a culture environment is 3-6%, the culture rotating speed is 100-200 rpm, and the culture medium is high-sugar DME containing 5-10% by mass of bovine serumM culture medium, wherein oxygen is introduced in the culture medium at 0.25-0.75L/min, so that the mammalian cell strain reaches 1-3 x 10⁷The number of cells of (a);

b. replacing all the culture medium with a serum-free culture medium added with 0.5% by mass of vitamin C to maintain the mammalian cell strain at 1-3 × 10⁷Cell number of (4), continuous expression.

2. The recombinant adiponectin antigen of claim 1, further comprising c.replacing half of the volume of the culture medium every 2-3 days, wherein the culture medium is serum-free with 0.5% by weight vitamin C added thereto.

3. The recombinant adiponectin antigen of claim 2, wherein the recombinant human full-length adiponectin protein employs an expression vector of pRSET A; the oxygen introduction amount is 0.5L/min, the concentration of carbon dioxide is 5%, the mass percentage of bovine serum is 10%, the rotating speed is 120rpm, and the continuous expression time is 4-10 days.

4. The recombinant adiponectin antigen of claim 3, wherein the sustained expression is for a period of 7 days; and/or the high expression is that the expression amount is more than 20 mg/L.

5. The recombinant adiponectin antigen according to claim 3, wherein the high expression is an expression level of 40mg/L or more.

6. An adiponectin antibody produced using the recombinant adiponectin antigen according to claim 1.

7. The adiponectin antibody of claim 6, wherein the adiponectin antibody is a polyclonal antibody.

8. The adiponectin antibody of claim 7, wherein the polyclonal antibody is a goat or rabbit polyclonal antibody.

9. A method for producing the adiponectin antibody according to claim 6, comprising the steps of:

a. emulsifying the recombinant adiponectin antigen with equivalent Freund's complete adjuvant, and injecting goat or rabbit subcutaneously at multiple points on the back;

c. extracting blood, and purifying to obtain serum.

10. The method of claim 9, wherein the rabbit is a New Zealand white rabbit.

11. A method for preparing adiponectin antibody nano latex particles is characterized by comprising the following steps: mixing the latex particle solution with the adiponectin antibody according to claim 2, and performing a crosslinking reaction.

12. The method of claim 11, wherein the latex particles are polystyrene microspheres obtained by polymerizing styrene and acrylic acid, and carboxyl groups are present on the surface of the latex particles.

13. The method of claim 12, wherein the polystyrene microspheres are polystyrene microspheres from Bangslab.

14. The method according to claim 13, wherein the latex particles have an average particle size in the range of 120 to 200 nm.

15. The production method according to any one of claims 11 to 14, wherein the crosslinking agent used for the crosslinking is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the final concentration of the adiponectin antibody is 0.5-5 mg/ml.

16. The method of claim 15, wherein the adiponectin antibody is provided at a final concentration of 3 mg/ml; the final concentration of the cross-linking agent is 200 mM; and/or, the crosslinking is carried out at room temperature for 4-12 hours.

17. An adiponectin antibody nano latex particle obtained by the production method according to any one of claims 11 to 16.

18. A kit for detecting adiponectin, comprising the adiponectin antibody nanoemulsion particle of claim 17.

19. The kit of claim 18, wherein the kit is a nanolatex enhanced immunoturbidimetry kit further comprising the recombinant adiponectin antigen of claim 1 as a calibrator, an R1 reagent, and an R2 reagent, wherein the adiponectin antibody nanolatex particles are in the R2 reagent, wherein the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative, and a buffer, and wherein the R2 reagent further comprises an electrolyte, a stabilizer, a surfactant, a preservative, and a buffer.

20. The kit of claim 19, wherein the R1 reagent comprises the following components: 0.5-2% of sodium chloride, 5-30 g of PEG8000, 0.05-1% of sodium azide and 100-300 mmol of MOPS buffer solution, wherein the pH value is 7.0-8.0; the R2 reagent contains the following components: 1-3 mg/mL adiponectin antibody nano latex particles, 1-2% of sodium chloride, 1-2% of BSA, 0.5-1% of triton X-100, 0.05-0.5% of sodium azide and 100-300 mmol of MOPS buffer solution, wherein the pH is 7.4-8.0, and the% is mass percent.

21. The kit of claim 20, wherein the R1 reagent comprises the following components: the sodium chloride is 1%, the PEG8000 is 15g, the sodium azide is 0.09%, and the MOPS buffer solution is 250mmol, and the pH value is 7.4; the R2 contains the following components: 1mg/mL of latex particles coated with anti-human adiponectin polyclonal antibody, 1% of sodium chloride, 1% of BSA, 0.5% of triton X-100, 0.09% of sodium azide, pH 8.0, and 250mmol of MOPS buffer, the% being mass%.

22. A method for detecting the concentration of adiponectin for non-diagnostic purposes, which is characterized by using the nano latex-enhanced immunoturbidimetry method using the kit according to any one of claims 18 to 21.

23. Use of the adiponectin antibody of any one of claims 6 to 8, the adiponectin antibody nanoemulsion particle of claim 17, or the kit of any one of claims 18 to 21 for the preparation of a reagent for predicting pre-or type 2 diabetes.

24. The use according to claim 23, wherein the pre-diabetic prediction criterion is a adiponectin content of 4-6.5 mg/L; the prediction standard of the type 2 diabetes is that the content of the adiponectin is less than 4 mg/L.