CN111007255A - Protein chip for detecting kidney injury marker and preparation method thereof - Google Patents

Protein chip for detecting kidney injury marker and preparation method thereof Download PDF

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CN111007255A
CN111007255A CN201911255012.8A CN201911255012A CN111007255A CN 111007255 A CN111007255 A CN 111007255A CN 201911255012 A CN201911255012 A CN 201911255012A CN 111007255 A CN111007255 A CN 111007255A
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antibody
urine
protein chip
protein
glass slide
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尤梦竹
泮锋纲
丁俊杰
施启尧
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Sunlant Biological Engineering Co ltd
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Sunlant Biological Engineering Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders

Abstract

The invention discloses a protein chip for detecting a kidney injury marker, which is prepared by the following steps of (1) black glass sheet pretreatment, (2) antibody solution spotting, (3) a sealing process, and the preparation method of the protein chip comprises the following steps of (1) protein chip preparation, wherein the protein chip preparation is used for detecting the kidney injury marker, the detection efficiency can be effectively improved and the detection cost can be reduced by the combined detection realized by the technology, and the protein chip diagnosis kit simultaneously containing 14 indexes of clusterin, cystatin C, kidney injury molecule-1, human plasminogen activator inhibitor 1, urine immunoglobulin, heat shock protein 72, β -catenin, β 2-microglobulin, urine transferrin, urine α 1-microglobulin, trefoil factor 3, urine trehalase, human neutrophil gelatinase-related lipid carrier protein and urine microalbumin, has the advantages of rapidness, high efficiency, low cost and the like.

Description

Protein chip for detecting kidney injury marker and preparation method thereof
Technical Field
The invention relates to the technical field of biology, in particular to a protein chip for detecting a kidney injury marker and a preparation method thereof.
Background
Acute Kidney Injury (AKI), which refers to a clinical syndrome arising from rapid decline in renal function due to multiple etiologies, increases in serum creatinine concentration by more than 50% over a basal value within 7 days, or within 48 hours, as defined by the KDIGO guidelines of 2012Serum creatinine concentration increased by 0.3 mg/dL. AKI is a relatively broad clinical pathophysiological state involving a variety of different etiologies, including some specific renal diseases (e.g., acute interstitial nephritis, acute glomerulonephritis, acute renal vascular disorder) and other non-specific etiologies (e.g., prerenal azotemia, acute post-renal obstructive nephropathy). The etiology of AKI involves many exposure high risk factors and associated risk factors. High risk factors include sepsis, severe systemic disease, cardiogenic shock, burns, trauma, cardiovascular surgery, nephrotoxic drugs, etc. Risk factors that may induce AKI include dehydration, advanced age, female, black, chronic kidney disease, chronic heart, lung and liver disease, diabetes, tumors and anemia[1]. According to statistics, the incidence rate of AKI of a patient in a hospital reaches 1-7%, and the mortality rate reaches 60.3%. In addition, AKI is also a significant cause of Chronic Kidney Disease (CKD) and chronic renal failure, with 20-50% of surviving patients developing CKD, while CKD is one of the major risk factors for AKI, with about 30% of patients with AKI having a history of CKD[2,3]。
More than 30 kidney injury related biomarkers are discovered and discovered in recent years, which mainly comprise ① markers reflecting glomerular function damage, such as cystatin C, urine immunoglobulin, urine transferrin, urine microalbumin and the like, ② markers reflecting renal tubular damage, such as neutrophil gelatinase-related lipocalin, kidney injury molecule-1, β 2-microglobulin, α 1-microglobulin and the like.
(1) Clusterin (clusterin)
Clusterin is a heterodimer glycoprotein with the molecular weight of 80kDa, and normal human kidneys only have trace expression, mainly distribute in the intima and renal tubules of internal arteries of the kidneys, and have no expression in glomeruli. Physiological state is unable to filter from glomerulus, and various renal injury states include urinary tract obstruction, ischemia/reperfusion injury, renal injuryToxic injury, etc., whose expression level in blood and urine is increased[4]Approved by the U.S. FDA as a biomarker for predicting drug-induced renal damage[5]。
(2) Kidney injury molecule-1 (KIM-1)
Kidney injury molecule-1A type I transmembrane glycoprotein secreted by the epithelium of the proximal tubule of the kidney, with a molecular weight of 104kD, contains a membrane-binding domain of 14kD and a soluble portion of 90 kD. According to the difference of the intracellular C-terminal structure, the protein can be divided into KIM-1a and KIM-1b, wherein the intracellular part of the protein lacks a tyrosine kinase phosphorylation region and is mainly expressed in the liver, and the intracellular part of the protein is rich in the tyrosine kinase phosphorylation region and is mainly expressed in the kidney. KIM-1 is also expressed on activated Th1 and Th2 cells and is therefore also called T-cell immunoglobulin and mucin-1 (TIM-1). KIM-1 has a high degree of tissue specificity, with its expression levels markedly upregulated in the proximal tubular epithelial cells that regenerate after injury, but not in normal kidney tissue. Under the mediation of metal matrix protease, the KIM-1 extracellular domain can be cracked, and the formed soluble molecules fall into the renal tubule lumen, so that the quantitative detection of the urine KIM-1 can be realized[6]. It has been found that, among diabetic early renal injuries, Kim-1 has the strongest sensitivity (77.2%), the strongest heat shock protein 72 specificity (90.0%), and the sensitivity and specificity of combined detection of KIM-1+ human plasminogen activator inhibitor 1+ heat shock protein 72 are 91.1% and 97.0%, respectively[7]. Because of its good specificity and sensitivity in identifying renal tissue injury, urine KIM-1 was approved by the FDA in 2014 for clinical use as an adjunct diagnosis for AKI[6]。
(3) Human plasminogen activator inhibitor 1(PAI-1)
Plasminogen activator inhibitor 1 is a glycoprotein with a molecular weight of 50kD, belongs to the family of serine protease inhibitors, and is the inhibitor of the most important urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in vivo. tPA and uPA degrade fibrin and extracellular matrix, and therefore PAI-1 can influence the process of thrombosis and fibrosis. PAI-1 plays an important role in the occurrence and development of renal diseases, under normal physiological conditions, the kidneyPAI-1 is expressed in very small amounts, but in acute or chronic pathological states, PAI-1 expression is markedly elevated, and PAI-1 levels decrease with reduced injury. The study of PAI-1 in renal disease has been mainly focused on atherosclerosis and hardened glomeruli, such as hardening of renal arteries, diabetic nephropathy and chronic graft nephropathy[8]。
(4) Heat shock protein 72(HSP72)
Heat shock proteins can be classified into HSP100, HSP90, HSP70, HSP60 and HSP40 according to their molecular weights. The HSP70 family, which mainly includes HSP72 and HSP73, is the most evolutionarily conserved and important member of the entire heat shock protein family, and is the most efficient buffer system for cellular stress responses. The amino acid sequences of the two proteins are 90% identical, HSP73 belongs to structural heat shock protein, the expression is basically fixed, HSP72 belongs to inducible heat shock protein, the expression is only a small amount in normal cells, and the expression can be large under the stimulation of various factors. HSP72 can be involved in folding of newly synthesized protein in vivo, degrading or repairing damaged protein, inhibiting synthesis and secretion of inflammatory factor, and reducing cell necrosis or apoptosis caused by stress injury[9]. HSP72 can be expressed in renal tubular epithelial cells, interstitial smooth muscle cells and perivascular nerve cells, and its protective effect on kidney has been proved in models of renal ischemia-reperfusion injury, nephrotoxic injury, obstructive renal injury, etc[10]。
(5) α 1-microglobulin (α 1-MG 1)
α 1-microglobulin is mainly synthesized by liver cells and lymph tissue, has a molecular weight of 26kDa, can be freely filtered by glomeruli, is almost completely reabsorbed by proximal tubules, has extremely small content in urine under normal conditions, and is frequently used for predicting and observing early renal injury and diabetic nephropathy complications if the reabsorption function of the tubules is damaged as the content is increased and is found in early renal tubule lesions, compared with β 2-MG, α 1-MG is not affected by malignant tumor and urine pH value, and is a very sensitive index for early renal tubule injury[11,12]The positive rate of α 1-MG and cystatin C in urine for combined detection of diabetic nephropathy is 79.8%[13] Urine α 1 in the case of acute tubular necrosisThe detection sensitivity of MG is 88% and the specificity is 81%[4]。
(6) β 2-microglobulin (β 2-MG 2)
β 2-microglobulin is produced by almost all nucleated cells in human body, has 11.8kDa molecular weight, is mainly filtered by glomeruli and is only excreted by kidney, the production rate in vivo is constant, and is not influenced by age, sex and muscle, β 2-MG in urine is increased, which indicates that the damage to the tubules or the filtration function is reduced, and is commonly seen in acute and chronic nephritis, renal failure, etc. β 2-MG is used in renal diseases, which mainly includes diagnosing early stage renal injury, identifying glomerular or tubular inflammation, predicting rejection reaction of renal transplantation, identifying upper and lower urinary tract infections, monitoring nephrotoxicity of certain drugs, etc. determination of blood and urine β 2-MG can be used as a sensitive index for diagnosing early stage diabetic nephropathy and hypertension, and using the ratio of Alb/β 2-MG in urine to identify renal damage of glomeruli or tubular kidney, if Alb/β 2-MG in urine is more than 1000, which indicates that primary glomerular disease, Alb/β 2 in urine is less than 40, indicating that renal disease[4]。
(7) β -catenin (β -catenin)
β -catenin mainly has the functions of mediating cell adhesion and participating gene expression, is widely distributed in endothelial cells, fibroblasts and the like and participates in proliferation, differentiation and apoptosis, the Wnt/β -catenin signal pathway is a key development pathway with evolutionary conservation and high complexity, and in normal adult kidneys, the Wnt/β -catenin signal pathway is relatively silent, but in various animal models and human kidney diseases, the Wnt/β -catenin signal is activated after kidney injury and participates in kidney injury and repair[14]Research shows that the Wnt/β -catenin signal channel plays an important role in the development of diabetic nephropathy, can aggravate insulin resistance and promote the occurrence and development of atherosclerosis, and is an independent risk factor influencing kidney functions[15]Urine β -catenin showed 96.6% and 90% sensitivity and specificity in diagnosing ANCA-associated small vessel inflammation renal damage, respectively[16]。
(8) Urine microalbumin (urine mAB)
The microalbuminuria isThe albumin content in urine is 30-300 mg/24h or the albumin concentration in a urine sample collected at regular time (20-200 mug/min) or random urine sample is 20-200 mg/L, namely the albumin content exceeds the upper limit of a normal reference value (30mg/24h or 20 mug/min or 20mg/L) and clinical proteinuria is not detected. The molecular weight of urine Microalbumin (MA) is 69kDa, has negative charge, is an early diagnosis index of renal dysfunction, and has higher specificity and sensitivity than total urine protein[17]. Normally, the majority of the fluid is unable to pass through the glomerular filtration membrane, and the amount of MA in urine increases when the permeability of the glomerular filtration membrane barrier is increased or damaged by various causes. KDIGO has included MA in CKD staging, and recommended urinary MA/urinary creatinine > 30mg/g for diagnosis of microalbuminuria[12]. The American diabetes Association recommends that all type I diabetics with a disease course of more than or equal to 5 years and all newly diagnosed type II diabetics detect the excretion of urinary albumin annually[18]。
(9) Urinary Transferrin (TRF)
The urinary transferrin is a single-chain glycoprotein with the molecular weight of 76.5kDa, is mainly synthesized by the liver and can not freely pass through a glomerular filtration membrane barrier under the general condition, but because the urinary transferrin has less negative charges than albumin, the TRF can be leaked out due to slight damage of the charge barrier on the glomerular filtration membrane, and when the urinary albumin level is normal at the early stage of renal injury, the TRF level of 70 percent of patients is remarkably increased, which indicates that the TRF can be used as an important diagnostic index for early diabetic nephropathy[19]. It has been shown that the sensitivity of urine TRF in early diagnosis of diabetic nephropathy is 50.0% and the specificity is 97.8%[20]。
(10) Urine immunoglobulin (IgG)
Urine IgG is an anionic plasma protein with molecular weight of 150kDa, which cannot pass through glomerular filtration barrier under physiological conditions, so its presence in urine is indicative of the destruction of glomerular filtration barrier and is a marker of glomerular injury[21]. IgG may appear before and at the stage of development of microalbuminuria, accompanied by elevation of urine TRF, urine plasma ceruloplasmin and the like[18]. There are studies showing that the IgG positive rate in patients with primary nephrotic syndrome is as high as 73.63％[22]。
(11) Human neutrophil gelatinase-associated lipocalin (NGAL)
NGAL is synthesized by activated neutrophils in the proximal kidney tubule, covalently linked to neutrophil gelatinase, and has a molecular weight of 25 kDa. Normally, it is low expressed in human tissues, but when nephrotoxic or ischemic injury occurs, NGAL is significantly up-regulated and highly expressed in damaged renal tubules. NGAL can resist the action of proteolytic enzyme, is convenient to detect in urine, and is an early sensitive and specific biological marker of nephrotoxicity or ischemic acute renal injury[12]. The sensitivity and specificity of the urinary NGAL for diagnosing ANCA-related small vasculitis renal damage are 82.8 percent and 40 percent respectively[16]。
(12) Cystatin C (CysC)
Cystatin C is a non-glycosylated basic protein with a molecular weight of 13359, is produced by all nucleated cells, has a stable production rate, is not affected by inflammation, freely passes through glomerular filtration membranes, is reabsorbed and metabolized in the proximal tubule, is not secreted from the tubule, has a molecular weight greater than creatinine, and is positively charged so as to more easily reflect the early change of the glomerular filtration rate, and the amount of CysC discharged from urine is not affected by age, sex, muscle mass, diet, exercise, infection and other factors, and once the function of the tubules is disturbed, the CysC content in urine can be increased by 200 times[13]And is therefore considered to be a novel biomarker which responds better than serum creatinine to early changes in glomerular filtration rate. Research reports that the detection sensitivity of urine CysC in acute tubular necrosis cases is 92 percent and the specificity is 83 percent[4]。
(13) Three-leaf factor 3(TFF3)
Trefoil factor 3 consists of 59 amino acids, has a molecular weight of about 6.7kDa, produces biological effects mainly in a dimeric form, and TFF3 has certain tissue cell specificity and is mainly present in small intestine, colon goblet cell, thyroid gland, endometrium, renal tubular epithelial cell and the like. Animal experiments show that compared with the current common clinical biomarkers of blood creatinine and urea nitrogen, the urine TFF3 can diagnose earlierThe renal tubule injury has high sensitivity and specificity, and is expected to become a novel marker of chronic nephropathy and glomerulonephritis[23,24]。
(14) Urinary trehalase
Urotrehalase (urory trehalase) is an extracellular glycoprotein with a molecular weight of 75kD, which is produced only in the epithelial tissue of the proximal renal tubule and small intestine mucosa in humans and has a high specificity in distribution. Trehalase is present in very low blood concentrations, generally cannot be filtered from the glomeruli due to its large molecular weight and low isoelectric point, and if trehalase activity is detected in urine, it is suggested that proximal tubular function is impaired. The biological activity of the trehalase is extremely stable and is not easy to degrade, and the activity of the trehalase is not obviously changed after the trehalase is placed at 4 ℃ for a week. Therefore, the trehalase can be used as an ideal index for reflecting the renal tubular injury[25]. The study indicates that the sensitivity and specificity of the urase for diagnosing ANCA-related small vessel inflammation renal damage are 93.1 percent and 80 percent respectively[16]。
The kidney injury related index is mostly detected by adopting an immunological method, and the most common detection methods in the market at present comprise an immunoblotting method (dot immunity and strip immunity), immunoturbidimetry, enzyme-linked immunosorbent assay, a chemiluminescence method and the like. The immunoblotting method is suitable for preliminary screening test, is simple, has poor sensitivity and specificity, and generally can only realize qualitative detection. Enzyme-linked immunosorbent assay (ELISA) and single-index chemiluminescence methods are the most common methods for immunodiagnosis, the sensitivity and specificity are high, the result reliability is higher than that of an immunoblotting method, but for a combined project, each project needs to be detected independently, the detection efficiency is low, and the cost is high. Moreover, the method needs independent calibration and quality control on more than ten indexes, is complex to operate and is not beneficial to quickly giving out a detection result.
The protein chip technology realizes combined detection, can detect a plurality of indexes at one time, effectively improves the detection efficiency, realizes quantitative detection, reduces the detection cost, and has the advantages of high sensitivity, good specificity, high full automation degree, wide application range and the like.
Disclosure of Invention
The invention provides a protein chip for detecting a kidney injury marker and a preparation method thereof, wherein the protein chip is used for detecting the kidney injury marker, joint detection can be realized by applying the protein chip technology originally invented by the company, so that the detection efficiency can be effectively improved, and the detection cost can be reduced, and the invention develops a protein chip diagnosis kit simultaneously containing 14 indexes of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urine mALB, urine TRF, urine IgG, NGAL, CysC, TFF3 and urine trehalase, and has the advantages of rapidness, high efficiency, low cost and the like.
The technical scheme of the invention is as follows:
a protein chip for detecting a kidney injury marker is prepared by the following steps:
(1) pretreating a black glass slide;
(2) spotting an antibody solution;
(3) and (3) sealing to obtain the protein chip.
The method for pretreating the black glass slide in the step (1) comprises the following steps:
①, soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 16-24 h, and then cleaning with purified water for 2-8 times;
② soaking the black glass slide in 0.05-1% silane solution (25% ethanol as medium) for 20-60 min;
③, blowing the soaked black glass slide with nitrogen, putting the black glass slide into an oven, and baking the black glass slide for 0.2 to 0.6h at the temperature of 100 to 180 ℃.
The antibody solution in step (2) comprises clusterin antibody, KIM-1 antibody, PAI-1 antibody, HSP72 antibody, α 1-MG antibody, β 2-MG antibody, β -catenin antibody, urinary mAB antibody, urinary TRF antibody, urinary IgG antibody, NGAL antibody, CysC antibody, TFF3 antibody and urinary trehalase antibody solution.
The spotting method in the step (2) is machine-automated spotting.
The sealing process in the step (3) is as follows: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.
The confining liquid is a buffer solution containing confining protein; the blocking protein is bovine serum albumin or ovalbumin; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPS buffer solution and MOPS buffer solution.
An application of the protein chip, and the protein chip is prepared into a kit.
The kit also includes a solution of a secondary antibody labeled with HRP enzyme or alkaline phosphatase, a chemiluminescent substrate sensitive to the label.
The concentration of the HRP enzyme-labeled secondary antibody solution is 0.5. mu.g/mL of clusterin secondary antibody, 0.5. mu.g/mL of KIM-1 secondary antibody, 0.1. mu.g/mL of PAI-1 secondary antibody, 0.1. mu.g/mL of HSP72 secondary antibody, 0.1. mu.g/mL of α 1-MG secondary antibody, 0.1. mu.g/mL of β 2-MG secondary antibody, 0.5. mu.g/mL of β -catenin, 0.5. mu.g/mL of urine mAB secondary antibody, 0.5. mu.g/mL of urine TRF secondary antibody, 0.5. mu.g/mL of urine IgG secondary antibody, 0.5. mu.g/mL of NGAL secondary antibody, 0.5. mu.g/mL of CysC secondary antibody, 0.1. mu.g/mL of TFF3 secondary antibody and 0.1. mu.g/mL of urine trehalase secondary antibody, and the chemiluminescent substrate is detection solution containing luminol, hydrogen peroxide, detection solution A and detection solution B, respectively.
The beneficial technical effects of the invention are as follows:
the protein chip is used for detecting 14 important kidney injury markers, joint detection is realized by applying a protein chip technology, the detection efficiency can be effectively improved, the detection cost is reduced, and meanwhile, the protein chip diagnosis kit comprises 14 indexes of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urine mALB, urine TRF, urine IgG, NGAL, CysC, TFF3 and urine trehalase, and has the advantages of rapidness, high efficiency, low cost and the like.
The invention adopts a classical immunological sandwich method. The capture antibodies are fixed on a chip substrate taking glass as a carrier, the antibodies can capture specific antigens in a detected sample, and the captured antigens are combined with a second antibody labeled with HRP enzyme or alkaline phosphatase to form a complex. Adding a chemiluminescent substrate sensitive to the marker to perform chemiluminescence, collecting an optical signal through a CCD camera, and judging the concentration of the specific marker antigen in the detected sample through the intensity of the optical signal.
The kit uses a kidney injury chip technology platform, and a single detection method of other domestic manufacturers uses a common chemiluminescence means, compared with the prior art, the kit has the advantages that ① has very low requirements on biological samples due to high specificity and strong affinity of the combination of the antigen and the antibody of the kit and low influence of other impurities, the pretreatment process of the samples can be simplified, ② can rapidly analyze a large number of samples in a high-flux and parallelization quantitative manner, ③ is simple to operate, the result accuracy is high, ④ patients only need to take one urine sample and can rapidly give a monitoring result, ⑤ needs few reagents and samples, and the price is low.
Drawings
FIG. 1 is a schematic representation of the spotting of antibodies of the invention;
in the figure, column 1 is a positive quality control column, column 2 is a blank, and columns 3 to 16 are clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urinary mAB, urinary TRF, urinary IgG, NGAL, CysC, TFF3 and urinary trehalase antibody, respectively.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Example 1
A protein chip for detecting a kidney injury marker is prepared by the following steps:
(1) pretreating a black glass slide;
① soaking the black glass slide in a glass slide pretreatment solution containing 2% NaOH for 16h, and then cleaning with purified water for 2-8 times;
② soaking the black glass in 0.05% silane solution (25% ethanol) for 60 min;
③ the soaked black glass slide is placed into an oven after being purged by nitrogen and baked for 0.2h at 180 ℃.
(2) Spotting an antibody solution;
referring to FIG. 1, a machine automated spotting of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urinary mAB, urinary TRF, urinary IgG, NGAL, CysC, TFF3 and urinary trehalase antibody solutions at a concentration of 0.1MG/mL at 20nL per spot was used, with the distribution of antibody spotting shown in FIG. 1.
(3) Sealing process;
and immersing the spotted black slide into a blocking solution (PBS buffer solution containing 1% bovine serum albumin) for 10 hours, then taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was co-packaged with a solution of a second antibody labeled with HRP enzyme (concentrations of clusterin second antibody 0.5. mu.g/mL, KIM-1 second antibody 0.5. mu.g/mL, PAI-1 second antibody 0.1. mu.g/mL, HSP72 second antibody 0.1. mu.g/mL, α 1-MG second antibody 0.1. mu.g/mL, β 2-MG second antibody 0.1. mu.g/mL, β -catenin 0.5. mu.g/mL, urine mAB second antibody 0.5. mu.g/mL, urine TRF second antibody 0.5. mu.g/mL, urine IgG second antibody 0.5. mu.g/mL, NGAL second antibody 0.5. mu.g/mL, CysC second antibody 0.5. mu.g/mL, TFF3 second antibody 0.1. mu.g/mL and urine trehalase second antibody 0.1. mu.g/mL), detection solution A (containing 1% minox and 2% hydrogen peroxide solution B) to give a kit.
Example 2
A protein chip for detecting a kidney injury marker is prepared by the following steps:
(1) pretreating a black glass slide;
① soaking the black glass slide in a glass slide pretreatment solution containing 2% NaOH for 24h, and then cleaning with purified water for 2-8 times;
② soaking black glass in 0.5% silane solution (25% ethanol as medium) for 30 min;
③ the soaked black glass slide is placed into an oven after being purged by nitrogen and baked for 0.5h at the temperature of 140 ℃.
(2) Spotting an antibody solution;
referring to FIG. 1, a machine automated spotting of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urinary mAB, urinary TRF, urinary IgG, NGAL, CysC, TFF3 and urinary trehalase antibody solutions at a concentration of 0.1MG/mL at 20nL per spot was used, with the distribution of antibody spotting shown in FIG. 1.
(3) Sealing process;
and immersing the spotted black slide into a blocking solution (PBS (phosphate buffer solution) containing 2% ovalbumin) for 24 hours, taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was co-packaged with a solution of a second antibody labeled with HRP enzyme (concentrations of clusterin second antibody 0.5. mu.g/mL, KIM-1 second antibody 0.5. mu.g/mL, PAI-1 second antibody 0.1. mu.g/mL, HSP72 second antibody 0.1. mu.g/mL, α 1-MG second antibody 0.1. mu.g/mL, β 2-MG second antibody 0.1. mu.g/mL, β -catenin 0.5. mu.g/mL, urinary mAB second antibody 0.5. mu.g/mL, urinary TRF second antibody 0.5. mu.g/mL, urinary IgG second antibody 0.5. mu.g/mL, NGAL second antibody 0.5. mu.g/mL, CysC second antibody 0.5. mu.g/mL, TFF3 second antibody 0.1. mu.g/mL and urinary trehalase second antibody 0.1. mu.g/mL), detection solution A (containing 1% minox and 2% hydrogen peroxide solution B) to give a kit.
Example 3
A protein chip for detecting a kidney injury marker is prepared by the following steps:
(1) pretreating a black glass slide;
① soaking the black glass slide in a glass slide pretreatment solution containing 2% NaOH for 20h, and then cleaning with purified water for 2-8 times;
② soaking the black glass in 1% silane solution (25% ethanol as medium) for 20 min;
③ the soaked black glass slide is placed into an oven after being purged by nitrogen and baked for 0.6h at the temperature of 100 ℃.
(2) Spotting an antibody solution;
referring to FIG. 1, a machine automated spotting of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urinary mAB, urinary TRF, urinary IgG, NGAL, CysC, TFF3 and urinary trehalase antibody solutions at a concentration of 0.1MG/mL at 20nL per spot was used, with the distribution of antibody spotting shown in FIG. 1.
(3) Sealing process;
and immersing the spotted black slide into a blocking solution (Tris buffer solution containing 3% bovine serum albumin) for 8 hours, then taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was co-packaged with a solution of a second antibody labeled with HRP enzyme (concentrations of clusterin second antibody 0.5. mu.g/mL, KIM-1 second antibody 0.5. mu.g/mL, PAI-1 second antibody 0.1. mu.g/mL, HSP72 second antibody 0.1. mu.g/mL, α 1-MG second antibody 0.1. mu.g/mL, β 2-MG second antibody 0.1. mu.g/mL, β -catenin 0.5. mu.g/mL, urine mAB second antibody 0.5. mu.g/mL, urine TRF second antibody 0.5. mu.g/mL, urine IgG second antibody 0.5. mu.g/mL, NGAL second antibody 0.5. mu.g/mL, CysC second antibody 0.5. mu.g/mL, TFF3 second antibody 0.1. mu.g/mL and urine trehalase second antibody 0.1. mu.g/mL), detection solution A (containing 1% minox and 2% hydrogen peroxide solution B) to give a kit.
Test example:
the SLXP-001 biochip reader produced by the company is used for detecting clinical urine samples, and the detection result is compared with each reference kit (the single index kit of a domestic mainstream manufacturer is used as a reference).
The working process of the SLXP-001 biochip reader is as follows:
the instrument automatically sucks 200 mu L of diluted sample to be detected (urine is diluted by 100 times) into a reaction cup, the instrument automatically puts the protein chip prepared in the embodiment of the invention into the sample to be detected, the sample is incubated for 40 minutes at 37 ℃, then the chip is taken out by an instrument clamping jaw, the chip is automatically washed by the instrument and then put into a second antibody solution (200 mu L, the instrument is automatically sucked in advance), the HRP enzyme is marked, the chip is taken out again by the instrument clamping jaw after the sample is incubated for 40 minutes again, the chip is automatically washed by the instrument and then put into a luminescent substrate solution (formed by mixing 100 mu L of detection solution A and 100 mu L of detection solution B, the chip is automatically sucked and mixed by the instrument), and finally, the protein chip is photographed and imaged, pictures are automatically analyzed by software, and an analysis result is given. The results of the measurements are shown in tables 1 to 5.
TABLE 1
Figure BDA0002309985170000071
TABLE 2
Figure BDA0002309985170000072
TABLE 3
Figure BDA0002309985170000081
TABLE 4
Figure BDA0002309985170000082
TABLE 5
Figure BDA0002309985170000091
As can be seen from the above table, the kit provided by the invention can simultaneously detect 14 indexes of clusterin, KIM-1, PAI-1, HSP72, α 1-MG, β 2-MG, β -catenin, urinary mAB, urinary TRF, urinary IgG, NGAL, CysC, TFF3 and urinary trehalase, can obtain results similar to those of a single-index kit (most of relative errors are within 10%), and has no significant difference in the aspects of sensitivity, linear range and the like.
Reference to the literature
[1] Yan Wen Juan, Bright, research progress of acute renal injury [ J ]. J.J.Clin and Pathology 2019,39(7):1571-1575.
[2] Chen Mulin, Yangchen, Han Huan Qin, et al, State of the research and New developments of biomarkers of acute renal injury [ J ] medical review, 2019,25(9):1761-1765.
[3] Establishment of a method for detecting a kidney injury marker by using a Maxi Hui.flow microsphere technology and primary clinical application [ D ]. Beijing, military medical science and academy of liberation of people of China, 2016.
[4] Zhao jin. evaluation of the effect of traditional Chinese medicine on renal function based on novel biomarkers [ D ]. Guangzhou: Guangzhou university of traditional Chinese medicine 2015.
[5]Petra V,Sofie S,Erio BT,et al.Tissue Kim-1and urinary clusterin asearly indicators of cisplatin-induced acute Kidney injury in rats[J].Toxicologic Pathology,2012,40(7):1049-1062.
[6] Zhang Yidan-kidney injury molecule-1 expression in primary membranous nephropathy and its meaning [ D ]. Jinan-Shandong university, 2018.
[7] Kim-1, PAI-1 and Hsp72 are combined to detect the diagnostic value of early renal damage of diabetes [ J ]. southwest defense medicine, 2017,27(3): 247-.
[8] PAI-1 was studied on the mechanism of reducing cardiac fibrosis and renal urinary protein by binding to vitronectin [ D.
[9] Expression and significance of heat shock protein 72 in rat kidney under acute hypoxic environment of Shili [ D ]. Xining, Qinghai university, 2014.
[10] Yanchungu, Zhu Dong, Wangfeng, Heat shock protein 72 and Toll-like receptor 4 in the renal tissue of rats with hepatorenal syndrome [ J ] Chinese Critical Emergency medicine 2007,19(12):731-734.
[11] Lemingyuan. an epidemiological study of acute kidney injury in intensive care unit patients [ D ]. Suzhou: Suzhou university, 2013.
[12] Jinyan, clinical value of urine microalbumin in early renal injury of critically ill patients is first explored [ D ]. Beijing, university of Chinese medicine, 2014.
[13] The diagnosis research of cystatin C and α -1 microglobulin in diabetic nephropathy [ J ]. J.China J.hygienic examination, 2016,26(12):1729-1733.
[14] Role of β -catenin in chronic transplanted kidney injury [ J ]. J.J.Clin & Pathology 2019,39(2): 268. one of 274.
[15] Evaluation of DN prognosis in conjunction with the detection of blood uric acid by Rhode, von Korea Red, Bolus, et al β -catenin and cadherin proteins [ J ] clinical transfusion and testing, 2019,21(2): 180-.
[16] Penweihua, Chenling boat, xuanmin, etc. detection and significance of urinary renal injury biomarkers in patients with renal injury caused by ANCA-related microangiotis [ J ] J.J.nephrosis, 2017,18(10): 860) 865, Chinese and Western medicine.
[17] Li Xia, discussion of relationship between normal hypertension and urine microalbumin [ D ] Taiyuan, Shanxi university of medical science, 2009.
[18] Zhengmao, investigation and study of urinary protein excretion of type 2 diabetes patients in hospitalization [ D ]. Combined fertilization, university of medical science in Anhui, 2018.
[19] Linchuanhui urinary transferrin assay has clinical significance in early diagnosis of diabetic nephropathy [ J ]. New world of diabetes 2015, (4): 125-.
[20] The research on the relativity of the WangJing, the serum cystatin C of diabetic nephropathy, urinary transferrin and the glomerular filtration rate [ D ]. Shijiazhuang, Hebei university of medicine, 2013.
[21] The value research of the Xuehao urine protein marker in the diagnosis, progression and prognosis evaluation of diabetic nephropathy [ D ]. Shanghai, China civil liberation army medical university, navy, 2018.
[22] Zhangxia. Primary nephrotic syndrome complement, immunoglobulin and clinical pathological analysis [ D ]. Yanan university, 2014.
[23]ASTOR B C.KOTTGEN A.HWANG S J.et al.Trefoil factor 3predictsincident chronic kidney disease:a case-control study nested within theAtherosclerosis Risk in Communities(ARIC) study[J].Am J Nephrol,2011,34(4):291-297.
[24]DU T Y.LUO H M.QIN H C.et al.Circulating serum trefoil factor 3(TFF3)is dramatically increased in chronic kidney disease[J].PLoS One.2014,8(11):e80271.
[25] Zhangshan urinary trehalase has clinical significance to proximal tubular injury [ D ]. Changsha, university of Central and south, 2004.

Claims (8)

1. A protein chip for detecting a kidney injury marker is characterized in that the preparation method of the protein chip comprises the following steps:
(1) pretreating a black glass slide;
(2) spotting an antibody solution;
(3) and (3) sealing to obtain the protein chip.
2. The protein chip according to claim 1, wherein the black glass slide pretreatment method in step (1) is:
①, soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 16-24 h, and then cleaning with purified water for 2-8 times;
②, soaking the black glass slide in a silane solution with the mass concentration of 0.05-1% for 20-60 min;
③, blowing the soaked black glass slide with nitrogen, putting the black glass slide into an oven, and baking the black glass slide for 0.2 to 0.6h at the temperature of 100 to 180 ℃.
3. The protein chip according to claim 1, wherein the antibody solution in step (2) comprises an anti-clusterin antibody, an anti-cystatin C antibody, an anti-kidney injury molecule-1 antibody, an anti-human plasminogen activator inhibitor 1 antibody, an anti-urine immunoglobulin antibody, an anti-heat shock protein 72 antibody, an anti- β -catenin antibody, an anti- β 2-microglobulin antibody, an anti-urine transferrin antibody, an anti-urine α 1-microglobulin antibody, an anti-trefoil factor 3 antibody, an anti-urine trehalase antibody, an anti-human neutrophil gelatinase-associated lipocalin antibody, and an anti-urine microalbumin antibody solution.
4. The protein chip according to claim 1, wherein the spotting method in step (2) is machine automated spotting.
5. The protein chip according to claim 1, wherein the blocking process in step (3) is: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.
6. The protein chip according to claim 5, wherein the blocking solution is a buffer solution containing blocking protein; the blocking protein is bovine serum albumin or ovalbumin; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPS buffer solution and MOPS buffer solution.
7. The use of the protein chip according to any one of claims 1 to 6, wherein the protein chip is used to prepare a kit.
8. The use according to claim 7, characterized in that the kit further comprises a solution of a secondary antibody labeled with HRP enzyme or alkaline phosphatase, a chemiluminescent substrate sensitive to the label.
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