CN114578064A

CN114578064A - Application of reagent for detecting CAF22 in preparation of composition for evaluating renal tubular injury

Info

Publication number: CN114578064A
Application number: CN202210483504.8A
Authority: CN
Inventors: 唐明镜; 郑旻亮; 席强
Original assignee: Beijing Watson Saiser Biotechnology Co ltd
Current assignee: Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2022-06-03
Anticipated expiration: 2042-05-05
Also published as: CN114578064B

Abstract

The invention discloses an application of a detection reagent for a C-terminal fragment (CAF 22) of collectin in preparing a renal tubular injury diagnostic kit and the kit thereof, and provides a new biomarker for diagnosing renal tubular injury by using CAF22 as a diagnostic marker of renal tubular injury; the invention also combines the CAF22 detection with other kidney injury biomarkers, can improve the accuracy and sensitivity of early diagnosis of renal tubular injury, and has higher application value for early diagnosis and treatment of renal injury.

Description

Application of reagent for detecting CAF22 in preparation of composition for evaluating renal tubular injury

Technical Field

The invention relates to the field of biological diagnosis, in particular to application of a reagent for detecting CAF22 in preparing a composition for evaluating renal tubular injury.

Background

Tubular Injury (renal tubular Injury) is a common complication in critically ill patients and patients undergoing cardiovascular surgery, and has a high incidence and mortality rate, with a postoperative incidence of about 5% to 47% in patients undergoing cardiac surgery and a tubular Injury mortality rate of 50% to 70% in patients with ICU. Tubular injury, if not treated promptly, can progress to chronic kidney disease or require permanent dialysis. Therefore, the early diagnosis and timely intervention and treatment of the renal tubular injury can effectively prevent the further deterioration of the disease and improve the prognosis of patients. In order to improve the diagnosis and treatment level of the renal tubular injury and improve the global renal disease prognosis organization (KDIGO), a KDIGO renal tubular injury clinical practice guideline is published in 2012, and Serum Creatinine (SCR) and urine volume are used as diagnosis and staging standards of the renal tubular injury in the guideline. However, both of these indicators have many drawbacks in the diagnosis of renal tubular injury. First, SCr and urine volume are in fact biomarkers of renal function and not markers of renal damage. Second, Scr is a surrogate marker for glomerular filtration rate (GFP), the concentration of which indicates a balance between creatinine production and excretion, but is reflected in an increase in Scr when GFP is less than 50% of normal levels, and therefore Scr delays diagnosis of tubular injury; third, the SCr is affected by many external factors such as the physiological and pathological states of the patient and the conditions of the drug administration. Fourth, changes in urine volume may not be consistent with the extent of renal injury, for example, patients in dehydration or using diuretics may result in changes in urine volume that are less specific for the diagnosis of renal tubular injury.

The puncture biopsy as the kidney injury diagnosis gold standard has important meanings such as definite diagnosis, treatment guidance, prognosis judgment, pathogenesis discussion, curative effect judgment and mechanism thereof on kidney diseases in specific clinical work, can intuitively and clearly reflect the specific situation of kidney injury through pathological sections, but the current kidney puncture is relatively complex, the pain of a patient is large, and the kidney puncture detection cannot be adopted for the patient to any extent.

The routine items mLB, IgG, TRF, NAG, beta 2-microglobulin can be used as the index of whether the renal function is abnormal or not. However, the failure to reflect a specific renal status and identify the specific cause of renal dysfunction is likely to result in a loss of optimal treatment time due to failure to correctly determine the status of renal injury in the clinic, while intervention at an early stage of renal injury may reduce the mortality associated with renal tubular injury and shorten the treatment time for various types of renal tubular injury, including but not limited to ischemic and nephrotoxic renal injuries. The identification of reliable early biomarkers of renal tubular injury is useful for facilitating early therapeutic intervention and helps guide drug development by providing an indicator of renal toxicity. Therefore, a new biomarker which has good specificity to renal tubular injury, can directly indicate renal tubular injury and can respond in time in early diagnosis is urgently needed.

Disclosure of Invention

In view of the above, the present invention is directed to the use of an agent for detecting C-terminal fragment of Agrin, which is a large proteoglycan, a major heparin sulfate proteoglycan of glomerulus and renal tubular basement membrane, highly expressed in the kidney, for the preparation of a composition for evaluating renal tubular injury in a subject. Agrin is cleaved from the beta site by neurotrypsin, resulting in a 22kDa C-terminal Fragment, i.e.the C-terminal Fragment of Agrin (C-terminal Fragment of Agrin 22, CAF 22). CAF22 is cleared from the circulation primarily by glomerular filtration, and filtered CAF22 is reabsorbed by the proximal tubules. When an injury to renal function occurs, the rate of renal clearance of CAF22 decreases, resulting in elevated levels of CAF22 in the urine. Research finds that the change of CAF22 is in positive correlation with the glomerular filtration rate and the urinary albumin level, so that the invention uses CAF22 as a novel biomarker reflecting the renal tubular injury, and the change of CAF22 is detected to reflect the renal tubular cell injury condition.

In order to achieve the purpose, the invention provides the following technical scheme:

use of a capture agent that detects a C-terminal fragment of agrin in the preparation of a composition for assessing the state of renal tubular injury in a subject.

Preferably, the injury to the renal tubules is selected from ischemic renal injury, nephrotoxic renal injury, or other injury affecting renal tubular cells.

Preferably, the assessing comprises detecting the polyprotein C-terminal fragment in a sample of bodily fluid obtained from the subject to provide a detection result, and correlating the detection result with the renal tubular injury status of the subject.

The term "bodily fluid sample" as used herein refers to a bodily fluid sample obtained for the purpose of diagnosis, prognosis, classification or evaluation of the relevant subject, e.g., a patient or transplant donor. In some embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen for a condition. Body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, and the like. Furthermore, those skilled in the art will appreciate that some bodily fluid samples may be more readily analyzed after fractionation and purification steps, e.g., separation of whole blood into serum or plasma components.

Preferably, the body fluid sample is serum or urine. More preferably, the body fluid sample is a random urine or serum sample prior to renal puncture.

Preferably, the capture agent is an antibody or a derivative thereof that specifically recognizes the C-terminal fragment of agrin.

Preferably, said derivatives comprise a fragment capable of specifically recognizing the C-terminal fragment of agrin: at least one of an antibody functional fragment, an antigen binding fragment, a conjugated antibody, a humanized antibody, a recombinant antibody, a diabody, a chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab 'fragment, or an F (ab') 2 fragment.

Preferably, wherein the application further comprises:

detecting one or more of the following markers: neutrophil gelatinase-associated lipocalin, interleukin 18, kidney injury molecule-1, liver-type-fatty acid binding protein, metalloproteinase tissue inhibitor 2, insulin-like growth factor binding protein 7;

and assessing the renal tubular injury status of the subject in conjunction with the detection of the C-terminal fragment of the polyprotein.

Preferably, the assessment comprises at least one of a prediction, risk stratification, diagnosis, staging, prognosis or monitoring of the renal tubular injury of the subject.

Preferably, said use comprises detecting the concentration and/or activity of the C-terminal fragment of agrin.

Preferably, said use comprises measuring the concentration and/or activity of at least one of the following markers: neutrophil gelatinase-associated lipocalin, interleukin 18, kidney injury molecule-1, liver-type-fatty acid binding protein, metalloproteinase tissue inhibitor 2, insulin-like growth factor binding protein 7.

Preferably, the composition is an ELISA detection composition, an immunoturbidimetric detection composition, an immunochromatographic detection composition or a chemiluminescent immunoassay detection composition.

Preferably, the composition is a chemiluminescent immunoassay composition comprising a conjugate-labeled agrin C-terminal fragment antibody, a chemiluminescent-label-labeled agrin C-terminal fragment antibody, streptavidin magnetic particles, and a chemiluminescent substrate;

the conjugate label is a biotin ester label;

the chemiluminescence label is one or more of alkaline phosphatase, acridinium ester and derivatives thereof, horseradish peroxidase or ruthenium terpyridyl;

the chemiluminescence zymolyte is one or more of NaOH and H2O2, luminol, (adamantane) -1, 2-dioxyethane or derivatives thereof, APS-5 or tripropylamine.

As the preferable technical proposal of the invention, the working concentration of the conjugate labeled agrin C-terminal fragment antibody is 0.1-2.0 mug/mL; the working concentration of the agrin C-terminal fragment antibody marked by the chemiluminescent marker is 0.1-2.0 mug/mL; the working concentration of the streptavidin magnetic particles is 0.2-2.0 mg/mL, and the particle size is 1.0-5.0 μm.

In a preferred embodiment of the present invention, the antibody against the C-terminal fragment of agrin labeled with the conjugate is stored in a buffer containing NaCl, a protein, a surfactant and a preservative; the agrin C-terminal fragment antibody labeled with the chemiluminescence label is preserved in a buffer solution containing NaCl, protein, a surfactant and a preservative.

More preferably, the buffer component may be PBS, Tris, MES, TBS or HEPES; the surfactant can be one or more of Tween, Brij and Triton; the preservative is one or a combination of phenol, sodium benzoate, proclin and sodium azide.

In another aspect, the present invention provides a kit comprising the above composition.

As the preferable technical scheme of the invention, the kit also comprises a collectin C-terminal fragment calibrator and a collectin C-terminal fragment quality control product.

In another aspect, the present invention discloses the use of a C-terminal fragment of agrin as a synergistic indicator for assessing the status of renal tubular injury in a subject, the synergistic indicator being capable of enhancing the diagnostic effect of a renal injury marker on renal tubular injury in combination with a renal injury marker, the renal injury marker being a marker capable of indicating renal injury, the assessment comprising detecting the C-terminal fragment of agrin and at least one of the renal injury markers in a sample of bodily fluid obtained from the subject to provide a combined detection result;

and correlating the combined test result with the renal tubular injury status of the subject;

preferably, the kidney injury marker is selected from one or more of the following: NGAL, IL-18, KIM-1, L-FABP, TIMP2 or IGFBP 7.

The synergistic index and the kidney injury marker are combined to improve the diagnosis effect of the kidney injury marker on renal tubular injury, and the effect refers to that: the combination of the C-terminal fragment of the agrin and other kidney status markers can improve the accuracy and sensitivity of other kidney injury markers to renal tubular injury detection.

The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are useful for human and veterinary disease. Further, the present invention can also be used for post mortem analysis when the subject is preferably a living organism. Preferably the subject is a human, and most preferably is a "patient", as used herein refers to a living human receiving medical care for a disease or condition. This includes persons without established disease who are being examined for pathological phenomena. Preferably, the subject is selected from patients with abnormal renal status.

The term "diagnosis" as used herein refers to a method by which one of skill in the art can estimate and/or determine the likelihood of whether a patient has a given disease or condition, as an aid to diagnosis. In the context of the present invention, "diagnosis" includes the use of the results of detection, most preferably immunodetection, of the kidney injury markers used in the present invention, optionally together with other clinical features, to obtain samples and tests from patients to achieve diagnosis (i.e., the occurrence or non-occurrence) of renal tubular injury. Such a diagnosis is "determined", which is not meant to imply that the diagnosis is 100% accurate. Thus, a measured level of a biomarker on one side of the predetermined diagnostic threshold indicates a greater likelihood of the subject developing a disease relative to a measured level on the other side of the predetermined diagnostic threshold.

Similarly, prognostic risk represents the likelihood that a given process or outcome will occur. A change in the level or level of a prognostic indicator, which in turn is associated with an increased likelihood of disease (e.g., renal tubular injury will occur), refers to "indicating an increased likelihood" of an adverse outcome in a patient.

The term "antibody" as used herein refers to a peptide or polypeptide derived from substantially an immunoglobulin based on coding or post-immunoglobulin gene simulation or immunoglobulin genes or fragments thereof, capable of specifically binding an antigen or epitope.

The invention has the beneficial effects that: the invention takes CAF22 as a diagnosis marker of renal tubular injury, provides a new biomarker for the diagnosis of renal tubular injury, can reflect the integrity of proximal tubules, and has higher application value in the aspect of early diagnosis or dynamic detection of renal tubular injury. And the invention is based on CAF22 combined with other biomarkers, which can further improve the sensitivity and accuracy of detection and provide more possibilities for diagnosis. In clinical treatment, once an indicator of tubular cell injury or acute renal failure is detected, intervention and treatment of the disease or condition is initiated, and the clinician can monitor the course of the treatment or intervention using the methods and kits of the invention. Typically, when treatment of renal injury begins and continues, one or more subsequent post-treatment urine samples are obtained and analyzed for the presence of biomarkers. The degree of improvement can be expressed as a corresponding decrease in the measured level of CAF22 in the sample.

Therefore, the CAF22 can be combined with other renal tubular injury biomarkers to be used for diagnosing the renal tubular injury, can improve the sensitivity and accuracy of detection, and has important significance for early diagnosis or dynamic detection of the renal tubular injury.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a CAF22 kit dilution line;

FIG. 2 is a renal tubular injury grading;

FIG. 3 is a ROC curve for CAF22 diagnosis of renal tubular injury;

FIG. 4A is a ROC curve for diagnosis of renal tubular injury by NGAL;

FIG. 4B is a ROC curve for CAF22 in combination with NGAL for the diagnosis of renal tubular injury;

FIG. 5A is a ROC curve for TIMP2 diagnosis of renal tubular injury;

FIG. 5B is a ROC curve of KIM-1 for diagnosis of renal tubular injury;

FIG. 5C is a ROC curve for CAF22 in combination with TIMP2 and KIM-1 for diagnosis of renal tubular injury;

FIG. 6A is a ROC curve for L-FABP diagnosis of renal tubular injury;

FIG. 6B is a ROC curve for CAF22 in combination with NGAL, L-FABP and TIMP2 for the diagnosis of renal tubular injury;

FIG. 7A is a ROC curve for diagnosis of renal tubular injury by IGFBP 7;

FIG. 7B is a ROC curve for IL-18 diagnosis of renal tubular injury;

FIG. 7C is a ROC curve for CAF22 in combination with IGFBP7 and IL-18 for diagnosis of renal tubular injury;

FIG. 7D is a ROC curve for CAF22 in combination with IGFBP7 and IL-18 for diagnosis of renal tubular injury;

FIG. 8 is a ROC curve for diagnosis of renal tubular injury with TIMP2 in combination with IGFBP 7;

FIG. 9 is a ROC curve for diagnosis of urinary albumin on renal tubular injury;

FIG. 10 is a ROC curve for urinary IgG versus renal tubular injury diagnosis;

FIG. 11 is a ROC curve for urinary NAG for diagnosis of renal tubular injury;

FIG. 12 is a ROC curve for the diagnosis of renal tubular injury from urinary TRF;

FIG. 13 is a ROC curve for urinary α MG diagnosis of renal tubular injury;

FIG. 14 is a ROC curve for renal function markers and renal tubular injury diagnosis.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1: sample collection

A total of 157 urine samples with abnormal renal status were collected.

The abnormal renal status is represented by: the indexes of albumin, immunoglobulin G, transferrin, alpha 1 microglobulin, lysosome enzyme and creatinine in urine are abnormal.

Example 2 preparation method of CAF22 chemiluminescence detection kit

The CAF22 chemiluminescence detection kit mainly comprises a CAF22 antibody marked by a conjugate, a CAF22 antibody marked by a chemiluminescence marker, streptavidin magnetic particles, a chemiluminescence substrate, a CAF22 standard substance and a quality control substance.

The preparation method of the CAF22 chemiluminescence detection kit mainly comprises the following steps:

step 1) preparation of conjugate labeled CAF22 antibody

Adding an activating agent biotin ester into the desalted CAF22 antibody according to the molar ratio of 1:10, uniformly mixing, and reacting for 4 hours at room temperature in a dark place. And adding the solution after reaction into a pre-wetted PD-10 purification column for purification. The purified conjugate was assayed for concentration using A280 and stored at-20 ℃ after addition of an equal volume of glycerol. When used, the reagent was diluted to a concentration of 0.1 to 2.0. mu.g/mL with a buffer solution containing 50 mM PBS, pH 7.4, 0.15M NaCl, 1g/L bovine serum albumin, 5g/L Tween 20, and 0.1% by mass sodium azide, and the solution was used as R1.

In this step, the Biotin ester label may be Sulfo-NHS-Biotin, Sulfo-NHS-LC-Biotin; preferably, the Biotin ester marker is Sulfo-NHS-LC-LC-Biotin; PBS can be replaced by one of Tris, MES, TBS and HEPES; tween 20 can be one or more of other Tween, Brij, and Triton; sodium azide may be used with one or more combinations of other preservatives such as phenol, sodium benzoate and proclin.

Step 2) preparation of chemiluminescent marker labeled CAF22 antibody

And (3) activating the desalted CAF22 antibody by using 2-IT according to the molar ratio of 1:5, and desalting the activated CAF22 antibody by using a G-25 column. Activating alkaline phosphatase with sulfol-SMCC at a molar ratio of 1:20, and desalting CAF22 antibody through G-25 column after activation. The activated CAF22 antibody and the activated alkaline phosphatase were mixed well, and the mixture was left to stand still at 4 ℃ for 24 hours, and the alkaline phosphatase-labeled antibody was purified by AKTA PRIME system. The purified alkaline phosphatase label was diluted to a concentration of 0.01 to 2.0. mu.g/mL with a buffer solution containing 50 mM pH 7.4 PBS, 0.1% casein, 0.05% Tween 20, and 0.1% sodium azide, and used as R2.

In this step, the alkaline phosphatase may also be replaced with other chemiluminescent markers, such as one or more of acridinium ester and its derivatives, horseradish peroxidase or ruthenium terpyridyl; PBS can be replaced by one of Tris, MES, TBS and HEPES; tween 20 can be one or more of other Tween, Brij, and Triton; sodium azide may be used with one or more combinations of other preservatives such as phenol, sodium benzoate and proclin.

Step 3) preparation of streptavidin magnetic particles

The magnetic particles of high-concentration streptavidin are diluted to 0.2-2.0 mu g/mL by magnetic bead diluent, the magnetic bead diluent contains buffer solution of 50 mM pH 7.4 PBS, 0.1% bovine serum albumin, 0.05% Tween 20 and 0.1% sodium azide, and the particle size of the magnetic bead is 1.0-5.0 mu m.

Step 4) preparation of a chemiluminescent substrate

In this step, the substrate of the chemiluminescent detection kit may be NaOH or H₂O₂One or more of luminol, 1, 2-dioxyethane (adamantane) -1, 2-dioxyethane or derivatives thereof, APS-5 or tripropylamine.

Step 4) kit Assembly

And respectively filling the diluted conjugate labeled CAF22 antibody, the diluted chemiluminescent label labeled CAF22 antibody, streptavidin magnetic particles, a chemiluminescent substrate, a CAF22 calibrator and a quality control product into a kit matched with a full-automatic chemiluminescent immunoassay analyzer to form the CAF22 chemiluminescent detection kit.

Calibration products: recombinant CAF22 protein was diluted to 1000.0 ng/mL, 800.0 ng/mL, 400.0 ng/mL, 200.0 ng/mL, 100.0 ng/mL, 50.0 ng/mL, 10.0 ng/mL, 0.0 ng/mL with 20 mM PBS buffer pH 7.4 containing a mass fraction of 1% BSA.

Quality control product: recombinant CAF22 protein was diluted to 100.0 ng/mL and 500.0 ng/mL with 20 mM PBS buffer pH 7.4 containing 1% BSA.

Example 4 method of detecting CAF22 with CAF22 chemiluminescence detection kit

The chemiluminescence detection kit for detecting CAF22 comprises a reagent R1, a reagent R2, streptavidin magnetic beads, a chemiluminescence substrate, a CAF22 calibrator and a quality control product.

R1 is biotin-conjugated CAF22 antibody, stored in 50 mM PBS pH 7.4 and 0.15M NaCl, and buffer solution also contains mass fraction of 0.1% sodium azide, 0.5 g/L Tween 20 and 1g/L bovine serum albumin.

R2 is alkaline phosphatase-labeled CAF22 antibody, stored in 0.05M PBS and 0.15M NaCl, and further containing 0.1% sodium azide, 0.5 g/L Tween 20 and 1g/L sodium caseinate in the buffer.

The concentration of streptavidin magnetic beads is 1 mg/mL, and the particle size of the magnetic beads is 2.8 mu M.

The chemiluminescent substrate is APS-5.

Taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool, taking a detection mode as a sandwich method, adding 5 muL of a test sample or a calibrator or a quality control product, 50 muL of a biotin-labeled CAF22 antibody and 50 muL of a basic phosphatase-labeled CAF22 antibody into a reaction cup of the analyzer, feeding the mixed solution into an incubation disc, and incubating for 10min at 37 ℃; then adding 30 mu L of streptavidin coated magnetic beads, uniformly mixing, sending into an incubation disc, and carrying out magnetic separation after incubation for 5min at 37 ℃; and adding 200 muL of luminescent substrate APS-5 into the mixture, and fully mixing uniformly to test the luminescence value after reaction.

In this example, the working concentration of the biotin-labeled CAF22 antibody was 1. mu.g/mL, the working concentration of the alkaline phosphatase-labeled CAF22 antibody was 0.05. mu.g/mL, and the working concentration of the streptavidin-coated magnetic beads was 1 mg/mL for detection.

Example 5 CAF22 chemiluminescence detection kit basic Properties

(1) CAF22 kit sensitivity

Calibrators (CALA) at 0ng/mL were tested 20 times with the CAF22 kit, the mean and standard deviation were calculated, and adjacent Calibrators (CALB) were tested, with the results shown in Table 1. The A, B light values are regressed at two points to obtain an equation. The value of CAL A average plus two standard deviations is substituted into the regression equation, and the corresponding concentration value is the sensitivity.

Through test and calculation, the sensitivity of the CAF22 chemiluminescence detection kit is 0.3 ng/mL.

(2) CAF22 kit Linearity

Diluting a sample with the concentration of 1000 ng/mL by using a sample diluent according to the dilution ratios of 1/1, 3/4, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and 1/128, testing the diluted sample by using a CAF22 kit, recording the test concentration and the corresponding dilution ratio of the sample, wherein the CAF22 kit dilution linearity is shown in figure 1, and the result shows that the calibration curve R is 7.75 ng/mL-500 ng/mL²>0.99。

(3) CAF22 kit repeatability

The mean and coefficient of variation were calculated for 10 samples at 92 ng/mL and 642 ng/mL, respectively, and the results are shown in Table 2.

The results show that the intra-batch-CV of sample 1 and sample 2 are 2.62% and 2.39%, respectively, indicating that the stability of the kit of the present invention is good.

Example 6 application of CAF22 detection kit contained in the invention in early diagnosis of renal tubular injury

The sample of this example is the sample of example 1.

The CAF22 chemiluminescence detection kit comprises the following detection steps of a body fluid sample of a patient:

step 1): sample collection and processing: collecting urine of a patient, centrifuging at 3000 rpm for 10min, transferring the supernatant into a centrifuge tube, and storing the treated urine sample at 4 ℃ for no more than one week or placing at-20 ℃ without repeated freeze thawing.

Step 2): as an example, the kit used R1 was a biotin-labeled CAF22 antibody, R2 was an alkaline phosphatase-labeled antibody, and the magnetic beads were streptavidin-coated magnetic beads.

Step 3): calibration, the kit and CAF22 master curve barcode were scanned on a fully automated chemiluminescent immunoassay analyzer, the calibrator was placed at the sample site, and the instrument could then calibrate the CAF22 project.

Step 4): and (4) quality control, namely detecting the light value of a quality control product on an instrument, automatically calculating the concentration through the standard curve, if the concentration of the quality control product is within the identification range, passing the test, otherwise, testing again, and testing the sample after the quality control is qualified.

Step 5): and (3) sample testing, namely putting a urine sample in a sample position, measuring the light value by using the instrument, automatically calculating the concentration by using a standard curve, and judging the result according to the detection concentration and the reference range.

Grading according to the result obtained by pathological biopsy, wherein the grading standard is as follows:

degree of renal tubular injury: 1-no denaturation; 2-denaturation; 3-brush edge drop and/or flattening; 4-naked membrane and/or necrosis

Area of renal tubular injury (cumulative degree of injury): 1-small oven; 2-a local range; 3-multiple cooking ranges; 4-Dispersion

The grading standard corresponds to the detection result.

The results of the grading are shown in FIG. 2;

and (2) drawing an ROC curve according to the detection result, (wherein the drawing tool is medcalc, the kit detection result of the sample is used for comparing with the pathological detection standard of the renal tubular injury, and the software can calculate the threshold value of the renal tubular injury degree and compare with the threshold value, so that the renal tubular injury can be predicted, graded in risk, diagnosed, staged, prognosed, classified and monitored according to the detection result).

As shown in fig. 3, the area under the curve (AUC) was calculated to be 0.916 with 95% confidence intervals of 0.8871-0.9549. When AUC is greater than 0.5, the prediction method is effective, and the prediction method is very effective, so that the CAF22 has good specificity and sensitivity for diagnosing the renal tubular injury, and has higher use value and diagnostic value clinically.

Example 7 marker combination assay

This example mainly illustrates that the sensitivity of detection can be further improved by combining CAF22 with other renal function indicators for detection.

(a) CAF22 in combination with NGAL

ROC curves were plotted for NGAL concentration versus renal tubular injury diagnosis and for NGAL in combination with CAF22 versus renal tubular injury diagnosis, and the results are shown in fig. 4A and 4B. The results show that the AUC for NGAL diagnosis of renal tubular injury is 0.8048; AUC increased to 0.934 after NGAL in combination with CAF 22. The result shows that the effectiveness of diagnosing the renal tubular injury is improved by combining the index CAF 22.

(b) CAF22 combined with TIMP2 and KIM-1

The results are shown in fig. 5A, 5B, 5C, and show that TIMP2 has an AUC of 0.7721 for the diagnosis of renal tubular injury; the AUC of KIM-1 for the diagnosis of renal tubular injury was 0.7267; the ROC of CAF22 in combination with TIMP2, KIM-1 increased to 0.947.

(c) CAF22 in combination with NGAL, L-FABP, TIMP2

The results are shown in fig. 5A, 4A, 6A, and 6B, and show that TIMP2 has an AUC of 0.7721 for the diagnosis of renal tubular injury; AUC for diagnosis of renal tubular injury by NGAL is 0.8048; AUC of L-FABP for diagnosis of renal tubular injury was 0.8264; the AUC of CAF22 combined with NGAL, L-FABP, TIMP2 increased to 0.948.

(d) CAF22 combined with IGFBP7 and IL-18

The results are shown in fig. 7A, 7B, 7C, and 7D, and show that AUC of IGFBP7 for renal tubular injury diagnosis is 0.6138; the AUC of IL-18 in the diagnosis of tubular injury was 0.6229, and the AUC of CAF22 in combination with IGFBP7 and IL-18 increased to 0.935.

(e) TIMP2 in combination with IGFBP7

The results are shown in fig. 7A, 5A, and 8, and show that AUC of IGFBP7 for diagnosis of renal tubular injury is 0.6138, and AUC of TIMP2 for diagnosis of renal tubular injury is 0.7721; the AUC for diagnosis of renal tubular injury by IGFBP7 in combination with TIMP2 was 0.7365.

The combination of the (a) - (e) shows that the combination of other indexes with common effects with CAF22 can obviously improve the sensitivity for detecting the renal tubular injury, and the combination of other indexes with common effects cannot improve the sensitivity for detecting the renal tubular injury. In addition, differences exist between different indexes combined by the CAF22, the more indexes which are not combined, the higher the sensitivity is, a complex calculation relationship exists, and compared with a single index of the CAF22, the detection accuracy can be further improved by combining other indexes.

Example 8 correlation of routine markers for detecting renal tubular injury

To prove that the renal function marker can not be used as a renal tubular injury marker or a renal tubular cell injury marker, ROC curves are drawn between the renal function conventional indexes of mALB, IgG, TRF, NAG and beta 2-microglobulin and the renal tubular injury, and the results are shown in FIGS. 9-14. The results show that the relevance of mLB, IgG, TRF, NAG and beta 2-microglobulin is lower than 0.8, and some indexes are even only about 0.6, and the relevance is not high.

The correlation of CAF22 of the present invention is best compared to these conventional indicators and can be effectively used to indicate and diagnose the status of renal tubular injury.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. Use of a capture agent that detects a C-terminal fragment of agrin in the preparation of a composition for assessing the state of renal tubular injury in a subject.

2. The use of claim 1, wherein the assessment comprises detecting the C-terminal fragment of the polyprotein in a sample of bodily fluid obtained from the subject to provide a detection result, and correlating the detection result with the renal tubular injury status of the subject.

3. The use of claim 1, wherein said capture agent is an antibody or derivative thereof that specifically recognizes a C-terminal fragment of agrin.

4. Use according to claim 3, said derivative comprising a fragment capable of specifically recognizing the C-terminal fragment of agrin: at least one of an antibody functional fragment, an antigen binding fragment, a conjugated antibody, a humanized antibody, a recombinant antibody, a diabody, a chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab 'fragment, or an F (ab') 2 fragment.

5. The application according to any one of claims 1-4, wherein the application further comprises:

6. The use of any one of claims 1-4, wherein the assessment comprises at least one of a prediction, risk stratification, diagnosis, staging, prognosis or monitoring of the renal tubular injury in the subject.

7. Use according to any one of claims 1 to 4, wherein the use comprises detecting the concentration and/or activity of the C-terminal fragment of agrin.

8. Use according to claim 5, comprising measuring the concentration and/or activity of at least one of the following markers: neutrophil gelatinase-associated lipocalin, interleukin 18, kidney injury molecule-1, liver-type-fatty acid binding protein, metalloproteinase tissue inhibitor 2, insulin-like growth factor binding protein 7.

9. Use according to claim 2, wherein the body fluid sample is serum or urine.

10. The use according to any one of claims 1 to 4, 8 or 9, wherein the composition is an ELISA test composition, an immunoturbidimetric test composition, an immunochromatographic test composition or a chemiluminescent immunoassay composition.

11. Use according to claim 10, wherein said composition is a chemiluminescent immunoassay composition comprising a conjugate labeled agrin C-terminal fragment antibody, a chemiluminescent label labeled agrin C-terminal fragment antibody, streptavidin magnetic particles and a chemiluminescent substrate;

the conjugate label is a biotin ester label;

the chemiluminescent substrate is NaOH and H₂O₂One or more of luminol, 1, 2-dioxyethane (adamantane) -1, 2-dioxyethane or derivatives thereof, APS-5 or tripropylamine.

12. Use of a C-terminal fragment of agrin as a synergistic indicator for assessing the state of renal tubular injury in a subject, the synergistic indicator in combination with a renal injury marker being capable of enhancing the diagnostic effect of the renal injury marker on renal tubular injury, the renal injury marker being a marker capable of indicating renal injury, the assessment comprising detecting the C-terminal fragment of agrin and at least one of the renal injury markers in a sample of bodily fluid obtained from the subject to provide a combined detection result;

and correlating the combined test result with the renal tubular injury status of the subject.

13. The use of claim 12, wherein the kidney injury marker is selected from one or more of: NGAL, IL-18, KIM-1, L-FABP, TIMP2 or IGFBP 7.