CN114578057A - Application of monomer C-reactive protein (mCRP) in preparation of kit and kit - Google Patents
Application of monomer C-reactive protein (mCRP) in preparation of kit and kit Download PDFInfo
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- CN114578057A CN114578057A CN202011377237.3A CN202011377237A CN114578057A CN 114578057 A CN114578057 A CN 114578057A CN 202011377237 A CN202011377237 A CN 202011377237A CN 114578057 A CN114578057 A CN 114578057A
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- 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
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Abstract
The invention provides a kit for detecting an anti-mCRP antibody, which comprises monomer C reactive protein (mCRP) and application of the monomer C reactive protein (mCRP) in preparation of the kit, wherein the kit is used for detecting the content of the anti-mCRP antibody in a sample to be detected. The inventor finds that when the monomer CRP (mCRP) is used for detecting the anti-mCRP antibody in a sample to be detected, compared with the prior art, the detection accuracy and sensitivity are both obviously improved.
Description
Technical Field
The invention relates to the field of biological detection, in particular to application of monomer C-reactive protein (mCRP) in preparation of a kit and the kit.
Background
Acute tubulointerstitial nephropathy (ATIN) is an important cause of acute kidney injury, accounts for 1-3% of all renal biopsies and 15-27% of Acute Kidney Injury (AKI) of renal parenchyma. The etiology mainly comprises medicine, infection, tubulointerstitial nephritis-uveitis (TINU) syndrome, autoimmunity, specific incidence and the like.
C-reactive protein (CRP) is a highly conserved acute phase reactive protein in evolution, and in humans there are two natural forms, pentameric CRP (pcrp) and monomeric CRP (mcrp). In our earlier studies, an Enzyme-linked Immunosorbent Assay (ELISA) is firstly applied to establish a detection method of an anti-mCRP antibody in China, and the anti-mCRP antibody is found to be closely related to tubulointerstitial injury in lupus nephritis. Biological function studies suggest that anti-mCRP antibodies may inhibit the binding of C1q to mCRP, may also inhibit the binding of mCRP to factor H, and may have inhibitory effects on the opsonization of mCRP to macrophages, which suggests that anti-mCRP antibodies may be associated with immune tubulointerstitial injury.
In order to better define the effect of the anti-mCRP antibody in the tubulointerstitial lesion, TINU is selected as a disease model, and the TINU not only takes the renal interstitial injury as a main symptom, but also belongs to autoimmune diseases in the aspect of the pathogenesis of the renal interstitial injury, and can be used as a better tubulointerstitial lesion model. We find that the positive rate of the anti-mCRP antibody in the patients with TINU syndrome is very high and can reach 100 percent, which is obviously higher than the rate in the general interstitial nephritis. Further studies have found that anti-mCRP antibodies are associated with disease activity in patients with TINU syndrome, with antibody titers increasing at the onset of acute interstitial nephritis and uveitis and decreasing at remission. Meanwhile, mCRP is mainly expressed in renal tubular epithelial cells and renal interstitial cells, and the glomerulus is basically free from the expression of mCRP. High level expression of local CRP was suggested to occur when tubulointerstitial involvement was involved and was mCRP with cryptic epitopes rather than pentameric CRP. Compared with other disease controls, the expression level of mCRP of the TINU syndrome patient is obviously higher than that of other glomerulonephritis and drug-related acute interstitial nephritis, and the mCRP has certain specificity. We also performed a co-localization study of mCRP with patient IgG, suggesting that mCRP may be one of the common target antigens for eyes and kidneys of patients with TINU syndrome.
To further verify the clinical value of anti-mCRP, the applicant has performed prospective detection of anti-mCRP antibodies in patients with acute interstitial nephritis confirmed by renal biopsy and performed long-term follow-up on these patients, and found that anti-mCRP antibody positive can predict late-onset TINU, which plays an important role in defining the cause of acute interstitial nephritis. The research on mCRP epitope also indicates that the mCRP35-47 can indicate the prognosis of lupus nephritis, and further indicates that the mCRP35-47 plays an important role in the occurrence and development process of diseases.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
when pentamer CRP (pCRP) is used for detecting the anti-mCRP antibody in a blood sample to be detected, the sensitivity and the accuracy of detection are not ideal, and the early screening and diagnosis of interstitial nephritis patients are limited. The inventor of the application unexpectedly finds that when the monomer CRP (mCRP) is adopted to detect the anti-mCRP antibody in the blood sample to be detected, the sensitivity and the accuracy of the detection are both obviously improved, and a powerful basis is provided for early screening, diagnosis and typing of patients with interstitial nephritis.
In a first aspect of the invention, the invention proposes the use of monomeric C-reactive protein (mCRP) for the preparation of a kit for detecting the content of anti-mCRP antibodies in a sample to be tested. As described above, the inventors found that when a monomer crp (mCRP) is used to detect an anti-mCRP antibody in a sample to be tested, the accuracy and sensitivity of the detection are significantly improved compared to the prior art.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to an embodiment of the present invention, the sample to be tested is serum or plasma. When the monomer C reactive protein (mCRP) is adopted to detect the anti-mCRP antibody in the serum or the plasma, the non-specific binding of the mCRP and other non-anti-mCRP antibodies in the serum or the plasma is greatly reduced, and the detection sensitivity and the detection accuracy of the anti-mCRP antibody in the serum or the plasma are further improved.
According to an embodiment of the present invention, the sample to be tested is derived from a human.
In a second aspect of the invention, the invention proposes the use of monomeric C-reactive protein (mCRP) for the preparation of a kit for the early screening, diagnosis or typing of tubulointerstitial renal disease. The invention discovers that the mCRP antibody has strong correlation with the tubulointerstitial nephropathy, and the kit prepared by the monomer C reactive protein (mCRP) can be used for more accurately and reliably screening, diagnosing or typing the tubulointerstitial nephropathy at the early stage.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to an embodiment of the invention, the tubulointerstitial kidney disease is acute tubulointerstitial kidney disease or TINU syndrome. According to the detection statistics, the inventor finds that the positive rate of TINU (tubular interstitial nephropathy and uveitis) patients is about 57.1% in the renal biopsy patients who are definitely diagnosed with the tubular interstitial nephropathy, the positive probability is high, the kit provided by the embodiment of the invention also realizes early screening of TINU syndrome, and the positive anti-mCRP antibody can predict late tubular interstitial nephritis-uveitis (TINU) syndrome.
According to an embodiment of the invention, a unilateral detection value of greater than 32.5% is indicative of a positive tubulointerstitial kidney disease. According to the embodiment of the invention, the kit prepared by using the monomer C reactive protein (mCRP) adopts an indirect enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) to detect the anti-mCRP antibody in the sample to be detected, the positive rate of the anti-mCRP antibody in the sample to be detected can be calculated according to the following formula, and when the single-side detection value is more than 32.50% (two decimal parts are reserved for the single-side detection value), the positive indication of the anti-mCRP antibody in the sample to be detected is provided, so that the renal tubular interstitial nephropathy patient can be preliminarily screened and determined.
In a third aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit contains monomeric C-reactive protein (mCRP). The kit provided by the embodiment of the invention is more sensitive and accurate in detection of the anti-mCRP antibody in the sample to be detected.
According to an embodiment of the present invention, the kit may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the monomeric C-reactive protein is provided in a form dissolved in a carbonate buffer comprising sodium carbonate, sodium bicarbonate and water.
According to an embodiment of the present invention, the carbonate buffer has a pH of 9.0 to 10.0, preferably a pH of 9.6. Thereby allowing better binding of monomeric C-reactive protein to the ELISA plate.
According to the embodiment of the invention, the concentration of the sodium carbonate in the carbonate buffer solution is 1-2 g/L, and the concentration of the sodium bicarbonate in the carbonate buffer solution is 2.5-3 g/L;
according to an embodiment of the present invention, the concentration of the monomeric C-reactive protein in the carbonate buffer is 7-8 mg/mL, preferably 7.5 mg/mL. The inventor finds that when the concentration of the monomer C reactive protein in the carbonate buffer solution is 7-8 mg/mL, the detection sensitivity of the anti-mCRP antibody in a sample to be detected can be further improved on the premise of controlling the low dosage of the antigen-monomer C reactive protein.
According to an embodiment of the invention, the kit further comprises the carbonate buffer. And then convenience of customers directly adopts the carbonate buffer solution to dilute the monomer C reaction protein for subsequent detection reaction, so that the experimental error caused by human factors is further reduced.
According to an embodiment of the present invention, the kit further comprises a second antibody. The kit provided by the embodiment of the invention is suitable for detecting the content of the anti-mCRP antibody in a sample to be detected by adopting an ELISA method. Specifically, the second antibody is goat anti-human IgG. In particular, the second antibody carries a detectable label. Specifically, the marker is horseradish peroxidase.
According to an embodiment of the invention, the kit further comprises: non-protein blocking solution and 0.1% PBST. The inventor finds that if a protein blocking solution, such as BSA, is used as the blocking solution, BSA can have cross reaction with mCRP to influence the sensitivity and accuracy of detection, and the use of a non-protein blocking solution can avoid the risk of mCRP cross reaction and further improve the sensitivity and accuracy of detection. 0.1% PBST can be used directly as a sample diluent, secondary antibody diluent and wash in ELISA assays.
According to an embodiment of the present invention, the kit further comprises a substrate coloring liquid;
according to an embodiment of the invention, the substrate coloration solution comprises 9.7% by volume of diethanolamine and MgCl2·6H2O。
According to an embodiment of the invention, the kit further comprises: positive control serum and negative control serum. Specifically, the positive control serum is a serum which is positive for detecting anti-mCRP antibody by using ELISA and Western blot methods, for example, the serum can be from the serum of a patient with acute tubulointerstitial renal disease or the plasma replacement fluid of a patient with other diseases; specifically, the negative control serum is normal human serum with the anti-mCRP antibody expression level detected as the average content of normal human serum antibodies by using an ELISA (enzyme-Linked immuno sorbent assay) and a Western blot method.
In the fourth aspect of the invention, the invention provides a kit for early screening, diagnosis or typing of tubulointerstitial renal disease or provides a kit for detecting an anti-mCRP antibody. According to an embodiment of the invention, the kit contains monomeric C-reactive protein (mCRP). The kit provided by the embodiment of the invention is more sensitive and accurate in detection of the anti-mCRP antibody in a blood sample to be detected. Positive anti-mCRP antibody may predict late tubulointerstitial nephritis-uveitis (TINU) syndrome, and strong correlation between anti-mCRP antibody and tubulointerstitial nephritis. The kit containing the monomer C-reactive protein (mCRP) provided by the embodiment of the invention can be used for more accurately and reliably early screening, diagnosing or typing the tubulointerstitial nephropathy.
According to the embodiment of the invention, the kit for screening, diagnosing or typing the early stage of the tubulointerstitial renal disease has the same or corresponding additional technical characteristics as the kit, and the technical effect is the same as the additional technical characteristics of the kit, so the details are not repeated.
Drawings
FIG. 1 is a schematic diagram of indirect enzyme-linked immunosorbent assay.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.
The invention prepares an anti-mCRP antibody detection ELISA kit for early screening of acute tubulointerstitial nephropathy according to the principle of indirect enzyme-linked immunosorbent assay. The indirect enzyme-linked immunity method is characterized in that an antigen is connected to a solid phase carrier, an antibody to be detected in a sample is combined with the solid phase carrier to form a solid phase antigen-detected antibody compound, an enzyme-labeled secondary antibody is combined with the antibody in the solid phase antigen-detected antibody compound to form the solid phase antigen-detected antibody-enzyme-labeled secondary antibody compound, and then the chromogenic degree after adding a substrate is measured to determine the content of the antibody to be detected (see figure 1).
It is noted that the monomeric C-reactive protein (mCRP) employed in the present application may be obtained commercially or by preparation, as described in patent U.S. patent 5,874,238.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in the event of conflict, the definitions set forth herein shall control.
The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Unless otherwise specified, the percentages (%) in this specification are all weight percentages (% by weight).
All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". The term "about" when referring to a quantity or a numerical range means that the quantity or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the quantity or numerical range may vary, for example, between ± 5 of the quantity or numerical range.
All ranges directed to the same component or property are inclusive of the endpoints, and the endpoints are independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.
When the present invention is directed to a physical property, such as molecular weight, or to a range of chemical properties, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "comprising" (and related terms such as "comprising" or "including" or "having" or "including") includes embodiments that are, for example, any combination of materials, components, methods, or processes that "consist of or" consist essentially of the recited features.
As used in this specification and claims, "and/or" should be understood to mean "either or both" of the associated components, i.e., the components may be present in combination in some instances and separately in other instances. A plurality of components listed with "and/or" should be understood in the same way, i.e., "one or more" of the associated component. In addition to the "and/or" clause-specific components, other components may optionally be present, whether related or unrelated to those specifically identified components. Thus, as a non-limiting example, reference to "a and/or B," when used in conjunction with open ended words such as "comprising," may refer in one embodiment to a alone (optionally including components other than B); in another embodiment, reference may be made to B alone (optionally including components other than a); in yet another embodiment, refers to a and B (optionally including other components), and the like.
It is to be understood that, unless explicitly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
The abbreviations used in the present invention have the usual meanings in the biological and chemical fields.
mCRP: monomeric C-reactive protein;
0.1% PBST: adding Tween-20 into the PBS solution to make the final concentration be 0.1%;
PBS: phosphate buffer saline (phosphate buffer saline).
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
EXAMPLE 1 preparation of the kit
1. Experimental materials and reagents:
(1) homemade monomeric C reactive protein (mCRP, 7.5mg/ml), preparation method refer to patent U.S. patent 5,874,238;
(2) an enzyme label plate: 3590(costar. us);
(3) carbonate buffer: 0.477g of sodium carbonate, 0.879g of sodium bicarbonate and 300ml of deionized water, wherein the pH value is 9.6, and the sodium bicarbonate is separated and frozen by passing through a membrane;
(4) PBST solution: 0.1% PBST;
(5) non-protein blocking solution: 2% blocking reagent (bio-organism, PW040-500 ml);
(7) second antibody: goat anti-human IgG (sigma, cat # a 3188);
(9) positive control serum: from one patient with acute tubulointerstitial nephropathy (anti-mCRP antibodies were positive in the serum as measured by ELISA and Western blot);
(10) negative control serum: serum of normal people (the expression level of the anti-mCRP antibody in the serum is detected by using an ELISA (enzyme-Linked immuno sorbent assay) and Western blot method, and the serum content is the average content of the serum antibody of the normal people);
(11) substrate color development solution: 97ml diethanolamine, 0.1g MgCl2·6H2O, using ddH2O is added to 1000ml with pH 9.8;
(13) stopping liquid: 2mol/L sulfuric acid;
(14) an enzyme-labeling instrument: star Fax 2100 (aware. us).
2. Preparing an antigen-coated ELISA plate:
(1) dilute 7.5mg/ml mCRP to 5 μ g/ml with carbonate buffer and mix well.
(2) Coating an enzyme label plate:
adding the prepared mCRP antigen into a reaction hole of a 96-hole enzyme label plate, wherein the sample adding amount is 100 mu l/hole; and adding mCRP antigen solution into the positive control hole and the negative control hole, adding coating solution into the blank control hole, incubating in a constant-temperature incubator at 37 ℃ for 1h, removing the coating solution after overnight at 4 ℃, and washing for 3 times with washing solution, wherein each time is 3 min.
(3) And (3) sealing:
adding a sealing solution (the sample adding amount is 300 mu l/hole) into the reaction holes of the coated 96-hole enzyme label plate, incubating for 2 hours at room temperature, removing the sealing solution, and washing for 3 times and 3min each time by using a washing solution.
(4) And (3) drying and packaging:
and (3) placing the 96-hole enzyme label plate subjected to sealing treatment in a drying box at 37 ℃, drying, and packaging to obtain the antigen-coated 96-hole enzyme label plate, and storing at 4 ℃ for later use.
3. The kit comprises the following components:
(1) the 96-hole enzyme label plate coated by the antigen prepared in the step 2;
(2) sample diluent: PBST buffer containing 1% (W/V) BSA;
(3) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;
(4) enzyme-labeled secondary antibody: horseradish peroxidase-labeled goat anti-human IgG (sigma, cat # a 3188);
(5) color development liquid: 97ml diethanolamine, 0.1g MgCl2·6H2O, using ddH2O is metered to 1000ml, and the pH value is 9.8;
(6) stopping liquid: 1mol/L sulfuric acid;
(7) washing liquid: PBST (phosphate tween) buffer containing 0.2% tween 20;
(8) positive control serum: anti-mCRP antibody positive control serum;
(9) negative control serum: anti-mCRP antibody negative control sera.
And the reagents (2) - (9) are packaged respectively and then form a kit with the 96-hole enzyme label plate coated with the antigen.
Example 2 method of Using the kit
2.1 dilution of mCRP (7.5mg/ml) with carbonate buffer to 5. mu.g/ml and mix well. Mu.l of mCRP diluent is added into the antigen well, 100 mu.l of carbonate buffer is added into the non-antigen coated well, and the plate is coated overnight at 4 ℃ or 2h at 37 ℃. (the specific number of wells is determined by the number of standard samples, and auxiliary wells are made, and positive control, negative control and blank control wells are simultaneously made)
2.2PBST washing plate, each hole of 300 u L, standing for 1min, pat dry, total three times.
2.3 preparation of non-protein blocking solution (one well, 200. mu.l by volume, 50% volume of commercial sterile PBS, 50% volume of commercial non-protein blocking solution).
2.4 non-protein blocking solution blocking, 200. mu.L per well, 37 ℃, 200rpm, 2 h.
2.5PBST washing plate, each hole of 300 u L, standing for 1min, pat dry, total three times.
2.6PBST dilutes the samples of blood sample to be tested and positive control, etc., with the volume ratio of 1:100, 100 mul/hole, 37 ℃ and 1 h.
2.7PBST washing plate, each hole of 300 u L, placed for 1min, pat dry, total three times.
2.8 preparing goat anti-human IgG secondary antibody by PBST, diluting at a ratio of 1:5000, mixing well, 100 μ l/well, 37 deg.C, 30min.
2.9PBST washing plate, each hole of 300 u L, placed for 1min, pat dry, total three times.
2.10 color development: and (3) preparing a substrate color developing solution, adding 5mg of color developing substrate into every 5mL of substrate buffer solution, uniformly mixing and dissolving in a dark place, developing at room temperature for 30min at a concentration of 100 mu l/hole (slightly shaking the microporous plate before color comparison to uniformly disperse the liquid).
2.11 reading: the absorbance (A) was read at 405nm using a microplate reader.
2.12 calculate:
positive interpretation: unilateral > 32.5% positive (two decimal places for result retention)
Remarking:
positive, blank and negative controls must be set for each experiment;
reagent storage conditions and expiration date: storing at 2-8 deg.C, storing at room temperature for 2 years from production day before unsealing the non-protein sealing liquid, storing in 2-8 deg.C refrigerator after opening the reagent, and if the solution becomes turbid or is polluted, it can not be used.
Care must be taken to ensure that the plate is washed cleanly every time, and the plate is particularly non-protein sealing liquid.
Example 3
The inventor uses the kit prepared in example 1 to detect anti-mCRP antibody in 388 patients suspected of acute tubulointerstitial renal disease, the detection process is as described in example 2, and the detection results show that 312 patients are negative to anti-mCRP antibody, and 76 patients are positive to anti-mCRP antibody. These 76 positive patients were initially diagnosed with acute tubulointerstitial nephropathy.
Furthermore, the inventors confirmed that these 76 positive patients had acute tubulointerstitial nephropathy by further renal biopsy, and the biopsy results were consistent with the primary diagnosis results of anti-mCRP antibody detection.
The kit prepared in the embodiment 2 is used for detecting the mCRP antibody in a suspected acute tubulointerstitial nephropathy patient so as to carry out early screening and accurate diagnosis of the tubulointerstitial nephropathy, and the reliability is high.
The contents of all references (including literature references, issued patents, published patent applications, and copending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims.
Claims (12)
1. Use of monomeric C-reactive protein (mCRP) in the preparation of a kit for detecting the content of an anti-mCRP antibody in a sample to be tested.
2. The use according to claim 1, wherein the sample to be tested is serum or plasma.
3. The use according to claim 1, wherein the sample to be tested is of human origin.
4. Use of monomeric C-reactive protein (mCRP) in the preparation of a kit for early screening, diagnosis or typing of tubulointerstitial renal disease.
5. The use according to claim 4, wherein the tubulointerstitial kidney disease is acute tubulointerstitial kidney disease or TINU syndrome.
6. Use according to claim 4, wherein a unilateral detection value greater than 32.5% is indicative of a positive tubulointerstitial kidney disease.
7. A kit comprising a monomeric C-reactive protein (mCRP).
8. The kit of claim 7, wherein the monomeric C-reactive protein is provided in a form dissolved in a carbonate buffer comprising sodium carbonate, sodium bicarbonate, and water;
optionally, the pH of the carbonate buffer is 9.0-10.0, preferably, the pH is 9.6;
preferably, the concentration of the sodium carbonate in the carbonate buffer solution is 1-2 g/L, and the concentration of the sodium bicarbonate in the carbonate buffer solution is 2.5-3 g/L;
preferably, the concentration of the monomer C reactive protein in the carbonate buffer solution is 7-8 mg/mL, preferably 7.5 mg/mL.
9. The kit of claim 8, further comprising the carbonate buffer.
10. The kit of claim 7, wherein the kit further comprises a second antibody;
optionally, the second antibody is goat anti-human IgG;
optionally, the second antibody carries a detectable label;
optionally, the label is horseradish peroxidase.
11. The kit of claim 7, wherein the kit further comprises: non-protein blocking fluid and 0.1% PBST;
optionally, the kit further comprises a substrate developing solution;
preferably, the substrate color developing solution comprises 9.7% by volume of diethanolamine and MgCl2·6H2O。
12. The kit of claim 7, wherein the kit further comprises: a positive control serum and a negative control serum,
optionally, the positive control serum is acute tubulointerstitial nephropathy patient serum with positive anti-mCRP antibody detected by ELISA and Western blot method;
optionally, the negative control serum is normal human serum with the expression level of anti-mCRP antibodies detected by ELISA and Western blot method as the average content of normal human serum antibodies.
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| CN205333654U (en) * | 2016-01-21 | 2016-06-22 | 深圳市博卡生物技术有限公司 | Early detection test strip of kidney damage based on biomarker |
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