WO2019075769A1

WO2019075769A1 - Method for detecting hypersensitive c-reactive protein

Info

Publication number: WO2019075769A1
Application number: PCT/CN2017/107928
Authority: WO
Inventors: 涂策; 陶俊; 沈杰; 张金林
Original assignee: 苏州长光华医生物医学工程有限公司
Priority date: 2017-10-16
Filing date: 2017-10-27
Publication date: 2019-04-25
Also published as: CN107632163A

Abstract

Disclosed is a method for detecting a hypersensitive C-reactive protein, comprising the steps of: 1) preparing the standard test sample from a standard substance by a sample diluent, obtaining the luminous signal value thereof, and then plotting a standard curve with the concentration of the standard substance as the horizontal coordinate and the signal value as the vertical coordinate; 2) preparing streptavidin magnetic beads, a marker buffer, biotin-labeled hs-CRP antibody and acridinium ester-labeled hs-CRP antibody; 3) then adding an appropriate amount of the serum sample to be tested into the cuvette of a fully automatic luminescence immune analyzer, and adding an appropriate amount of the biotin-labeled antibody, the acridinium ester-labeled antibody and the streptavidin magnetic beads prepared in step 2) in a warm bath, obtaining the signal value of the serum sample to be tested, and meanwhile adding an appropriate amount of the standard test sample as control into the cuvette for testing; 4) and then comparing the signal value of the sample to be tested to the standard curve in step 1) to obtain the concentration of the hypersensitive C-reactive protein in the serum sample to be tested.

Description

Method for detecting hypersensitive c-reactive protein

Technical field

[0001] The present invention relates to a method for detecting a C-reactive protein, and particularly to a method for detecting a hypersensitive C-reactive protein, which belongs to the field of biodetection technology.

Background technique

[0002] C-reactive protein (CRP) is named for its ability to precipitate with the C-polysaccharide of the cell wall of Pneumococci, and is a serum β-globulin with a relative molecular mass of 115-140 KD. The continuous increase in CRP indicates chronic inflammation or autoimmune disease in the body. CRP does not increase in viral infection, and its changes are not affected by individual differences in the patient, the state of the body, and the therapeutic drug. In recent years, with the advancement of detection technology, the CRP detected by the ultra-sensitive method is called hypersensitive CRP (hs-CRP). A large number of articles have shown that it plays an increasingly important role in the diagnosis and prediction of diseases such as coronary heart disease, stroke, and peripheral vascular embolism, and is even considered to be the "gold standard" for cardiovascular disease risk assessment.

[0003] In many countries, coronary heart disease (CHD) remains the disease with the highest morbidity and mortality, but routine risk factor diagnosis will miss a significant number of patients with myocardial infarction. In fact, only 50% of the patients with myocardial infarction treated at the Fmmingham Heart Research Center had significantly elevated levels of cholesterol in their serum. Fortunately, in the past 10 years, the diagnosis of coronary artery disease caused by atherosclerosis has been greatly improved, and there is increasing evidence that C-reactive protein is diagnosed in coronary artery disease. Very good indicator. Previously, we thought that coronary artery disease was caused only by fat deposition on the arterial wall. However, today, this disease is considered to be a process in which multiple factors are involved, and chronic inflammation plays an important role. Recent studies have shown that C-reactive protein is a very good indicator of inflammation, and it is a good indicator of coronary artery disease when it is significantly increased. In fact, several prospective studies in the United States and Europe have shown that the concentration of C-reactive protein is within the normal reference range, and in a clearly healthy individual, the likelihood of future coronary artery disease can be predicted.

[0004] High-sensitivity C-reactive protein is a highly sensitive detection technology used in clinical laboratories. It can accurately detect low-concentration C-reactive protein, improve the sensitivity and accuracy of the test, and is a sensitive indicator for distinguishing low-level inflammatory states. Serum hs-CRP levels are closely related to the occurrence, severity and prognosis of atherosclerosis and acute cerebral infarction (ACI). Studies have shown that in elderly patients with acute cerebral infarction, patients with elevated CRP have a poor prognosis; hs-CRP levels are associated with infarct size and neurological deficits.

It is one of the indicators of the degree of lesions in patients with cerebral infarction; and CRP is also involved in the pathogenesis of thrombosis and arteriosclerosis, and is one of the risk factors for stroke. The inflammatory response of atherosclerotic plaque is an important cause of plaque rupture and instability. During the formation of atherosclerotic plaque, CRP, complement complex and foam cells are deposited in the arterial wall, and CRP can Lipoprotein binding, activates the complement system, produces a large number of inflammatory mediators, releases oxygen free radicals, causes endometrial damage, vasospasm and unstable plaque detachment, aggravates luminal stenosis caused by atherosclerosis and the occurrence of ACI. In recent years, there is increasing evidence that low levels of CRP are closely related to other risk factors for cardiovascular disease, such as hypertension and hyperlipidemia. Similarly, elevated CRP may increase heart disease and stroke in hypertensive patients. Incidence; therefore, CRP is a pro-inflammatory factor associated with the development, progression, and progression of atherosclerosis. Epidemiological surveys have also shown that patients with elevated hs-CRP levels are twice as likely to develop acute stroke as those in normal healthy individuals, and three times more likely to develop myocardial infarction. The European Guidelines for the Prevention and Treatment of Hypertension (ESH/ESC) was officially recommended in 2003.

Hypertensive patients need to detect hs-CRP levels. The clinical guiding role of hypersensitive C-reactive protein is mainly in the aspects of cardiovascular disease, neonatal bacterial infection, and kidney transplantation.

[0005] However, the current detection method of high-sensitivity C-reactive protein, similarly using various instruments to test various methodological reagents, enzyme-linked immunosorbent assay for about 30-120 min, latex immunoturbidimetry for about 15-30 min, enzymatic chemistry The luminescence method is about 15-60 min; the intercondylar length is detected, and the sensitivity is not high, and the patient's high-sensitivity C-reactive protein content cannot be accurately detected, so that the disease condition cannot be accurately judged, and the downtime is delayed.

technical problem

The technical problem to be solved by the present invention is to provide a method for detecting a hypersensitive C-reactive protein, thereby improving detection sensitivity, shortening detection time, and accurately determining the content of hypersensitive C-reactive protein in a patient.

Problem solution

Technical solution

[0007] A method for detecting a hypersensitive C-reactive protein, comprising the steps of:

[0008] 1) preparing a standard test sample by using a sample diluent to obtain a luminescence signal value, and then The concentration of the standard is the abscissa, and the signal value is plotted on the ordinate as a standard curve;

[0009] 2) preparing streptavidin magnetic beads, a marker buffer, a biotinylated hs-CRP antibody, and an acridinium ester labeled hs-CRP antibody, the marker buffer being 2-(N- Morpholine) ethanesulfonic acid buffer;

[0010] 3) taking an appropriate amount of the serum sample to be tested into a reaction cup of a fully automated luminescence immunoassay analyzer, and adding an appropriate amount of the biotin-labeled antibody, acridinium ester-labeled antibody, streptavidin prepared in step 2) Magnetic bead bath

, testing the luminescence value, obtaining the signal value of the serum sample to be tested, and adding an appropriate amount of the standard test sample to the reaction cup as a control for testing; preferably, tempering at 37 ° C for 3-9 min, preferably 5-6 min;

[0011] 4) The signal value of the sample to be tested is then compared with the standard curve of step 1) to obtain the concentration of the hypersensitive C-reactive protein in the serum sample to be tested.

[0012] Preferably, in step 1), the active agent is added to the 2-(N-morpholine) ethanesulfonic acid buffer, the active agent is lauryl polyoxyethylene ether, and the active agent lauryl alcohol Polyoxyethylene ether (Brig35) is added to the reaction system to effectively inhibit non-specific reactions and reduce the background signal, thereby greatly improving the sensitivity.

Preferably, the 2-(N-morpholine)ethanesulfonic acid buffer has a pH of 6.0 and a concentration of 0.05 Mol/L.

[0014] Preferably, the 2-(N-morpholine) ethanesulfonic acid buffer contains the following components: 0.5 volume < 3⁄4 bovine serum albumin (BSA), 1 mass < 3⁄4 sucrose, 0.1 volume < 3⁄4 Tween -20 (Tween-20), 0.1 volume <3⁄4 lauryl polyoxyethylene ether (Brig35), 0.1 volume <3⁄4 biopreservative (ProClin300).

[0015] Preferably, in step 2), the streptavidin magnetic beads have a particle diameter of 0.5 to 3.0 μm, preferably Ι.Ομ m, and a magnetic bead of a suitable diameter is selected as the solid phase for capture. Easier and more capture antibodies in the phase solution

, to get more signals.

[0016] Preferably, in step 2), the concentration of the biotin-labeled hs-CRP antibody is 0.1 to 0.5 mg/L, preferably 0.3 mg/L.

Preferably, the biotinylated hs-CRP antibody is made by a method comprising the steps of:

[0018] The hs-CRP antibody is first dialyzed with phosphate buffer, and the activated biotin is added to pure water to dissolve to obtain a biotin solution; then the dialyzed hs-CRP antibody is mixed with the biotin solution, and then After the mixture was allowed to stand at room temperature, it was dialyzed against phosphate buffer; then the dialyzed mixture was diluted with a marker buffer for use.

[0019] Preferably, the activated biotin is mixed with the hs-CRP antibody in a mass ratio of 1:5 to 1:20, preferably [0020] Preferably, in step 2), the concentration of the acridinium ester-labeled hs-CRP antibody is 0.05 to 0.5 mg/L, preferably 0.1 mg/L.

Preferably, the acridinium ester-labeled hs-CRP antibody is made by a method comprising the steps of:

[0022] The hs-CRP antibody is first dialyzed against a phosphate buffer, and the activated acridinium ester is added to pure water to dissolve to obtain an acridinium ester solution; then the dialyzed hs-CRP antibody is mixed with the acridinium ester solution. Thereafter, the mixture was allowed to stand at room temperature to obtain a mixed solution, which was dialyzed against phosphate buffer to obtain a mixed solution after dialysis; and then the dialyzed mixture was diluted with a marker buffer for use.

Preferably, the activated acridinium ester and the hs-CRP antibody are mixed at a mass ratio of 1:10 to 1:50, preferably at a mass ratio of 1:20.

[0024] Preferably, in step 2), the concentration of the streptavidin magnetic beads is 0.3 to 1.0 g/L, preferably 0.45 g/L.

[0025] The beneficial effects of the present invention are: using a magnetic separation system using a fully automatic chemiluminescence immunoassay analyzer, only need to add a sample, 6~15min can obtain test results, convenient and fast; using streptavidin biotin The amplification system allows each streptavidin on the magnetic beads to bind to four biotin antibodies, greatly improving the capture capacity, thereby enhancing the signal obtained; by using MES (2-(N-morpholine) B As a reaction system, the sulfonic acid buffer can effectively increase the reaction intensity of the antigen and antibody, thereby enhancing the signal; and the test shows that the detection method of the present invention is very sensitive, and the effective detection limit can reach 0.005 mg/L. Sensitivity can be up to 0.001 mg / L.

Advantageous effects of the invention

Brief description of the drawing

DRAWINGS

The invention is further described below in conjunction with the drawings and embodiments.

1 is a test standard curve of the hypersensitive C-reactive protein of the present invention;

2 is a test result of detecting a low concentration of hs-CRP serum sample after the activated biotin and the hs-CRP antibody are mixed in different mass ratios in the detection method of the present invention;

3 is a test result of detecting a high concentration of hs-CRP serum sample after the activated biotin and the hs-CRP antibody are mixed in different mass ratios in the detection method of the present invention;

4 is a method in which the activated acridinium ester and the hs-CRP antibody are mixed in different mass ratios in the detection method of the present invention. Post-test results of low concentrations of hs-CRP serum samples;

5 is a test result of detecting a high concentration of hs-CRP serum sample after the activated acridinium ester and the hs-CRP antibody are mixed in different mass ratios in the detection method of the present invention;

6 is a test result of detecting a low concentration of hs-CRP serum sample by streptavidin magnetic beads of different particle diameters in the detection method of the present invention;

7 is a test result of detecting a high concentration of hs-CRP serum samples with streptavidin magnetic beads of different particle diameters in the detection method of the present invention.

Detailed ways

The invention will now be described in further detail with reference to the drawings. The drawings are simplified schematic diagrams, and only the basic structure of the invention is illustrated in a schematic manner, and thus only the configurations related to the present invention are shown.

[0035] Unless otherwise indicated, the reagents in the following examples are commercially available from regular sources.

[0036] Unless otherwise specified, the standards in the following examples are international standard materials (NIBSC code: 85/506

Embodiment 1

[0038] 1) Streptavidin magnetic bead preparation: Take a certain amount of magnetic beads, and separate the supernatant in a magnetic field, and then use 0.1 volume <3⁄4 BSA, 1 mass <3⁄4 sucrose, 0.15 volume <3⁄4 Tween-20 0.1 volume of <3⁄4 biological preservative (Pr oClin300) phosphate buffer (concentration 0.01Mol / L, pH 7.4) was washed 3 times, and then the magnetic beads were reconstituted to 0.45g / L with this buffer.

[0039] 2) Preparation of the marker buffer: 0.05 Mol/L of 2-(N-porphyrin) ethanesulfonic acid (MES) buffer, adjusted to pH 6.0, and adjusted to the following weight percentages , 0.5 volume <3⁄4 bovine serum albumin (BS A), 1 mass <3⁄4 sucrose, 0.1 volume <3⁄4 Tween-20 (Tween-20), 0.1 volume <3⁄4 lauryl polyoxyethylene ether (Brig35), 0.1 volume <3⁄4 Biopreservative (ProClin300), filtered through a 0.2μηι filter membrane and stored at 2-8 °C until use.

[0040] 3) Biotin-labeled antibody preparation: The hs-CRP antibody was dialyzed against 0.02 M phosphate buffer at 2-8 ° C overnight; the activated biotin was dissolved in pure water to make a final concentration of 20 mg / Ml; mix hs-CRP antibody and biotin in a mass ratio of 1:10, leave it at room temperature for 0.5 h, then use 0.2 M, 0.1 M, 0.05 M, 0.02 M phosphate buffer (pH=7.4) at 2- Gradient dialysis was performed at 8 ° C, and each buffer was dialyzed for 4 h. Rear Collect and dilute to 0.3 mg/L with labeling buffer.

[0041] 4) Preparation of acridinium ester-labeled antibody: The hs-CRP antibody was dialyzed against 0.02 M phosphate buffer at 2-8 ° C overnight; the activated acridinium ester was dissolved in pure water to dissolve to a final concentration of 20 mg/ml; hs-CRP antibody and acridinium ester were mixed at a mass ratio of 1:20, and allowed to stand at room temperature for 0.5 h, then 0.2 M, 0.1 M, 0.05 M, 0.02 M phosphate buffer (pH=7.4). Gradient dialysis was performed at 2-8 ° C and each buffer was dialyzed for 4 h. After the collection, dilute with labeling buffer to O. lmg / L for use.

[0042] 5) Preparation of sample diluent: Prepare 0.05Mol/L phosphate buffer with pH 7.4, add 5 volumes of <3⁄4 bovine serum, 0.1 volume <3⁄4Tween-20, 1 mass <3⁄4 sucrose, 0.1 volume <3⁄4Ρπχ ^η300, after mixing, it was filtered through a 0.2 μm filter membrane and stored at 2-8 ° C until use.

[0043] 6) Preparation of calibrator: International standard substance (NIBSC code:

85/506) Dissolve in pure water to a concentration of 20mg/L, then dilute to 10, 2, 0.4, 0.1, 0.02, 0.005, 0.0mg/L with sample dilution, and store at 2-8 °C. The standard concentration is plotted on the abscissa and the signal value is plotted on the ordinate as a standard curve, as shown in Figure 1.

[0044] 7) Sample test: The above prepared good reagents are divided into corresponding reagent bottles, assembled and loaded in Suzhou Changguanghua Medical Medicine Co., Ltd. (HYBIOME) to produce fully automatic fluorescent immunoassay analyzer (Model AE-180) On the same, load the calibrator and the hs-CRP serum sample to be tested (two sets of parallel samples, one set of high-concentration hs-CRP serum samples, one set of low-concentration hs-CRP serum samples). The reagent test was set as follows: ΙΟΟμΙ biotin-labeled antibody, ΙΟμΙ sample, ΙΟΟμ-aziridin-labeled antibody, 25μ1 streptavidin magnetic beads were added to the reaction cup, and then incubated at 37 ° C for 5 min, finally washed and tested. Luminous value. After loading and setup is complete, start testing.

[0045] 8)

The results of the test showed that the analytical sensitivity was <0.001 mg/L, the correlation linearity was R>0.9995, the intra-assay variation was <10%, and the blood sample correlation was R>0.9995.

Example 2

[0047] The test method is the same as that of Example 1, except that the activated biotin and the hs-CRP antibody are different in masses of 1:2, 1:5, 1:10, 1:15, and 1:20, respectively. The results are shown in Fig. 2-3. It can be seen from the figure that when biotin is mixed with hs-CRP antibody at a mass ratio of 1:10, the detection signal obtained is the strongest.

Embodiment 3 [0049] The test method is the same as in Example 1, except that the activated acridinium ester and the hs-CRP antibody are different at 1:1 0, 1:20, 1:30, 1:40, and 1:50, respectively. The mass ratio is mixed, and the results are shown in Fig. 4-5. It can be seen from the figure that when the acridinium ester and the hs-CRP antibody are mixed at a mass ratio of 1:20, the detection signal obtained is the strongest.

Embodiment 4

[0051] The test method is the same as that of Example 1, except that the streptavidin magnetic beads having the particle diameters of 0.2 μm, 0.5 μm, 1.0 μm, and 3.0 μm are detected, and the results are shown in FIG. 6-7. It can be seen that when the particle size of the streptavidin magnetic beads is Ι.Οηηι吋, the detection signal obtained is the strongest.

Example 5

[0053] Analytical sensitivity test of high-sensitivity C-reactive protein: test blank sample 20 times and detection lower limit sample 5 times, calculate the signal mean value and standard deviation of the blank sample, and make the minimum of the blank sample and the lower limit sample concentration and signal mean value. Multiply fitting, and then adding the standard deviation of the blank sample signal to the equation by 2 times, the calculated value is the analytical sensitivity. The results are shown in Table 1 below. The results show that the sensitivity is less than 0.001 mg/L in the description.

Table 1

[0055]

Example 6-7

[0057] The test method is the same as in Example 1, except that in step 1), the concentration of streptavidin magnetic beads is 0.3 g/L, 1.0 g/L; in step 3), the biotin-labeled antibody The concentration is 0.1 mg/L, 0.5

In the step 4), the acridinium ester antibody was used at a concentration of 0.05 mg/L and 0.5 mg/L.

[0058] The results show that the effective detection limit can reach 0.005mg / L, the analytical sensitivity can be up to 0.001mg / L

Comparative Example 1

[0060] The reagent prepared by the invention and the high-sensitivity C-reactive protein (hs) produced by Beijing Lederman Biochemical Co., Ltd. -CRP) Quantitative determination kit (magnetic particle chemiluminescence method) for comparison, test calibrator and serum sample, and take a serum sample for gradient dilution to investigate the measured value. The results are as follows:

Table 2

[0062] [Number]

[0063] The results show that: Lidman's hs-CRP can only detect about 0.1 mg / L, and the reagent of the present invention can detect 0

.005mg/L, the sensitivity is far beyond.

[0064] In addition, the inventors detected the hs-CRP serum samples by adjusting the test parameters and data in each step, and the results showed that the effective detection limit of these detection methods can reach 0.005 mg/L, and the analytical sensitivity can be up to 0.

.OOlmg/Lo

[0065] The above-described embodiments of the present invention are intended to be variously modified and modified without departing from the scope of the present invention. Ben The technical scope of the invention is not limited to the contents of the specification, and the technical scope thereof must be determined according to the scope of the claims.

Claims

Claim

[Claim 1] A method for detecting a hypersensitive C-reactive protein, comprising the steps of:

1) The standard product is made into a standard test sample by sample dilution, and the luminescence signal value is obtained.

Then, the concentration of the standard is plotted on the abscissa, and the signal value is plotted on the ordinate as a standard curve;

2) Preparation of streptavidin magnetic beads, label buffer, biotinylated hs-CRP antibody and acridinium ester labeled hs-CRP antibody, the label buffer is 2-(N-morpholine) Acetate buffer;

3) Take an appropriate amount of the serum sample to be tested and add it to the reaction cup of the fully automatic luminescence immunoassay analyzer, and add the appropriate amount of step 2) to prepare the biotin-labeled antibody, the acridinium ester-labeled antibody, and the streptavidin magnetic bead bath. , testing the luminescence value, obtaining the signal value of the serum sample to be tested, and simultaneously adding an appropriate amount of the standard test sample to the reaction cup as a control; preferably, the temperature is 37 ° C for 3-9 min, preferably 5-6 min;

4) Then compare the signal value of the sample to be tested with the standard curve of step 1) to obtain the concentration of the hypersensitive C-reactive protein in the serum sample to be tested.

[Claim 2] The method for detecting a hypersensitive C-reactive protein according to claim 1, wherein in the step 1), the 2-(N-morpholine) ethanesulfonic acid buffer is added with an activity The active agent is lauryl polyoxyethylene ether.

[Claim 3] The method for detecting a hypersensitive C-reactive protein according to claim 2, wherein the 2-(N-morpholine)ethanesulfonic acid buffer has a pH of 6.0 and a concentration of 0.05 Mol/ L.

[Claim 4] The method for detecting a hypersensitive C-reactive protein according to claim 3, wherein the 2-(N-morpholine)ethanesulfonic acid buffer contains the following components: 0.5 volume < 3⁄4 Bovine serum albumin (BSA), 1% sucrose, 0.1 volume <3⁄4 Tween-20, 0.1 volume <3⁄4 lauryl polyoxyethylene ether (Brig35), 0.1 volume <3⁄4 biopreservative (ProClin300) .

[Claim 5] The method for detecting a hypersensitive C-reactive protein according to any one of claims 1 to 4, wherein, in the step 2), the particle size of the streptavidin magnetic bead is 0.5 to 3.0 μm, preferably 1.0 μm.

[Claim 6] The method for detecting a hypersensitive C-reactive protein according to any one of claims 1 to 5, wherein, in the step 2), the concentration of the biotin-labeled hs-CRP antibody is 0.1~

0.5 mg/L, preferably 0.3 mg/L.

The method for detecting a hypersensitive C-reactive protein according to claim 6, wherein the biotin-labeled hs-CRP antibody is produced by a method comprising the steps of: first treating a hs-CRP antibody with a phosphate buffer solution After dialysis, the activated biotin is dissolved in pure water to obtain a biotin solution; then the dialyzed hs-CRP antibody is mixed with the biotin solution to obtain a mixed solution, and the mixture is allowed to stand at room temperature, and then phosphate is used. Dialysis by buffer to obtain a mixed solution after dialysis; then diluting the dialyzed mixture with a marker buffer for use;

Preferably, the activated biotin is mixed with the hs-CRP antibody at a mass ratio of 1:5 to 1:20, preferably at a mass ratio of 1:10.

The method for detecting a hypersensitive C-reactive protein according to any one of claims 1 to 7, wherein in the step 2), the concentration of the acridinium ester-labeled hs-CRP antibody is 0.05 to 0.5 mg. /L, preferably 0.1 mg/L.

The method for detecting a hypersensitive C-reactive protein according to claim 8, wherein the acridinium ester-labeled hs-CRP antibody is produced by a method comprising the steps of: first buffering the hs-CRP antibody with phosphate After liquid dialysis, the activated acridinium ester is dissolved in pure water to obtain an acridinium ester solution; then the dialyzed hs-CRP antibody is mixed with the acridinium ester solution, and then the mixture is allowed to stand at room temperature, and then phosphoric acid is used. Dialysis with salt buffer; then dilute the dialyzed mixture with the marker buffer for use;

Preferably, the activated acridinium ester and the hs-CRP antibody are mixed at a mass ratio of 1:10 to 1 :50, preferably at a mass ratio of 1:20.

The method for detecting a hypersensitive C-reactive protein according to any one of claims 1 to 9, wherein in the step 2), the concentration of the streptavidin magnetic beads is 0.3 to 0.9 g/L. Preferably, it is 0.45 g/L.