CN112485446A - Kit for measuring full-range C-reactive protein and preparation method thereof - Google Patents
Kit for measuring full-range C-reactive protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kit for measuring full-scale C-reactive protein, which comprises: fluorescence immunochromatography test paper strip and sample diluent, fluorescence immunochromatography test paper strip includes: the detection device comprises a bottom lining, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein a quality control line and a detection line which are parallel to each other are arranged on the end, close to the water absorption pad, of the nitrocellulose membrane, and a trace particle labeled antibody line is arranged on the end, close to the sample pad, of the nitrocellulose membrane. According to the invention, the tracer particle labeled antibody is directly scratched on the nitrocellulose membrane in a special complex solution preparation mode, so that a marking mode and a conventional combined pad structure in the prior art are replaced, the detection sensitivity and accuracy are improved, and the preparation process of the test strip is simplified. Meanwhile, the sample is pretreated before the sample is added by preparing the sample diluent, so that the hemolysis phenomenon can be generated, the interference of background noise is avoided, and the stability of detection is further improved.
Description
Technical Field
The invention belongs to the field of medical examination, and particularly relates to a kit for measuring full-range C-reactive protein and a preparation method thereof.
Background
CRP is used as a routine test item in hospitals, generally used as an index of inflammatory response of bacterial infection and virus infection and used for guiding antibiotics. And has important guiding significance for evaluating the severity of coronary heart disease and the risk stratification of coronary heart disease: research shows that the CRP level of blood is helpful for evaluating the coronary stenosis degree and clinical stability, the rise of CRP concentration of blood can obviously increase the incidence rate of acute coronary syndrome of a coronary heart disease patient, and the detection after coronary heart disease stent operation can also be effectively evaluated; the CRP detection also has important clinical significance for patients with cerebral apoplexy: CRP level can reflect the severity of cerebral apoplexy patients, and can also reflect the cerebral infarction degree of patients, and can be used as nonspecific index for distinguishing cerebrovascular disease risk and prognosis; monitoring CRP level distribution and influencing factors thereof of diabetes population can predict and discuss future cardiovascular events of diabetes patient population, and has certain reference significance for reducing CRP level and improving prognosis of diabetes population. In conclusion, in clinical work, CRP monitoring is not only suitable for inflammation patients, but also has important clinical significance for diabetes patients, coronary heart disease patients, cerebral apoplexy patients and the like.
The relatively early CRP detection methods include latex agglutination experiments, immunoturbidimetry, radioimmunoassays, etc., but have various defects of low sensitivity, small detection range, isotope pollution, etc., and are gradually eliminated at present. In recent years, immunochromatography has been rapidly developed. The immunochromatography method for rapidly detecting CRP has become a trend, and patent CN103226143A discloses a ball-scribing process, wherein a marker is scribed on a nitrocellulose membrane, so that the test strip has a simple structure and stable reaction time, and the detection precision is improved. Meanwhile, in the prior art, when a CRP detection sample is whole blood, hemolysis is easy to occur, and the problems of poor test background and the like are caused.
Disclosure of Invention
The invention provides a kit for measuring full-range C-reactive protein, which simplifies the preparation process of a test strip by directly scratching an antibody marker on a nitrocellulose membrane, and simultaneously provides a diluent for CRP detection, thereby solving the hemolysis problem in the whole blood detection.
In order to achieve the purpose, the invention adopts the following technical means: a sample diluent for detecting full-scale C-reactive protein is characterized by comprising 2-6mmol/L K +, 80-180mmol/L cl-, 90-190mmol/L Na +, 2-9mmol/L HPO42-, 0.7-2.5mmol/L H2PO4-, 0.5-2g/L preservative and 5-20g/L surfactant.
Preferably, the sample diluent comprises 1-3g/L disodium hydrogen phosphate dodecahydrate, 0.1-0.3g/L potassium dihydrogen phosphate, 5-10g/L sodium chloride, 5-20g/L surfactant, 0.5-2g/L preservative, and 0.1-0.3g/L potassium chloride.
Preferably, the surfactant is selected from at least one of Tween, Triton, Tetronic 1307 or Brij 58, the Tween is preferably Tween-20 or Tween-80, the Triton is preferably Triton X-305 or Triton100, and the preservative is selected from at least one of proclin-300 or sodium azide.
A kit for measuring full-scale C-reactive protein, which comprises a fluorescence immunochromatographic test strip and is characterized by further comprising the sample diluent of claim 1.
Preferably, the fluorescence immunochromatographic test strip comprises: end liner, sample pad, nitrocellulose membrane, absorbent pad, nitrocellulose membrane overlap joint is on the end liner, sample pad overlap joint is in the one end of nitrocellulose membrane, absorbent pad overlap joint is at the other end of nitrocellulose membrane, it is provided with the quality control line and the detection line that are parallel to each other to be close to absorbent pad end on the nitrocellulose membrane, nitrocellulose membrane is close to sample pad end and is provided with tracer particle mark antibody line.
Preferably, the tracer particle is one of fluorescein, carboxyfluorescein, 2-methoxyfluorescein, rhodamine, phycoerythrin or quantum dot.
A preparation method of a full-scale C-reactive protein assay kit is characterized by comprising the following steps:
(1) preparation of fluorescent antibody complex: adding the CRP monoclonal antibody into the fluorescent microspheres, stirring at room temperature for reaction, adding BSA, sealing and stirring, centrifuging, removing supernatant, and recovering the volume of the precipitate with a fluorescent antibody complex solution;
(2) preparation of nitrocellulose membranes
a. Preparation of a detection line: diluting the mouse anti-human C-reactive protein monoclonal antibody with a PBS buffer solution, and marking a detection line at the left end of the nitrocellulose membrane;
b. preparing a quality control line: marking a quality control line on the right end of the nitrocellulose membrane for the goat anti-chicken IgG antibody, wherein the line is parallel to the detection line, and then drying in a drying oven;
c. preparing a tracer particle labeled antibody line: coating the fluorescence-antibody compound prepared in the step (1) at the left end of the nitrocellulose membrane, drying in a drying oven, sealing, drying and storing;
(3) assembling: and the bottom lining is sequentially and tightly lapped with the sample pad, the nitrocellulose membrane and the water absorption pad, and the strip is cut.
Preferably, the fluorescent antibody complex solution comprises: 5-15mmol/L buffer solution, 20-40g/L protein, 5-40g/L surfactant, 20-50g/L saccharide, 1-5g/L macromolecular substance and 0.5-1 g/L preservative, wherein the buffer solution is selected from at least one of MOPSO buffer solution, HEPES buffer solution, Tris buffer solution or phosphate buffer solution, the protein is selected from at least one of bovine serum albumin or casein, the surfactant is selected from at least one of Tween, triton or Brij 58, the Tween is preferably Tween-80, the saccharide is selected from one of sucrose or trehalose, the macromolecular substance is selected from at least one of PEG or PVP, and the preservative is selected from at least one of proclin-300 or sodium azide.
Preferably, the method further comprises preparing a sample diluent comprising: 1-3g/L disodium hydrogen phosphate dodecahydrate, 0.1-0.3g/L potassium dihydrogen phosphate, 5-10g/L sodium chloride, 5-20g/L surfactant, preferably Tween-20 or Tween-80, 0.5-2g/L preservative, preferably Triton X-305 or Triton100, and 0.1-0.3g/L potassium chloride, wherein the surfactant is selected from at least one of Tween, Triton, Tetronic 1307 or Brij 58, and the preservative is preferably selected from at least one of proclin-300 or sodium azide.
The use method of the kit is characterized by comprising the following steps:
(1) pretreating a sample by using the sample diluent to obtain a sample to be detected;
(2) detecting the sample to be detected by using the test strip to obtain a detected test strip;
(3) and analyzing the detected test strip by using a fluorescence immunoassay analyzer to obtain a detection result.
The invention has the beneficial effects that:
(1) according to the invention, the tracer particle labeled antibody is directly scribed on the nitrocellulose membrane in a special complex solution preparation manner, and a closed protein line auxiliary scribing manner and a conventional binding pad coated with the tracer particle labeled antibody are replaced, so that on one hand, the preparation process of the test strip is simplified, the preparation time is greatly shortened, the yield of the test strip preparation is improved, and the material cost is reduced; on the other hand, the specificity and the stability of the test strip are improved, and the accuracy of a detection result is further improved.
(2) According to the invention, the special sample diluent is adopted, and the sample is pretreated before sample detection, so that blood leakage does not occur in whole blood test, the background detection is uniform and clean, the background interference is reduced, and the precision and the accuracy of the detection are improved.
Drawings
FIG. 1 is a schematic structural diagram of a C-reactive protein fluorescent detection test strip in example 1;
FIG. 2 is a standard curve of signal values for the calculation of CRP standard concentration-CRP concentration in example 2;
FIG. 3 is a graph showing the correlation between CRP measurement values and sample values provided in example 2;
fig. 4 is a graph showing the correlation between the CRP measurement value and the sample value provided in example 3.
The reference signs are:
detecting a line 1; a quality control line 2; labeling the antibody line 3 with a tracer particle; a sample pad 4; a nitrocellulose membrane 5; a water absorbent pad 6; a bottom lining 7.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
EXAMPLE 1C preparation of test paper for fluorescent detection of reactive protein
1. Preparation of the Diluent
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
Diluting liquid:
2. preparation of fluorescent composite solution
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
Fluorescence labeling complex solution:
3. preparation of fluorescent-antibody complexes
Adding 60ug of CRP monoclonal antibody into 300ug of fluorescent microspheres, stirring at room temperature for 40 min, adding 5% BSA, blocking and stirring for 20min, then 14000rpm, centrifuging for 20min, discarding supernatant, recovering volume of the precipitate with fluorescent antibody preservation solution, and preserving at 4 ℃.
And (3) suspending the prepared substance in a fluorescent redissolution to obtain a fluorescent antibody-compound.
4. Assembly of immunochromatographic test strip
(1) Treatment of nitrocellulose membranes
a. Preparation of detection line
The mouse anti-human C-reactive protein monoclonal antibody was diluted to 2.0mg/ml with 20mmol/L PBS buffer pH7.2, and a test line was scribed at the left end from the nitrocellulose membrane in an amount of 0.6 ul/cm.
b. Preparation of quality control line
A quality control line is scribed at the right end of the nitrocellulose membrane according to the concentration of 2mg/ml and the amount of 0.6ul/cm of the goat anti-chicken IgG antibody, the line is parallel to the detection line, and then the goat anti-chicken IgG antibody is dried for 24 hours at the temperature of 45 ℃ in a drying oven.
c. Fluorescent antibody coating line
The fluorescent-antibody complex prepared in the first step was coated on the left end of the nitrocellulose membrane in an amount of 0.5ul/cm, and then dried in a drying oven at 45 ℃ and stored hermetically.
(2) Assembly
The plastic bottom lining, the sample pad and the absorbent pad are materials commonly used in the field, and the sample pad, the nitrocellulose membrane and the absorbent pad are tightly overlapped on the plastic bottom lining in sequence (as shown in figure 1). The pasted intermediate was cut into strips of 3.72mm wide by a cutter.
Example 2 method of Using test strips
(1) Test results of CRP standard substance
CRP antigen standards with different concentrations (seven different concentrations, 0.5mg/L, 1.0mg/L, 10.0mg/L, 50.0mg/L, 100mg/L, 150mg/L, 200mg/L, 3 replicates for each concentration) were added to the sample pad of the test strip prepared in example 1, and the signal was read by an immunoassay analyzer (Yuan Biotechnology Ltd. in Chongqing, Q8 pro). The results are shown in the following table:
TABLE 1 CRP Standard test results
The CRP antigen standard substance concentration is used as a horizontal coordinate, the measured average concentration is used as a vertical coordinate, a logarithmic equation is established and a standard curve is fitted, the result is shown in figure 2, the result shows that the test strip is used for detection, the linearity of the detection result is good, and the CRP protein concentration in the sample can be quantitatively analyzed through the standard curve.
(2) Precision degree
CRP standard substance solutions of 0.5mg/L of a low-value sample, 40.0mg/L of a medium-value sample and 200.0mg/L of a high-value sample are prepared, test strips prepared in example 1 and dispensed in example 2 are adopted for measurement, each concentration is repeatedly measured for 10 times, the measurement mean value and the standard deviation are respectively calculated, the coefficient of variation is calculated for repeated investigation, and the detection results are shown in the following table:
TABLE 2 CRP fluorescence detection test paper strip precision
The coefficient of variation is 2.21%, 2.18% and 2.04% respectively, and meanwhile, the accuracy examination is carried out by calculating the relative deviation by (1-mean/standard value) × 100%, and the results show that the relative deviation is 0.20%, -1.30% and-1.01% respectively, and the experimental results show that the test strip obtained by adopting the preparation scheme of the embodiment has better precision and repeatability.
(3) Determination of the difference between different times
Preparing a sample with the concentration of 10mg/L, repeatedly measuring the sample at 1min, 3min, 5min, 10min, 20min, 30min and 60min after sample addition by using an immune quantitative analyzer (Q8 pro, Chongqing, Zhongyuan Biotechnology Co., Ltd.), comparing the difference of the detection results at different times, and calculating the relative deviation (the concentration value measured at different time points/the concentration value measured at the first time), wherein the detection results are shown in the following table:
TABLE 3 difference of CRP fluorescence detection test paper strip in different time
| Time | 1min | 3min | 5min | 10min | 20min | 30min | 60min |
| Concentration of | 10.06 | 10.22 | 9.86 | 9.77 | 10.26 | 10.22 | 10.25 |
| Relative deviation of | -0.60％ | -2.20％ | 1.40％ | 2.30％ | -2.60％ | -2.20％ | -2.50％ |
The result shows that the test strip prepared in the embodiment 1 of the invention has small detection difference within 1-60min after sample addition, which indicates that the test strip has good time stability.
(4) Clinical sample testing
Selecting 200 clinical definite value whole blood samples, uniformly mixing 5ul samples with 995ul of diluent prepared in example 1, sucking 60ul of mixed solution, dropwise adding the mixed solution on a sample pad of the test strip prepared in example 1 for sample adding detection, wherein the result shows that no blood leakage phenomenon occurs in 200 samples in the detection process, the whole blood background is clean, then detecting by using an immune quantitative analyzer (Q8 pro, Zhongqing, Biotech Co., Ltd.), and performing linear analysis on the detection result and the sample value, the result is shown in figure 3, R of the result is R of the linear analysis, and the test result is a sample with a concentration of the sample as shown in figure 32The test strip has high accuracy and is suitable for clinical detection, because the test strip is 0.989.
According to the invention, the tracer particle labeled antibody is directly scribed on the nitrocellulose membrane in a special complex solution configuration mode, so that a scribing mode and a conventional bonding pad structure in the prior art are replaced, on one hand, the preparation process of the test strip is simplified, and on the other hand, the test sensitivity and accuracy of the test strip are improved. Meanwhile, the sample diluent is added, and the sample is pretreated before sample adding (especially a whole blood sample), so that the background noise of detection can be obviously reduced, and the stability and accuracy of detection can be further improved.
Example 3
In this embodiment, the CRP test strip does not contain a diluent component, and the rest of the preparation method is the same as that of embodiment 1.
Selecting 200 clinical fixed value whole blood samples, mixing 5ul samples with 995ul PBS buffer solution, sucking 60ul mixed solution, dripping the mixed solution on the sample pad of the test strip prepared in the example 1 for sample adding detection, and displaying that 63 samples of 200 samples have hemolysis in the detection process, and then using an immune quantitative analyzer (Chongqing Zhongyuan biotechnology Co., Ltd., Q8)pro), linear analysis is performed on the detection result and the sample value, the detection result is shown in figure 4, and R is20.7187, the results show that when the whole blood sample is not pretreated with a diluent, hemolysis is very likely to occur in the test strip, which in turn causes background interference and even affects the precision of the test.
Example 4
In this example, except that the preparation method of the fluorescent antibody coated wire on the nitrocellulose membrane is different from that in example 1, the other preparation methods are the same as in example 1, and the specific preparation process of the fluorescent antibody coated wire is as follows:
(1) preparation of blocking protein solution
The preparation method of the blocking protein solution I comprises the following steps: casein and BSA in a mass ratio of 2:1 were dissolved in triple distilled water, and then prepared into a 2% strength solution.
The preparation method of the blocking protein liquid II comprises the following steps: casein and BSA in a mass ratio of 1:5 were dissolved in triple distilled water, and then prepared into a solution having a concentration of 6%.
(2) Preparation of fluorescent antibody coated wire
a. The blocking protein solution I is coated at the position 6mm from the left end of the nitrocellulose membrane, and the coating amount is 1.5 ul/cm.
b. Coating 1.5mm of the left end of the nitrocellulose membrane with blocking protein solution II, drying at 25 deg.C for 2h, coating with fluorescent-antibody complex at 0.5ul/cm position of blocking protein II, drying, and sealing for storage.
(3) CRP standard test results
The 7 standards from example 1 were also tested using the method described in example 1. CRP antigen standard substances with different concentrations are added into a test strip sample pad, after 3min, signals are read by an immune quantitative analyzer (Chongqing Zhongyuan Biotechnology Co., Ltd., Q8 pro), and the detection results are shown in the following table:
TABLE 5 CRP Standard test results
The detection result shows that the CRP detection test strip prepared by the method in the embodiment 2 has lower detection precision than the CRP detection test strip prepared by the method in the embodiment 1, meanwhile, when the CRP detection test strip is prepared by the method in the embodiment 2, the blocking protein solution is coated on the nitrocellulose membrane, the marker-antibody complex is coated on the same position after drying, secondary drying is carried out, that is, the method additionally adds a scribing and drying process compared to example 1, and complicates the entire manufacturing process, and it is known that, in the preparation process of the test strip, the precision requirement of the marking process is extremely high, and in the actual operation process, errors often appear due to marking, the detection performance of the test strip is influenced, and even the whole waste of the test strip is caused, so that the efficiency is greatly reduced, and the material cost is increased. In the invention, by improving the coating process of the marker antibody, on one hand, the detection precision and stability of the reagent strip are improved, and simultaneously, the scribing step is reduced, the error in the preparation process of the test strip is avoided to the greatest extent, and the efficiency is greatly improved.
(4) Background interference
The result shows that the blood leakage phenomenon does not occur in all detection items, the whole blood background is clean, the detection background interference is reduced, and the diluent is suitable for detection test strips in different forms.
Example 5
In this example, five sets of experiments were set up, wherein each set of experiments was carried out using a kit differing from example 1 only in the concentration of the surfactant Tween-20 in the diluent, and the remaining preparation method was the same as in example 1. The specific grouping is shown in the following table:
TABLE 6
Note: in the embodiment, the content of Tween-20 in the group A diluent is 0.1 percent; group B is 0.5%; group C is 2%; group D is 4%; the E group is 10%, and the rest components and the preparation process are the same as those of the example 1.
Selecting 50 samples of fresh EDTA whole blood samples, respectively taking 5ul samples and uniformly mixing the samples with the diluent prepared by the five technical schemes, sucking 60ul mixed solution, dropwise adding the mixed solution on the sample pad prepared by the test strip prepared in the embodiment 1, and carrying out sample adding detection, wherein the results show that when the content of the surfactant Tween-20 in the diluent is 0.5% and 2%, the blood leakage phenomenon does not occur in all samples in the detection process, and when the content of the surfactant Tween-20 in the diluent is 0.1%, 4% and 10%, the blood leakage phenomenon occurs in the part of the detected samples. The experimental result shows that when the content of the surfactant Tween-20 in the diluent is 0.5-2%, the effect is better, the background of whole blood detection is clean, and the detection background interference is reduced.
Example 5
In this example, five sets of experiments were set up, wherein each set of experiments was carried out using a kit differing from example 1 only in the concentration of the surfactant Tween-20 in the diluent, and the remaining preparation method was the same as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
TABLE 7
Note: in the embodiment, the content of Tween-20 in the group A fluorescent complex solution is 1 g/L; the group B is 5 g/L; group C is 20 g/L; the group D is 40 g/L; group E was 100g/L, and the remaining components and preparation process were the same as in example 1.
Experimental results show that when the content of the surfactant Tween-20 in the fluorescent antibody redissolution is 5-20g/L, the precision is high.
Example 6
In this example, five sets of experiments were set up, wherein each set of experiments was carried out using a kit differing from example 1 only in the kind of surfactant in the diluent, and the remaining preparation methods were the same as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
TABLE 8
Note: in the group A diluent of the present example, the surfactant was 10g/L Tetronic 1307; group B is 10g/L Triton 100; group C is 10g/L Brij 58; group D is 10 g/LTween-80; group E was 10g/L Triton X-305, and the remaining components and preparation were the same as in example 1.
50 samples of fresh EDTA whole blood samples are selected, 5ul samples are respectively taken and mixed with the diluent prepared by the five technical schemes, 60ul of mixed solution is sucked and dripped on the sample pad of the test strip prepared in the embodiment 1 for sample adding detection, and the result shows that when the surfactant in the diluent is the five surfactants, the whole blood detection background of the blood leakage phenomenon of all the detection sample parts is clean, so that the detection background interference is reduced.
Example 7
In this example, a total of five sets of experiments were set, wherein each set of experiments used a kit different from example 1 only in the concentration of the surfactant Tween-80 in the fluorescent reconstituted solution, and the rest of the preparation method was the same as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
TABLE 9
Note: in the embodiment, the content of Tween-80 in the group A fluorescent complex solution is 1 g/L; the group B is 5 g/L; group C is 20 g/L; the group D is 40 g/L; group E was 100g/L, and the remaining components and preparation process were the same as in example 1.
Experimental results show that when the content of the surfactant Tween-80 in the fluorescent antibody redissolution is 5-40g/L, the precision is high.
Example 8
In this example, three sets of experiments were performed, wherein each set of experiments was performed using a kit different from example 1 only in the kind of surfactant in the fluorescent antibody complex solution, and the rest of the preparation method was the same as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
watch 10
Note: in the embodiment, the surfactant in the group A diluent is 10g/L Tween-20; group B is 10g/L triton; group C was 10g/LBrij 58, and the remaining components and preparation process were the same as in example 1.
Experimental results show that the detection precision of the test strip is high by adopting the three surfactants in the fluorescent antibody complex solution.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. The sample diluent for detecting the full-scale C-reactive protein is characterized by comprising 2-6mmol/L K+、80-180mmol/L cl-、90-190mmol/L Na+、2-9mmol/L HPO4 2-、0.7-2.5mmol/L H2PO4 -0.5-2g/L preservative and 5-20g/L surfactant.
2. The sample diluent for detecting full-scale C-reactive protein according to claim 1, wherein the sample diluent comprises 1-3g/L disodium hydrogen phosphate dodecahydrate, 0.1-0.3g/L potassium dihydrogen phosphate, 5-10g/L sodium chloride, 5-20g/L surfactant, 0.5-2g/L preservative and 0.1-0.3g/L potassium chloride.
3. The full-scale C-reactive protein detection sample diluent as claimed in claim 1 or 2, wherein the surfactant is at least one selected from Tween, Triton, Tetronic 1307 or Brij 58, the Tween is preferably Tween-20 or Tween-80, the Triton is preferably Triton X-305 or Triton100, and the preservative is at least one selected from proclin-300 or sodium azide.
4. A kit for measuring full-scale C-reactive protein, which comprises a fluorescence immunochromatographic test strip and is characterized by further comprising the sample diluent of claim 1.
5. The kit for measuring full-scale C-reactive protein according to claim 4, wherein the fluorescence immunochromatographic strip comprises: end liner, sample pad, nitrocellulose membrane, absorbent pad, nitrocellulose membrane overlap joint is on the end liner, sample pad overlap joint is in the one end of nitrocellulose membrane, absorbent pad overlap joint is at the other end of nitrocellulose membrane, it is provided with the quality control line and the detection line that are parallel to each other to be close to absorbent pad end on the nitrocellulose membrane, nitrocellulose membrane is close to sample pad end and is provided with tracer particle mark antibody line.
6. The kit for detecting full-scale C-reactive protein according to claim 5, wherein the tracer particle is one of fluorescein, carboxyfluorescein, 2-methoxyfluorescein, rhodamine, phycoerythrin or quantum dot.
7. A preparation method of a full-scale C-reactive protein assay kit is characterized by comprising the following steps:
(1) preparation of fluorescent antibody complex: adding the CRP monoclonal antibody into the fluorescent microspheres, stirring at room temperature for reaction, adding BSA, sealing and stirring, centrifuging, removing supernatant, and recovering the volume of the precipitate with a fluorescent antibody complex solution;
(2) preparation of nitrocellulose membranes
a. Preparation of a detection line: diluting the mouse anti-human C-reactive protein monoclonal antibody with a PBS buffer solution, and marking a detection line at the left end of the nitrocellulose membrane;
b. preparing a quality control line: marking a quality control line on the right end of the nitrocellulose membrane for the goat anti-chicken IgG antibody, wherein the line is parallel to the detection line, and then drying in a drying oven;
c. preparing a tracer particle labeled antibody line: coating the fluorescence-antibody compound prepared in the step (1) at the left end of the nitrocellulose membrane, drying in a drying oven, sealing, drying and storing;
(3) assembling: and the bottom lining is sequentially and tightly lapped with the sample pad, the nitrocellulose membrane and the water absorption pad, and the strip is cut.
8. The method for preparing a kit for measuring full-scale C-reactive protein according to claim 7, wherein the fluorescent antibody complex solution comprises: 5-15mmol/L buffer solution, 20-40g/L protein, 5-40g/L surfactant, 20-50g/L saccharide, 1-5g/L macromolecular substance and 0.5-1 g/L preservative, wherein the buffer solution is selected from at least one of MOPSO buffer solution, HEPES buffer solution, Tris buffer solution or phosphate buffer solution, the protein is selected from at least one of bovine serum albumin or casein, the surfactant is selected from at least one of Tween, triton or Brij 58, the Tween is preferably Tween-80, the saccharide is selected from one of sucrose or trehalose, the macromolecular substance is selected from at least one of PEG or PVP, and the preservative is selected from at least one of proclin-300 or sodium azide.
9. The method for preparing a kit for measuring full-scale C-reactive protein according to claims 7-8, characterized by further comprising the preparation of a sample diluent, wherein the sample diluent comprises: 1-3g/L disodium hydrogen phosphate dodecahydrate, 0.1-0.3g/L potassium dihydrogen phosphate, 5-10g/L sodium chloride, 5-20g/L surfactant, preferably Tween-20 or Tween-80, 0.5-2g/L preservative, preferably Triton X-305 or Triton100, and 0.1-0.3g/L potassium chloride, wherein the surfactant is selected from at least one of Tween, Triton, Tetronic 1307 or Brij 58, and the preservative is preferably selected from at least one of proclin-300 or sodium azide.
10. The method of using the kit according to any one of claims 4 to 6, comprising the steps of:
(1) pretreating a sample by using the sample diluent to obtain a sample to be detected;
(2) detecting the sample to be detected by using the test strip to obtain a detected test strip;
(3) and analyzing the detected test strip by using a fluorescence immunoassay analyzer to obtain a detection result.
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