CN113759129A - Rapid detection card for leucocyte-bound C-reactive protein and preparation method thereof - Google Patents
Rapid detection card for leucocyte-bound C-reactive protein and preparation method thereof Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
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- G01N2800/32—Cardiovascular disorders
Abstract
The invention belongs to the technical field of protein immunodetection, in particular to a rapid detection card for leucocyte-bound C-reactive protein, and further discloses a preparation method and application thereof. According to the rapid detecting component for the leucocyte combined with the C-reactive protein, the structure of the leucocyte bin and the structure of the C-reactive protein test paper card are respectively arranged, and the rapid quantitative detection of the leucocyte and the C-reactive protein in a whole blood sample can be simultaneously realized by utilizing the hemocyte staining counting and the immunochromatography reaction principle of the C-reactive protein, so that the accuracy of infection diagnosis is greatly improved, and the rapid detecting component has important significance for identifying bacterial and viral infection.
Description
Technical Field
The invention belongs to the technical field of protein immunodetection, in particular to a rapid detection card for leucocyte-bound C-reactive protein, and further discloses a preparation method and application thereof.
Background
C-reactive protein (C-reactive protein CRP) is a typical acute phase protein, and the content of CRP is very small under normal conditions, but the blood concentration of the CRP is increased sharply during acute trauma and infection. In general, in healthy people, the serum CRP of newborn is less than 2mg/L, the serum CRP of children and adults is less than or equal to 10mg/L, and the CRP value can be sharply increased to about 20-500mg/L within 4-8 hours when the human body is in inflammatory diseases or tissues are ulcerated or necrotic, and the CRP content value can also be restored to a normal level along with the restoration of the tissue structure and function. Thus, based on the level of CRP in blood, it can be used to assess the severity of infection, tissue damage or inflammatory disease, and to assess the progress, prognosis and therapeutic efficacy.
In addition, hypersensitive C-reactive protein (hs-CRP) can be used as an important index of cardiovascular diseases, and the level of the hypersensitive C-reactive protein can predict the risk of myocardial infarction and stroke in the future. It is reported that the reference range of hypersensitive CRP for healthy people is basically 0.58-1.13mg/L, and that for people with CRP content more than 2.1mg/L and less than or equal to 1.0mg/L than CRP, the risk of myocardial infarction is 2.9 times of the latter, and the risk of ischemic stroke is 1.9 times of the latter. Therefore, the combined measurement of the hypersensitive CRP and the blood fat can indicate the risk of the occurrence of cardiovascular and cerebrovascular diseases more than other risk factors.
Leukocytes, commonly known as white blood cells, are a very important class of blood cells in human blood. It has the functions of phagocytizing foreign body and producing antibody, and has the functions of healing injury, resisting pathogen invasion, resisting disease immunity, etc. As the human body becomes ill-timed, it is often manifested by a significant change in the number of leukocytes. It has been studied that the increase and decrease of leukocytes are classified into physiological factors and pathological factors, wherein the physiological factors include:
age: neonate meterHigher number, up to (15-30) x 109L, usually reduced to 10X 10 in 3-4d9/L；
Change in the daytime: generally, the white blood cells are lower during resting relaxation, higher after activity and eating; the morning is low, the afternoon is high, and the difference can be 1 time within one day;
motor, pain and mood effects: acute exercise, severe pain, extreme fear, etc. all can cause the white blood cells to rise for a short time;
and (3) pregnancy and delivery: the leukocyte increase during pregnancy, especially in the last 1 month, can reach 34 × 10 during parturition9L, recovering to normal 2-5 days after delivery; the female menopause and menstruation can be reduced;
alcohol consumption, smoking, and cold bath can also be increased, and the white blood cell count of the same test object can fluctuate by even 50% due to physiological factors.
Elevated white blood cell counts, mainly neutrophil increases within the differential counts, are also seen with glucocorticoids.
Pathological factors can be subdivided into factors causing the increase of white blood cells and factors causing the decrease of white blood cells, wherein the factors causing the increase of white blood cells include:
acute infection and suppurative inflammation caused by various cocci: otitis media, tonsillitis, appendicitis, abscesses, and the like;
systemic infection: pneumonia, septicemia, scarlet fever, etc.;
poisoning: uremia, diabetic acidosis, mercury poisoning, lead poisoning;
acute hemorrhage, acute hemolysis, post-operative;
malignant tumors, granulocytopathies, and the like; sixthly, leukemia reaction.
Factors causing leukopenia:
viral infection: severe hepatitis, influenza, measles, etc.;
some infectious diseases: such as typhoid, paratyphoid, malaria, etc.;
certain hematological disorders such as aplastic anemia, leukopenia, agranulocytosis;
chemical and radiation damage such as X-ray irradiation, radium irradiation, advanced arsenic poisoning, etc.;
autoimmune diseases and hyperfunction of spleen.
By analyzing the above physiological and pathological factors that cause changes in the white blood cell count, we have no difficulty in concluding that the white blood cell count is either increased or decreased, neither of which is the gold standard for determining whether an infection is present in the body. That is, infection cannot be determined to have occurred based only on an increase or decrease in white blood cell count, which is normal and cannot be completely excluded. Therefore, in the actual clinical work, the diagnosis of the infection of the body cannot be simply given according to the change of the white blood cell count, so that the combined detection of the white blood cells and other items is often adopted for judging.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a rapid detection card component for combining leucocytes with C-reactive protein, which utilizes the principles of hemocyte staining counting and immunochromatography reaction to realize rapid quantitative detection of the leucocytes and the C-reactive protein in a whole blood sample and has the advantages of simple operation and rapid diagnosis;
the invention aims to provide a preparation method and application of the rapid detection card component for the leucocyte-bound C-reactive protein.
In order to achieve the above object, the present invention provides a rapid detection card assembly for leukocyte-bound C-reactive protein, comprising a detection card and a sample diluent; the detection card comprises a shell, and a C-reactive protein test paper card and a leukocyte bin which are arranged in the shell;
the C-reactive protein test paper card comprises an absorption pad, an NC membrane, a combination pad, a blood filtration pad and a sample pad which are sequentially fixed on a bottom plate;
the NC membrane surface is respectively sprayed with a first detection line, a second detection line and a quality control line which are formed by a crp-2 monoclonal antibody, a crp mixed antibody and a goat anti-mouse polyclonal antibody; the CRP mixed antibody comprises a mixture of CRP-2 monoclonal antibody and anti-human CRP antigen, and preferably 1: 1, mixing the components in equal amount;
the conjugate pad is coated with a colloidal gold labeled crp-1 antibody.
Specifically, the sample diluent comprises: 10-50mM PBS buffer solution +0.1-0.5mM acridine orange +1-5mM blood anticoagulant.
The invention also provides a method for preparing the rapid detection card component of the leucocyte-bound C-reactive protein, which comprises the following steps:
(1) respectively diluting the crp-2 monoclonal antibody, the crp mixed antibody and the goat anti-mouse polyclonal antibody by using an antibody coating solution, spraying the diluted antibodies on the surface of an NC (numerical control) membrane to respectively form a first detection line, a second detection line and a quality control line, and drying the NC membrane for later use;
preferably, the crp-2 monoclonal antibody, the crp mixed antibody and the goat anti-mouse polyclonal antibody are controlled to be coated on the surface of an NC membrane in a spraying amount of 1 ul/cm;
(2) pretreating a glass fiber membrane, spraying the colloidal gold-labeled crp-1 antibody on the surface of the glass fiber membrane in the amount of 3-8uL/cm to form the bonding pad, and drying for later use;
(3) cutting the absorption pad, the NC membrane, the combination pad, the blood filtration pad and the sample pad respectively, and then sequentially sticking and fixing the absorption pad, the NC membrane, the combination pad, the blood filtration pad and the sample pad on a bottom plate to form the C-reactive protein test paper card for later use;
(4) respectively placing the C-reactive protein test paper card and the leucocyte bin into preset positions in the shell, and packaging after welding;
(5) and (4) subpackaging the sample diluent at 240uL/test, and sealing and storing.
Specifically, in the step (1):
the antibody coating solution comprises: 10-50mM PBS buffer +1-5 wt% methanol;
the crp-2 monoclonal antibody, the crp mixed antibody and the goat anti-mouse polyclonal antibody are respectively diluted to be 0.1-0.5mg/mL in concentration.
Specifically, in the step (2), the preparation method of the colloidal gold-labeled crp-1 antibody is as follows: adding a pH value regulator into the colloidal gold solution, uniformly mixing, adding the crp-1 antibody solution, fully mixing uniformly, performing coupling reaction at room temperature, and continuously adding a sealing liquid after the reaction is finished to perform normal-temperature sealing reaction; centrifuging to precipitate colloidal gold marker, and adding gold-labeled antibody complex solution.
Specifically, the preparation method of the rapid detection card component for the leucocyte-bound C-reactive protein comprises the following steps:
the glass fiber pretreatment liquid comprises: 10-50Mm borate buffer + 0.1-0.5% TW20+ 0.1-5% BSA;
the gold-labeled antibody complex solution comprises: 10-50mM borate buffer solution + 1-10% sucrose + 0.1-5% BSA;
the pH regulator comprises: 1% potassium carbonate solution;
the confining liquid comprises: 10% BSA in water.
Specifically, the step (3) further comprises the step of soaking the C-reactive protein test paper card in a hydrophilic treatment solution;
the hydrophilic treatment liquid includes: 10-50mM PBS buffer + 0.5-2% TW 20.
Specifically, in the step (4), the C-reactive protein test paper card and the leukocyte bin are arranged in parallel, and a liquid separation channel is arranged at the position of the sample adding hole of the shell to control the sample to be detected to flow into the leukocyte bin and the sample pad of the C-reactive protein test paper card respectively.
The invention also discloses a using method of the rapid detecting card component for the leucocyte-combined C-reactive protein, which comprises the steps of diluting a whole blood sample to be detected by the sample diluent and adding samples, wherein the whole blood sample to be detected respectively flows into the leucocyte bin to carry out blood cell staining counting, flows into the C-reactive protein test paper card to carry out chromatography reaction, and reads a detection result after 15-20 min.
The invention also discloses application of the rapid detection card component for the leucocyte-combined C-reactive protein in the fields of diagnosis and identification of bacterial and viral infection.
The invention also discloses a method for diagnosing and identifying bacterial and viral infection, which comprises the step of detecting a whole blood sample by utilizing the leucocyte-combined C-reactive protein rapid detection card component.
Compared with the prior art, the rapid detecting component for the combination of the leucocytes and the C-reactive protein can simultaneously realize the rapid quantitative detection of the leucocytes and the C-reactive protein in a whole blood sample by respectively arranging the structures of the leucocyte bin and the C-reactive protein test paper card and utilizing the principles of blood cell staining counting and the immunochromatography reaction of the C-reactive protein. The detection assembly provided by the invention utilizes the combined detection of the leucocytes and the C-reactive protein, so that the accuracy rate of infection diagnosis is greatly improved, and the detection assembly has an important significance for identifying bacterial and viral infections; meanwhile, the method can effectively predict and evaluate infectious diseases, antibiotic curative effects and cardiovascular and cerebrovascular diseases, is suitable for all levels of hospitals, and is particularly beneficial to wide popularization in primary hospitals and clinics.
According to the rapid detecting component for the leucocyte combined with the C-reactive protein, a blood cell staining counting principle is adopted for the measurement of the leucocyte, and an accurate leucocyte counting result can be obtained through high-power amplification and a large number of picture acquisition and analysis of a leucocyte bin of a detection card, so that a rapid and accurate quantitative detection result is realized; the accurate leucocyte and C-reactive protein quantitative detection result can be obtained by combining the whole course C-reactive protein immunochromatography detection reagent.
The rapid detection assembly for the leucocyte combined with the C-reactive protein is simple in detection operation and sample treatment, after venous blood/fingertip blood is sampled, the blood sample does not need to be pretreated, the simultaneous detection of the leucocyte and the C-reactive protein can be realized only by one-step dilution and one-time sample addition, a matched small detection device is combined without specific environment and technical personnel, the detection result can be rapidly obtained within 15-20 minutes, the accuracy of infection diagnosis is greatly improved by combined detection, and the rapid detection assembly has important significance for identifying bacterial and viral infections.
Drawings
FIG. 1 is a schematic diagram of the structure of the test card of the present invention;
FIG. 2 is a schematic structural diagram of a C-reactive protein test paper card according to the present invention;
FIG. 3 is a fitting regression equation for C-reactive protein;
FIG. 4 is a fitted regression equation for leukocytes;
description of the main reference numerals:
1-detection card, 11-shell, 12-test paper card, 13-leucocyte bin, 14-liquid separation channel, 111-sample adding hole, 121-bottom plate, 122-absorption pad, 123-NC membrane, 124-combination pad, 125-sample pad and 126-blood filtering pad.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Example 1
In this example, the formulation solution involved included:
reagent card hydrophilic treatment fluid: 10mM PBS buffer + 1% TW 20;
gold-labeled antibody complex solution: 10mM borate buffer + 6% sucrose + 2% BSA;
glass fiber membrane pretreatment liquid: 50Mm borate buffer + 0.3% TW20+ 2% BSA;
antibody coating solution: 10mM PBS buffer + 3% methanol;
pH regulator: 1% potassium carbonate solution;
sealing liquid: 10% BSA in water.
The component for rapidly detecting the combination of the white blood cells and the C-reactive protein utilizes the principle of blood cell staining counting and the immunochromatography reaction of the C-reactive protein, and simultaneously realizes the rapid quantitative detection of the white blood cells and the C-reactive protein in a whole blood sample. The rapid detecting component for the leucocyte-combined C-reactive protein comprises a detecting card 1 and a sample diluent, wherein the sample diluent is arranged in a diluting bottle and is used for diluting a whole blood sample to be detected and performing hemocyte staining counting and immunochromatography of the C-reactive protein through the detecting card.
The sample diluent contained in the test card assembly of the present invention comprises: 10mM PBS buffer solution +0.1-0.5mM acridine orange +1-5mM blood anticoagulant. In this embodiment, the sample diluent is prepared by: 10mM PBS buffer solution +0.3mM acridine orange +3mM blood anticoagulant, the sample dilution is dispensed at 240uL/test, and the dilution bottle is sealed and stored.
As shown in fig. 1, the detection card 1 includes a housing 11, and a C-reactive protein test paper card 12 and a leukocyte bin 13 disposed in the housing 11, wherein the test paper card 12 and the leukocyte bin 13 are disposed in parallel, the leukocyte bin 13 is disposed at a middle position, and the test paper card 12 is disposed at any one side or both sides (as shown in fig. 1, the structure is only provided with the test paper card 12). A liquid distribution channel 14 is arranged between the test paper card 12 and the leukocyte bin 13, the liquid distribution channel 14 is formed on the inner surface of the shell 11, the middle position of the liquid distribution channel corresponds to the position of the sample adding hole 111, and microfluidic channels leading to the test paper card 12 and the leukocyte bin 13 are respectively formed by extending and forming the middle position of the liquid distribution channel, after a sample to be detected is added through the sample adding hole 111 at one time, the sample to be detected flows to the middle leukocyte bin 13 and the test paper cards 12 (flowing to the positions of the bonding pads 125) on the left side and the right side through internal micro-flow to carry out liquid distribution, the liquid distribution amount can be controlled to meet or be larger than the required detection amount, wherein the sample amount for leukocyte detection is 50ul, and the sample amount for CRP detection is 75-100 ul.
In the structure shown in fig. 1, the leukocyte bin 13 is a transparent bin body with a hollow middle part and sealed periphery, and a uniform liquid level with a thickness of about 1-2mm is formed after the sample liquid flows into the leukocyte bin 13 and is used for bearing the sample during leukocyte detection.
As shown in fig. 2, the C-reactive protein test paper card of the present invention includes a PS base plate 121, and an absorbent pad 122, an NC membrane 123, a binding pad 124, a sample pad 125 and a blood filter pad 126 sequentially fixed on the base plate 121, wherein the absorbent pad 122 may be selected from conventional absorbent filter paper, the NC membrane surface is respectively sprayed with a first detection line, a second detection line and a quality control line formed by a crp-2 monoclonal antibody, a crp mixed antibody and a goat anti-mouse polyclonal antibody, the binding pad is coated with a colloidal gold-labeled crp-1 antibody, the sample pad 125 may be selected from conventional polyester cellulose membrane, and the blood filter pad may be selected from models of GF-2, GF-5, and the like.
Respectively taking a CRP-2 monoclonal antibody, a CRP mixed antibody (a mixture of the CRP-2 monoclonal antibody and an anti-human CRP antigen in a ratio of 1: 1) and a goat anti-mouse polyclonal antibody, diluting the antibodies to a concentration of 0.3mg/mL by using an antibody coating solution, respectively coating the antibodies according to a spraying amount of 1ul/cm by using a biodo film spraying instrument, spraying the diluted solutions on the surface of an NC (numerical control) membrane to respectively form a first detection line (a T1 line), a second detection line (a T2 line) and a quality control line (a C line), controlling the width of the line to be 1-1.2mm, drying the coated NC membrane overnight at a distance of 4-4.5mm between lines to obtain an NC membrane 123 for later use.
Adding a 5 mu LpH value regulator into 1000uL of colloidal gold solution, and uniformly mixing; then, 10. mu.g of the crp-1 antibody solution was added thereto, and after mixing, the reaction solution was placed on a shaker and subjected to coupling reaction at room temperature for 4 hours. After the reaction is finished, 20-100uL of the confining liquid is added into the coupled colloidal gold-antibody solution, the colloidal gold-antibody solution is uniformly mixed and placed on a shaking table, and the confining reaction is carried out for 1 hour at room temperature. And centrifuging the sealed colloidal gold-antibody solution at a high speed at 4 ℃, removing supernatant, adding 50uL of the gold-labeled antibody complex solution into the lower colloidal gold-labeled crp-1 antibody precipitate, and shaking and uniformly mixing for later use. The obtained colloidal gold-labeled crp-1 antibody conjugate was sprayed on a pretreated glass fiber membrane (the glass fiber membrane was soaked in the pretreatment solution for 30min and dried at room temperature) at a usage amount of 5 μ L/cm using a biodot spray coater, dried at room temperature for 4 hours to obtain the conjugate pad 124, and cut for use.
The assembly sequence of the detection card is PS bottom plate → absorption pad (absorbent filter paper) → NC film (sequentially having C line, T1 line and T2 line) → combination pad adsorbed with colloidal gold labeled crp-1 antibody → blood filter pad → sample pad (polyester film) → shell (provided with sampling holes), and the specific operation is as follows:
cutting the membrane modules into the following sizes by a cutting machine respectively: an absorption pad: 18mm × 300mm, NC membrane: 25mm × 300mm, bond pad: 8mm × 300mm, sample pad: 18mm × 300mm, blood filter pad: 18mm by 300 mm. Sequentially adhering the membrane modules on a PS base plate (overlapped by about 2mm) according to the structure shown in figure 2, cutting into test strips with the width of 3mm by a numerical control high-speed cutting machine, drying and storing in dark place;
and (3) fully soaking the test paper card in the hydrophilic treatment liquid for 24 hours, taking out after soaking, drying in a forced air drying oven, and completely drying for later use.
The cut test paper card 12 and the leukocyte bin 13 are loaded into the housing according to the position shown in fig. 1, and the card housing is assembled and ultrasonically welded. And putting the prepared detection card into an aluminum foil bag, drying, sealing and storing.
As shown in fig. 1, the bottom of the detection card of the present invention is provided with a sample adding hole 111, after sampling, a whole blood sample is added into a diluent bottle and mixed with the sample diluent, then the sample is added from the sample adding hole 111 at the bottom, the whole blood sample to be detected respectively flows into the white blood cell chamber 13 at the middle position for blood cell measurement under the control of the formed liquid separating channel 14, and the sample pad 125 position of the C-reactive protein test paper card at the right position for immunochromatography, and after 15-20min, the result is read, and the white blood cell value and the CRP value are respectively read, which is the quantitative detection result.
The rapid detecting component for the leucocyte combined with the C-reactive protein adopts a blood cell staining counting principle for the measurement of the leucocyte, and can obtain an accurate leucocyte counting result by carrying out high-power amplification and a large number of picture acquisition and analysis on a leucocyte bin of a detection card, thereby realizing a rapid and accurate quantitative detection result.
Example 2 Performance test
In this embodiment, the performance indexes of the leukocyte-binding C-reactive protein rapid detection card, such as detection limit, analysis specificity, accuracy, hook effect, precision, and linear range, are detected respectively to evaluate whether the leukocyte-binding C-reactive protein rapid detection card meets the requirements of experimental design.
In this example, the standards used for performance evaluation were human hypersensitive C-reactive protein, Millipore No. 8C72, and quality Control for blood CELL analysis, CELL-DYN 29Plus Control, Yapei Co. All experiments required the experimenter to be familiar with the detection method and instrumentation; adopting a proper standard substance; the reagents used in the experiment should be within the expiration date.
1. Detection limit (the performance evaluation aims at the C reactive protein detection reagent in the kit)
The sample treatment solution in the kit of example 1 was used as a blank solution, and the blank solution should not contain the C-reactive protein as a test substance. The C-reactive protein standard was diluted sequentially with blank solutions to final concentrations of 1.0, 0.8, 0.6, 0.4, 0.2. mu.g/ml, the above solutions and blank solutions were tested with the above three diagnostic reagents, each solution was tested 5 times with each reagent, and the test results were recorded as in Table 1 below.
TABLE 1 detection results of three reagent detection limits
| Batches of | Blank liquid | 1.0μg/ml | 0.8μg/ml | 0.6μg/ml | 0.4μg/ml | 0.2μg/ml |
| 1 | -5/5 | +5/5 | +5/5 | +5/5 | +5/5 | +1/5 |
| 2 | -5/5 | +5/5 | +5/5 | +5/5 | +5/5 | +3/5 |
| 3 | -5/5 | +5/5 | +5/5 | +5/5 | +5/5 | +0/5 |
As can be seen, the detection limit of the C-reactive protein in the kit of the scheme is 0.4 mug/ml.
2. Analysis specificity (the performance evaluation aims at the C reactive protein detection reagent in the kit)
The sample treatment solution in the kit of example 1 was used as a blank solution, and the blank solution should not contain the C-reactive protein as a test substance. The interference response of 25mg/ml hemoglobin, 25mg/ml triglyceride solution, 50U/ml heparin sodium, 32mg/ml sodium citrate, 1.5mg/ml tripotassium EDTA was tested. When each solution is tested, the C-reactive protein with the final concentration of 0, 1 and 3 mug/ml is dissolved by the solution respectively for detection, each solution is detected three times by each batch of reagent, and the recorded detection results are shown in tables 2-6.
TABLE 2 results of three reagent batches for detecting 25mg/ml hemoglobin solution interference reaction
| Batches of | Contains no CRP | Contains |
Contains CRP 3 μ g/ml |
| 1 | -3/3 | +3/3 | +3/3 |
| 2 | -3/3 | +3/3 | +3/3 |
| 3 | -3/3 | +3/3 | +3/3 |
TABLE 3 results of three reagent batches for detecting 25mg/ml triglyceride solution interfering reaction
| Batch number | Contains no CRP | Contains |
Contains CRP 3 μ g/ml |
| 1 | -3/3 | +3/3 | +3/3 |
| 2 | -3/3 | +3/3 | +3/3 |
| 3 | -3/3 | +3/3 | +3/3 |
TABLE 4 results of interfering reaction of three reagent batches with 50U/ml heparin sodium solution
| Batch number | Contains no CRP | Contains |
Contains CRP 3 μ g/ml |
| 1 | -3/3 | +3/3 | +3/3 |
| 2 | -3/3 | +3/3 | +3/3 |
| 3 | -3/3 | +3/3 | +3/3 |
TABLE 5 results of three reagent batches for detecting 32mg/ml sodium citrate solution interference reaction
| Batch number | Contains no CRP | Contains |
Contains CRP 3 μ g/ml |
| 1 | -3/3 | +3/3 | +3/3 |
| 2 | -3/3 | +3/3 | +3/3 |
| 3 | -3/3 | +3/3 | +3/3 |
TABLE 6 results of three reagent batches for detecting 1.5mg/ml EDTA tripotassium solution interference reaction
| Batch number | Contains no CRP | Contains |
Contains CRP 3 μ g/ml |
| 1 | -3/3 | +3/3 | +3/3 |
| 2 | -3/3 | +3/3 | +3/3 |
| 3 | -3/3 | +3/3 | +3/3 |
In conclusion, three batches of the C-reactive protein test strips in the embodiment have no interference reaction in the hemoglobin solution of 25mg/ml, the triglyceride solution of 25mg/ml, the heparin sodium solution of 50U/ml, the sodium citrate solution of 32mg/ml and the EDTA tripotassium salt solution of 1.5 mg/ml.
3. Accuracy of
Reaction protein detection test paper
Sample processing liquid in the kit is used as blank liquid, and the blank liquid does not contain C-reactive protein of a detected object. Sequentially diluting the C-reactive protein standard substance with blank solution to final concentrations of 10, 3 and 1 μ g/ml, respectively detecting with the above three batches of diagnostic reagents for 10 times, recording the detection result and calculating the standard deviation.
Total number of leukocytes
Respectively adopting the leukocyte concentration of 3 × 109/L、7×109/L、10×109The quality control product is analyzed by the blood cells, the three batches of diagnostic reagents are used for detection, each batch of diagnostic reagents is used for 10 times, the detection result is recorded, and the standard deviation is calculated.
The experimental results are shown in tables 7-8 below.
Table 7 test results of hypersensitive C reactive protein test strip
TABLE 8 Total leukocyte count assay results
Therefore, the deviation of the accuracy of the combined rapid quantitative detection kit for the total number of the three batches of the white blood cells and the hypersensitive C reactive protein is not more than 15%.
4. Hook effect (the performance evaluation aims at the detection reagent of C-reactive protein in the kit)
The sample treatment solution in the kit of example 1 was used as a blank solution, and the blank solution should not contain the C-reactive protein as a test substance. The C-reactive protein standard substance is diluted into the concentrations of 100, 80, 50, 20, 10, 5, 0.5 mu g/ml and the like by using blank solutions, three batches of reagents are used for respectively detecting, the color development intensity of a detection line is judged, each batch of reagent of each solution is detected three times, and the detection results are recorded as shown in the following table 9.
TABLE 9 hook Effect test results for three batches of reagents
| Batch number | Blank liquid | 1μg/ml | 5μg/ml | 10μg/ml | 20μg/ml | 50μg/ml | 80μg/ml | 100μg/ml |
| 1 | -3/3 | +3/3 | ++3/3 | +++3/3 | +++3/3 | ++++3/3 | ++++3/3 | +++++3/3 |
| 2 | -3/3 | +3/3 | ++3/3 | +++3/3 | +++3/3 | ++++3/3 | ++++3/3 | +++++3/3 |
| 3 | -3/3 | +3/3 | ++3/3 | +++3/3 | +++3/3 | ++++3/3 | ++++3/3 | +++++3/3 |
Note: the number of plus signs is represented by the coloration intensity of the detection line, and the more the number of plus signs is, the darker the coloration is.
As can be seen, the three batches of the reagent of the embodiment can be normally detected within the range of the concentration of the hypersensitivity C-reactive protein of 1-100 mug/ml, and the hook effect does not occur.
5. Precision-repeatability
C reaction protein test paper
Sample processing liquid in the kit is used as blank liquid, and the blank liquid does not contain C-reactive protein of a detected object. Diluting the C-reactive protein standard substance to a concentration of 3. mu.g/ml and 1. mu.g/ml with a blank solution, detecting, repeating the detection 10 times, and recording the detection result.
Total number of leukocytes
Adopting the leukocyte concentration of 3 × 109/L、7×109the/L blood cell analysis quality control material is detected by using a reagent, and the measurement is repeated for 10 times.
The results are reported in tables 10-11 below.
TABLE 10C reaction protein reproducibility results
TABLE 11 reproducibility of total white blood cell count
Therefore, the repetitive hypersensitivity C-reactive protein of the kit meets CV < 15%, and the total number of leucocytes meets CV < 6%.
6. Precision-inter-batch Difference
C reaction protein test paper
Sample processing liquid in the kit is used as blank liquid, and the blank liquid does not contain C-reactive protein of a detected object. Diluting the C-reactive protein standard substance to a concentration of 3 mu g/ml by using a blank solution, respectively detecting by using three batches of reagents, repeatedly measuring for 10 times, and recording the detection result.
Total number of leukocytes
Adopting the leukocyte concentration of 7 × 109The quality control product is analyzed by the blood cells, three batches of reagents are used for detection respectively, and the measurement is repeated for 10 times.
The test results were recorded and the standard deviation was calculated as shown in tables 12-13.
TABLE 12C reaction protein batch-to-batch Difference results
TABLE 13 Total leukocyte count inter-batch Difference results
As can be seen, the difference of the C-reactive protein between batches of the three-batch kit meets CV < 15%, and the total number of the white blood cells meets CV < 6%.
7. Linear range
C reaction protein test paper
Sample processing liquid in the kit is used as blank liquid, and the blank liquid does not contain C-reactive protein of a detected object. Diluting the C-reactive protein standard substance into a solution with a blank concentration of 0.5. mu.g/ml, 1. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml and 8. mu.g/ml, respectively detecting with three batches of reagents, repeatedly measuring each concentration sample twice, wherein the detection results are shown in Table 14, the original concentration content in the diluted concentration is taken as an x axis, the machine reading value is taken as a Y axis, and fitting to obtain a regression equation, which is shown in figure 3.
Total number of leukocytes
Taking a leukocyte with a concentration of 40 × 109The quality control product for analyzing blood cells is prepared by diluting quality control blood with 0.01M PBS (pH7.4) as diluent to 1 × 109/L、2.5×109/L、5×109/L，10×109/L、20×109and/L, respectively detecting by using three batches of reagents, repeatedly measuring each concentration sample for three times, wherein the detection result is shown in table 15, and fitting to obtain a regression equation by taking the original concentration in the diluted concentration as an x axis and the computer reading value as a Y axis, which is shown in figure 3.
TABLE 14C reaction protein Linear assay results
TABLE 15 Linear measurement of total white blood cell count
| Theoretical concentration | High value serum content | Measured |
Measured value 2 | Measured value 3 | Mean value of |
| 1 | 0.025 | 1.54 | 1.80 | 1.59 | 1.64 |
| 2.5 | 0.0625 | 2.63 | 2.83 | 2.95 | 2.80 |
| 5 | 0.125 | 5.34 | 5.11 | 5.38 | 5.28 |
| 10 | 0.25 | 10.18 | 10.06 | 10.04 | 10.09 |
| 20 | 0.5 | 19.97 | 20.08 | 20.39 | 20.15 |
The above measured data show that the total leukocyte test interval (1.0-10.0) x 10 of the detection card9a/L, deviation of not more than 0.5X 109/L，(10.1-18.0)×109The deviation is not more than +/-5 percent, the linear correlation coefficient is more than 0.990, and the relative deviation is not more than +/-5 percent. The test interval (0.4-12) mg/L of the hypersensitive C reactive protein has a linear correlation coefficient of more than 0.990, and the relative deviation is not more than +/-10%.
In conclusion, the rapid detection card for the leucocyte combined with the C-reactive protein is detected, and the result shows that the verification results of various indexes of the product, such as detection limit, analysis specificity, accuracy, precision, linearity and the like, all meet the design requirements and can meet the actual clinical needs.
EXAMPLE 3 clinical serum sample testing
The experimenter should be familiar with the detection method and the operation of the instrument; reagents used for the experiments should be the same lot number and within the expiration date. Whole blood was used for the experiments as fresh collection and assigned values have been determined by clinical testing.
Randomly selecting 0.5mg/L<C reactive protein content<3mg/L、0.3×109/L<Total white blood cells<10mg/ml×10930/L Whole blood samples, 12mg/L>C reactive protein content>3mg/L、18×109/L>Total white blood cells>10mg/ml×10970/L whole blood samples were each dropped on the test card of example 1 for blind testing and the statistical results are shown in tables 16-17 below.
TABLE 16C reaction protein assay results
TABLE 17 Total leukocyte count assay results
Therefore, the detection card has the clinical accuracy CV of less than 15 percent and meets the clinical detection requirement.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A rapid detection card component for leucocyte-bound C-reactive protein is characterized by comprising a detection card (1) and a sample diluent; the detection card (1) comprises a shell (11), and a C-reactive protein test paper card (12) and a leukocyte bin (13) which are arranged in the shell (11);
the C-reactive protein test paper card (12) comprises an absorption pad (122), an NC membrane (123), a combination pad (124), a blood filtration pad (126) and a sample pad (125) which are sequentially fixed on a bottom plate (121);
the surface of the NC film (123) is respectively sprayed with a first detection line, a second detection line and a quality control line which are formed by a crp-2 monoclonal antibody, a crp mixed antibody and a goat anti-mouse polyclonal antibody;
the conjugate pad (124) is coated with colloidal gold labeled crp-1 antibody.
2. The rapid leukocyte binding C-reactive protein detection card assembly of claim 1 wherein the sample diluent comprises: 10-50mM PBS buffer solution +0.1-0.5mM acridine orange +1-5mM blood anticoagulant.
3. A method for preparing the rapid leukocyte-binding C-reactive protein detection card assembly according to claim 1 or 2, comprising the steps of:
(1) respectively diluting the crp-2 monoclonal antibody, the crp mixed antibody and the goat anti-mouse polyclonal antibody by using an antibody coating solution, spraying the diluted antibodies on the surface of an NC (numerical control) membrane to respectively form a first detection line, a second detection line and a quality control line, and drying the NC membrane (123) for later use;
(2) pretreating a glass fiber membrane, spraying the colloidal gold-labeled crp-1 antibody on the surface of the glass fiber membrane in the amount of 3-8uL/cm to form the bonding pad (124), and drying for later use;
(3) cutting the absorption pad (122), the NC membrane (123), the combination pad (124), the blood filtration pad (126) and the sample pad (125) respectively, and then sequentially adhering and fixing the cut absorption pad, the NC membrane, the combination pad (124), the blood filtration pad (126) and the sample pad (125) on a bottom plate (121) to form the C-reactive protein test paper card (12) for later use;
(4) respectively placing the C-reactive protein test paper card (12) and the leucocyte bin (13) into preset positions in the shell (11), and packaging after welding;
(5) and (4) subpackaging the sample diluent at 240uL/test, and sealing and storing.
4. The method for preparing a rapid leukocyte binding C-reactive protein detection card kit according to claim 3, wherein in the step (1):
the antibody coating solution comprises: 10-50mM PBS buffer +1-5 wt% methanol;
the crp-2 monoclonal antibody, the crp mixed antibody and the goat anti-mouse polyclonal antibody are respectively diluted to be 0.1-0.5mg/mL in concentration.
5. The method for preparing the rapid leukocyte binding C-reactive protein detection card assembly according to claim 3 or 4, wherein the colloidal gold-labeled crp-1 antibody in the step (2) is prepared as follows: adding a pH value regulator into the colloidal gold solution, uniformly mixing, adding the crp-1 antibody solution, fully mixing uniformly, performing coupling reaction at room temperature, and continuously adding a sealing liquid after the reaction is finished to perform normal-temperature sealing reaction; centrifuging to precipitate colloidal gold marker, and diluting with gold-labeled antibody redissolution.
6. The method for preparing a rapid leukocyte binding C-reactive protein detection card kit according to claim 5, wherein the kit comprises:
the glass fiber pretreatment liquid comprises: 10-50Mm borate buffer + 0.1-0.5% TW20+ 0.1-5% BSA;
the gold-labeled antibody complex solution comprises: 10-50mM borate buffer solution + 1-10% sucrose + 0.1-5% BSA;
the pH regulator comprises: 1% potassium carbonate solution;
the confining liquid comprises: 10% BSA in water.
7. The method for preparing a rapid leukocyte-binding C-reactive protein detection card assembly according to any one of claims 3 to 6, wherein the step (3) further comprises a step of immersing the C-reactive protein test card (12) in a hydrophilic treatment solution;
the hydrophilic treatment liquid includes: 10-50mM PBS buffer + 0.5-2% TW 20.
8. The method for preparing a rapid leukocyte-binding C-reactive protein detection card assembly according to any one of claims 3-7, wherein in the step (4), the C-reactive protein test card (12) and the leukocyte bin (13) are arranged side by side, and a liquid separation channel (14) is arranged at the position of the sample application hole (111) of the housing (11) to control the sample to be tested to flow into the positions of the leukocyte bin (13) and the sample pad (125) of the C-reactive protein test card (12), respectively.
9. The method for using the rapid leukocyte-binding C-reactive protein detection card assembly according to any one of claims 3-8, comprising the steps of diluting and loading a whole blood sample to be detected with the sample diluent, flowing the whole blood sample to be detected into the leukocyte bin (13) for blood cell staining counting, and flowing the whole blood sample into the C-reactive protein test card (12) for chromatography reaction, and reading the detection result after 15-20 min.
10. Use of the leukocyte binding C-reactive protein rapid detection card assembly according to any one of claims 3 to 8 in the field of diagnosis and identification of bacterial and viral infections.
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