CN204287198U - Omnidistance C reactive protein immunochromatographiassay assay quantitative detection test paper - Google Patents

Abstract

The utility model discloses a test strip for quantitative detection of C-reactive protein immunochromatography, which comprises a base plate, a sample pad, a fluorescent marker binding pad, nitrocellulose and a water-absorbing pad; the test strip consists of a sample pad, a fluorescent The marker binding pad, nitrocellulose, and water-absorbing pads are sequentially overlapped and pasted on the base plate to form a structure. The fluorescent marker binding pad contains streptavidin-labeled fluorescent protein and biotin-labeled whole C-reactive protein monoclonal antibody; there are detection lines and quality control lines on the nitrocellulose membrane, and the detection line is covered by the whole C-reactive protein. Reactive protein monoclonal antibody, which has a different recognition epitope from the above-mentioned biotin-labeled whole C-reactive protein monoclonal antibody. Compared with the current common methods for detecting the whole process of C-reactive protein (such as: chemiluminescence method/immunoenhanced turbidimetric method/colloidal gold method), the utility model not only greatly shortens the detection time, but also improves the detection sensitivity.

Description

Whole C-reactive protein immunochromatographic quantitative detection test strip

technical field

The utility model belongs to the field of immune detection, in particular to a high-sensitivity full-range C-reactive protein immune chromatography quantitative detection test strip.

Background technique

Human C-reactive protein (CRP) is the major acute-phase protein with rapid, dramatic rises in plasma concentrations upon infection and tissue injury. C-reactive protein can activate complement and strengthen the phagocytosis of phagocytes to play an opsonizing role, so as to remove pathogenic microorganisms and damaged, necrotic, and apoptotic tissue cells invading the body, and play an important protective role in the body's natural immune process. The research on C-reactive protein has a history of more than 70 years. The traditional view is that C-reactive protein is a non-specific inflammatory marker, but research in the past ten years has revealed that CRP is directly involved in inflammation and atherosclerosis, etc. Cardiovascular disease, and is the most powerful predictor and risk factor of cardiovascular disease.

The detection of C-reactive protein was widely used clinically as a non-specific marker of inflammation and tissue damage before the 1980s. However, because the detection method of C-reactive protein was relatively backward in the past, the false positive and false negative were high, which affected its clinical value, and it was gradually neglected in clinical practice. In recent years, due to the update of detection technology, a fast, simple and reliable method for measuring C-reactive protein has been rapidly established, which has greatly increased the clinical application of C-reactive protein.

The current common methods for detecting C-reactive protein are:

1 One-way immunodiffusion method (single-diffusion method for short)

The single expansion method is a classic antigen-antibody precipitation test. Its basic principle is to mix a certain amount of antibody in the agar gel, so that the antigen solution to be tested can diffuse freely from the local area to the agar, and form a visible precipitation ring in the precipitation area. The diameter or area of the precipitation ring is related to the amount of antigen. As a simple antigen quantification method, the single amplification method has the advantages of high specificity, good repeatability, simple operation, low price, and does not require special equipment for detection. Therefore, it is widely used in some small and medium hospitals. However, the biggest disadvantage of this method is that when the antigen is excessive, the reaction system does not precipitate, and when the CRP concentration is too high, there will be a high false negative. Therefore, when there is no precipitation ring in the detection of CRP by the one-way immunodiffusion method, the sample must be diluted and retested to avoid missed diagnosis. In addition, due to the poor sensitivity of this method, it restricts its wide clinical application.

2 latex agglutination method

Latex agglutination is a more commonly used clinical serological method, which belongs to indirect agglutination test. The latex reagent is sensitized with purified anti-human CRP antibody, which can react specifically with CRP in the patient's serum, and presents clear agglutinated particles within a few minutes. Those with agglutination are positive, and those without agglutination are negative. This method is simple, rapid, high sensitivity and specificity. However, it is susceptible to the interference of complement, rheumatoid factor (RF) and other factors, resulting in false positive results. Therefore, in order to improve the accuracy of the results, the samples to be tested should be pretreated to remove the interference factors.

3-rate nephelometry

The rate-scattering turbidimetry is a micro-immunoprecipitation method created by Sternberg in 1977, which is to determine the amount of antigen in the sample by measuring the degree of light scattering of the solution. The light of a certain wavelength is irradiated along the horizontal axis, and the small particles of the immune complex can cause light scattering, and the scattering intensity is proportional to the content of the antigen-antibody immune complex.

This method is a dynamic determination method of antigen-antibody binding reaction, which can quickly and accurately measure the content of antigen in the sample, and the results can be determined on a variety of automatic detectors. The most widely used automatic instrument is the automatic protein analyzer produced by Backman-Coulter Company. Velocity nephelometry has been used as a routine detection method of CRP in clinical practice. Studies have shown that the rate nephelometric method is superior to the single expansion method in terms of detection time, operation method, result accuracy, repeatability and sensitivity. Compared with the latex agglutination method, the sensitivity and accuracy also have significant differences (P<0.05), which are significantly higher than the latex agglutination method.

4 Immunoturbidimetry

Immunoturbidimetry is a commonly used method for detecting CRP in laboratories, and many manufacturers also use this method at present, such as the CRP of Yingke Xinchuang (Xiamen) Technology Co., Ltd. Immunoturbidimetry is also a microvolume immunoprecipitation assay. It differs from the rate nephelometric method by measuring the amount of light passing through the solution to reflect the content of the antigen to be tested. When the light passes through the reaction system, the antigen-antibody immune complex in the solution can absorb and reflect the light, reducing the transmitted light. The more immune complexes, the more light is absorbed and the less light is transmitted. This change can be expressed by absorbance. If the amount of antibody is fixed, the measured absorbance is proportional to the amount of the immune complex and also proportional to the amount of the antigen to be tested. Using a series of antigen standards of known concentration as a control, the content of the tested substance can be measured.

5 Latex-enhanced immunoturbidimetry

In the above-mentioned turbidimetric method, the sensitivity of detecting CRP is still not ideal. This just needs to establish more sensitive, more accurate, still has the detection method of higher accuracy under the lower situation of serum CRP concentration. So people improved and updated the detection method, and created the latex-enhanced immune transmission turbidimetric method.

The basic principle of latex-enhanced immune transmission turbidimetry is that the antibody is firstly adsorbed on a latex particle, and when the corresponding antigen is encountered, the antigen-antibody combination results in latex agglutination. The size of individual latex particles is within the wavelength of incident light through which light is transmitted. When two or more latex particles are aggregated, it can hinder the transmission of light and reduce the transmitted light. The degree of reduction is proportional to the degree of latex agglutination and also proportional to the amount of antigen. This method is a new type of high-sensitivity detection method for the determination of high-sensitivity C-reactive protein (Hs-CRP), which has the advantages of rapidity, convenience and accuracy.

6 Immunolabeling technology

Immunolabeling technology The immunolabeling methods used for CRP determination include radioimmunoassay, enzyme immunoassay, gold standard immunoassay, fluorescent labeling immunoassay, etc. Due to the shortcomings of radioimmunoassay such as short half-life of radioactive isotopes, difficult preservation of radioactive contamination, poor stability, etc., there are many inconveniences in use, especially the wide application of enzyme immunoassay, so this method is rarely used clinically. At present, the most clinically applied methods are enzyme-linked immunosorbent assay (ELISA)-based enzyme immunolabeling technology. This method is to combine the specificity of antigen-antibody reaction with the high-efficiency catalysis of the enzyme on the substrate. According to the color change after the enzyme acts on the substrate, the detection sample is quantitatively measured and analyzed by a microplate reader, and the CRP is calculated by comparing with the standard curve. content. ELISA has high sensitivity and specificity, and its reagents are relatively stable without radioactive contamination. In particular, the application of commercial kits and automatic microplate readers makes it a detection method suitable for inspection departments at all levels. At the same time, it is also one of the commonly used methods for measuring serum Hs-CRP in patients.

The detection of quantitative CRP by the gold standard method and the fluorescent labeling immunochromatography method has been relatively well unified in terms of detection sensitivity and test speed. The advantage is that the amount of test specimen is small, the test is fast, it can be tested together with blood routine, the specificity is high, and it also shows a good application prospect in clinical testing. At present, domestic products and products imported from South Korea and Canada have been applied in China to a certain extent.

Biotin-avidin system (biotinavidin system, BAS), is a new biological reaction amplification system. With the advent of various biotin derivatives, BAS was soon widely used in various fields of medicine. Applying this system to detection techniques such as immunohistochemistry, enzyme-linked immunosorbent, fluorescent immunoassay, and radioimmunoassay can significantly improve the sensitivity, specificity, and stability of the above technical methods, make the method more convenient, and contribute to rapid clinical Diagnosis, and facilitates large-scale epidemiological investigations, has become a powerful tool for studying immune responses. Because the BAS detection system is economical and fast, has no radioactive material pollution, and does not require complex instruments, it fully demonstrates the great potential and application possibility of this system.

Biotin-avidin system detection principle: BAS is a detection system established on the basis of the conventional ELISA principle, combined with the high amplification effect between biotin and avidin. Avidin is a basic glycoprotein extracted from ovalbumin, with a molecular weight of 68kDa, composed of 4 subunits, and has a very high affinity for biotin (the binding constant is as high as 1015M-1). Biotin is easily combined with proteins (such as antibodies, etc.) by covalent bonds. In this way, the avidin molecule bound to the enzyme reacts with the biotin molecule bound to the specific antibody, which not only plays a multi-stage amplification role, but also develops color due to the catalysis of the enzyme when it encounters the corresponding substrate, and achieves detection. The purpose of the unknown antigen (or antibody) molecule. Streptavidin is a protein with similar biological characteristics to avidin. Like avidin, each peptide chain in the molecule of streptavidin can bind a biotin, because almost all the markers used for labeling Substances can be combined with avidin or streptavidin. There is no report on the development of rapid diagnostic reagents for procalcitonin detection test using biotin-avidin system.

Utility model content

The purpose of this utility model is to provide a kind of whole C-reactive protein immune chromatography quantitative detection test strip, which is based on double-antibody sandwich method, fluorescent immunological lateral chromatography and biotin-streptavidin amplification system, providing High-sensitivity full-range C-reactive protein immunochromatographic quantitative detection test strip. When the utility model is used, the detection sensitivity and detection stability are improved, the non-specific combination is reduced, the detection time is not more than 10 minutes, and the diagnosis efficiency is greatly improved.

The solution of the utility model to solve the technical problem is: the whole C-reactive protein immunochromatography quantitative detection test strip, which includes a base plate, and the base plate is sequentially provided with a sample pad, a fluorescent marker binding pad, and a nitrocellulose membrane and a water-absorbing pad, the water-absorbing pad and the fluorescent marker-binding pad are respectively overlapped and pressed on the two ends of the nitrocellulose membrane to form a detection area on the surface of the nitrocellulose membrane, and the sample pad is overlapped and pressed on the fluorescent marker-binding pad. On the pad, biotin-labeled C-reactive protein monoclonal antibody and streptavidin-labeled fluorescent protein are immobilized on the fluorescent marker binding pad; -A detection line composed of a monoclonal antibody that reacts to another epitope of the protein and a quality control line composed of a goat anti-mouse IgG polyclonal antibody.

As a further improvement of the above technical solution, the fluorescent marker binding pad includes a stacked first fluorescent marker binding pad and a second fluorescent marker binding pad, one end of the first fluorescent marker binding pad is placed on the sample pad. Below, the second fluorescent marker-binding pad overlaps one end of the nitrocellulose membrane.

As a further improvement of the above technical solution, a positioning bar is provided at one end of the first fluorescent marker binding pad inserted below the sample pad, and a positioning groove capable of accommodating the positioning bar is provided on the lower end surface of the sample pad.

As a further improvement of the above technical solution, it also includes a clamping case, the bottom plate, the sample pad, the fluorescent marker binding pad, the nitrocellulose membrane and the water-absorbing pad are all placed in the clamping case, and the surface of the clamping case corresponds to the surface of the nitrocellulose membrane. An observation window is provided at the position, and a sample injection hole is provided at the position corresponding to the sample pad on the surface of the clamping shell.

As a further improvement of the above technical solution, the observation window is a rectangular hole.

As a further improvement of the above technical solution, the fluorescent protein may be one of green fluorescent protein and phycobiliprotein.

As a further improvement of the above technical solution, the sample pad is a glass fiber member that is soaked in a surfactant buffer solution and then dried.

As a further improvement of the above technical solution, the bottom plate is a polystyrene member or a polyethylene member.

As a further improvement of the above technical solution, the clamping case includes a plastic upper case and a plastic lower case, and the plastic upper case is fastened to the plastic lower case to form a clamping case.

Compared with the prior art, the utility model has the following advantages:

(1) The utility model uses fluorescent protein as a marker, which has good stability and is conducive to improving the detection stability.

(2) The utility model improves the detection sensitivity and reduces non-specific binding by utilizing the "streptavidin-biotin amplification system", which is beneficial to improve the performance of the kit.

(3) The detection time of procalcitonin by using the utility model is not more than 10 minutes, and the detection linear range is 0.10ng/ml-100.00ng/ml, which greatly improves the detection efficiency.

(4) The utility model can interpret the results through the fluorescent immunoassay instrument, which can realize automation, reduce the influence of subjective factors, and provide convenient, fast and reliable diagnostic results.

(5) The new clamping case is provided with a sampling hole for the sample to enter and an observation window for observing the result, and the determination result is accurate and reliable according to the analysis instrument.

(6) The utility model is easy to manufacture, small in size and easy to carry.

(7) The detection cost of the utility model is relatively low.

(8) The utility model can be mass-produced, and is suitable for rapid clinical diagnosis and on-site rapid diagnosis; it is easy to preserve and is beneficial to popularization in grassroots units.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following will briefly describe the accompanying drawings that are used in the description of the embodiments. Apparently, the drawings described are only some embodiments of the utility model, not all embodiments, and those skilled in the art can also obtain other designs and drawings according to these drawings without creative work.

Fig. 1 is the structural representation of the utility model embodiment one;

Fig. 2 is the structural representation of the second embodiment of the utility model;

Fig. 3 is the structural representation of the utility model embodiment three;

Fig. 4 is a schematic structural view of Embodiment 4 of the present utility model.

Detailed ways

Below in conjunction with embodiment and accompanying drawing, design of the present utility model, concrete structure and the technical effect that produce are clearly and completely described, to fully understand purpose, feature and effect of the present utility model. Apparently, the described embodiments are only some of the embodiments of the present utility model, rather than all embodiments. Based on the embodiments of the present utility model, other embodiments obtained by those skilled in the art without paying creative efforts, All belong to the protection scope of the utility model. In addition, all the connection/connection relationships mentioned in this article do not refer to the direct connection of components, but mean that a better connection structure can be formed by adding or reducing connection accessories according to specific implementation conditions.

Referring to Fig. 1, the test strip for quantitative detection of C-reactive protein immunochromatography in the whole process includes a bottom plate 5, on which a sample pad 1, a fluorescent marker binding pad 2, a nitrocellulose membrane 3 and an absorbent pad are sequentially arranged on the bottom plate 5 4. The water-absorbing pad 4 and the fluorescent marker binding pad 2 are respectively overlapped and pressed on the two ends of the nitrocellulose membrane 3 to form a detection area on the surface of the nitrocellulose membrane 3, and the fluorescent marker binding pad 2 is a glass fiber membrane , the sample pad 1 is overlapped and pressed on the fluorescent marker binding pad 2, and the biotin-labeled whole C-reactive protein monoclonal antibody (concentration 0.3-1.5 mg/mL) is immobilized on the fluorescent marker binding pad 2 Streptavidin-labeled (or avidin) fluorescent protein (excitation light wavelength 530nm, emission light wavelength 570nm, concentration 0.1-1.0mg/mL); fixed on the nitrocellulose membrane 3 in the detection area There is a test line T (concentration 0.5-3 mg/mL) composed of a monoclonal antibody that recognizes another epitope of the whole C-reactive protein and a quality control line C (concentration 0.2-2.0 mg/mL) composed of a goat anti-mouse IgG polyclonal antibody. ), the quality control line C is used to detect the validity of the test strip.

Immobilize the biotin-labeled C-reactive protein monoclonal antibody and streptavidin (or avidin)-labeled fluorescent protein on the fluorescent marker binding pad 2, which will recognize another epitope of C-reactive protein Monoclonal antibody and goat anti-mouse IgG polyclonal antibody were immobilized on nitrocellulose membrane as detection line T and quality control line C, respectively. When the sample to be tested is added to the sample pad 1, it moves forward through chromatography, and the C-reactive protein antibody (Mab -CRP*Fluoro) reacts to form a complex CRP-Mab-CRP*Fluoro, and when the reaction complex continues to move forward through the CRP antibody (detection line) coated on the nitrocellulose membrane under the action of chromatography, the reaction complex is coated The CRP antibody is captured to form a complex (Mab-CRP-CRP-Mab-CRP*Fluoro) (detection line), and the reaction signal of the detection line is read by a fluorescent immunoassay analyzer. Under the action of the excitation light source, the fluorescent substance emits a specific Fluorescent signal of the wavelength, the fluorescence immunoassay analyzer captures the fluorescent signal, automatically converts it into a quantitative value through signal conversion and the set standard curve, calculates the concentration of C-reactive protein in the sample, and obtains the detection result of C-reactive protein.

As a further improvement of the above technical solution, referring to FIG. 3 , the fluorescent marker binding pad 2 includes a laminated first fluorescent marker binding pad 20 and a second fluorescent marker binding pad 21, and the first fluorescent marker binding pad One end of the pad 20 is placed under the sample pad 1 , and the second fluorescent marker binding pad 21 is overlapped and pressed against one end of the nitrocellulose membrane 3 . The layered arrangement of the fluorescent marker binding pad 2 facilitates the installation of the fluorescent marker binding pad 2 between the sample pad 1 and the nitrocellulose membrane 3 .

As a further improvement of the above-mentioned technical solution, referring to FIG. 3, one end of the first fluorescent marker binding pad 20 inserted below the sample pad is provided with a positioning bar 22, and the lower end surface of the sample pad 1 is provided with a positioning bar 22. Locating slot. The positioning strip 22 facilitates the installation and fixation between the fluorescent marker binding pad 2 and the sample pad 1 .

As a further improvement of the above-mentioned technical solution, with reference to Fig. 2 and Fig. 4, it also includes a clamping case, the bottom plate 5, the sample pad 1, the fluorescent marker binding pad 2, the nitrocellulose membrane 3 and the water-absorbing pad 4 are all placed in the clamping case 6 An observation window 8 is provided on the shell surface of the cartridge 6 corresponding to the position of the nitrocellulose membrane 3 , and a sampling hole 7 is provided on the shell surface of the cartridge 6 corresponding to the position of the sample pad 1 .

As a further improvement of the above technical solution, the observation window 8 is a rectangular hole.

As a further improvement of the above technical solution, the fluorescent protein may be one of green fluorescent protein and phycobiliprotein.

As a further improvement of the above technical solution, the sample pad 1 is a glass fiber member soaked in a surfactant buffer solution and then dried. The sample pads are made of glass fibers soaked in surfactant buffer and dried before use. The cutting width of the sample pad 1 is 0.3-0.5 cm.

As a further improvement of the above technical solution, the bottom plate 5 is a polystyrene member or a polyethylene member.

As a further improvement of the above technical solution, the clamping case 6 includes a plastic upper case 60 and a plastic lower case 61 , and the plastic upper case 61 is snapped onto the plastic lower case 61 to form the clamping case 6 .

The above is a specific description of the preferred embodiments of the present utility model, but the invention is not limited to the described embodiments, those skilled in the art can also make various equivalent modifications without violating the spirit of the present utility model Or replacement, these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims (9)

1. The whole C-reactive protein immunochromatography quantitative detection test strip, is characterized in that: it comprises base plate, and described base plate is provided with sample pad, fluorescent marker binding pad, nitrocellulose membrane and water-absorbing pad successively, described The water-absorbing pad and the fluorescent marker binding pad are respectively overlapped and pressed on the two ends of the nitrocellulose membrane to form a detection area on the surface of the nitrocellulose membrane. The sample pad is overlapped and pressed on the fluorescent marker binding pad, and the fluorescent marker Biotin-labeled whole-range C-reactive protein monoclonal antibody and streptavidin-labeled fluorescent protein are immobilized on the object-binding pad; another surface that recognizes whole-range C-reactive protein is immobilized on the nitrocellulose membrane in the detection area. The detection line is composed of monoclonal antibody and the quality control line is composed of goat anti-mouse IgG polyclonal antibody. 2. The full-range C-reactive protein immunochromatography quantitative detection test strip according to claim 1, characterized in that: the fluorescent marker binding pad comprises a laminated first fluorescent marker binding pad and a second fluorescent marker As for the binding pad, one end of the first fluorescent marker binding pad is placed under the sample pad, and the second fluorescent marker binding pad overlaps and presses one end of the nitrocellulose membrane. 3. the whole process C-reactive protein immunochromatography quantitative detection test strip according to claim 2, is characterized in that: the end that inserts sample pad below in described first fluorescent marker binding pad is provided with positioning bar, and described The lower end surface of the sample pad is provided with a positioning groove capable of accommodating the positioning bar. 4. The full-range C-reactive protein immunochromatography quantitative detection test strip according to any one of claims 1 to 3, characterized in that: it also includes a jam, the base plate, sample pad, fluorescent marker binding pad, nitric acid Both the cellulose membrane and the water-absorbent pad are placed in the clamping case, and an observation window is provided on the clamping shell surface corresponding to the position of the nitrocellulose membrane, and a sampling hole is provided on the clamping shell surface corresponding to the position of the sample pad. 5. The test strip for quantitative detection of C-reactive protein immunochromatography for the whole process according to claim 4, characterized in that: the observation window is a rectangular hole. 6. The test strip for quantitative detection of C-reactive protein immunochromatography for the whole process according to claim 4, characterized in that: the fluorescent protein can be one of green fluorescent protein and phycobiliprotein. 7. The test strip for quantitative detection of C-reactive protein immunochromatography according to claim 4, characterized in that: the sample pad is a glass fiber member soaked in a surfactant buffer solution and then dried. 8. The C-reactive protein immunochromatographic quantitative detection test strip according to claim 4, characterized in that: the bottom plate is a polystyrene member or a polyethylene member. 9. The full-range C-reactive protein immunochromatographic quantitative detection test strip according to claim 4, characterized in that: the stuck case comprises a plastic upper case and a plastic lower case, and the plastic upper case is fastened to the plastic lower case Then form a jam.