CN204514932U - Myoglobins immunochromatographiassay assay quantitative detection test paper - Google Patents

Abstract

The utility model discloses a test strip for quantitative detection of myoglobin immunochromatography, which comprises a bottom 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 marker Combination pads, nitrocellulose, and water-absorbent pads are sequentially lapped and pasted on the bottom plate to form. The fluorescent marker binding pad contains streptavidin-labeled fluorescent protein and biotin-labeled myoglobin monoclonal antibody; there are detection lines and quality control lines on the nitrocellulose membrane, and the detection line is covered by myoglobin monoclonal antibodies. An antibody having a different recognition epitope from the above-mentioned biotin-labeled myoglobin monoclonal antibody. Compared with the current common methods for detecting myoglobin (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

Myoglobin immunochromatography quantitative detection test strip

technical field

The utility model belongs to the field of immune detection, in particular to a high-sensitivity myoglobin immune chromatography quantitative detection test strip.

Background technique

Myoglobin (MYO) is a binding protein composed of a peptide chain and a heme prosthetic group. It is a protein that stores oxygen in muscle and is also the main protein that composes skeletal muscle and cardiac muscle. When muscle damage occurs, MYO can leak from muscle tissue into circulating blood, increasing serum MYO concentration, which is used to judge whether muscle damage occurs. Determination of serum MYO can be used as the early and most sensitive index for the diagnosis of acute myocardial infarction (AMI), and can also be used as an index for observing muscle damage in surgical operations.

At present, there are deficiencies in many methods for detecting myocardial injury markers, such as: enzyme-linked immunoassay, solid-phase immunoassay, radioimmunoassay, cumbersome operation, long detection time, poor repeatability of results, nuclide contamination, certain The method can only be used for qualitative detection. Although chemiluminescent immunoassay has the advantages of accuracy, high sensitivity, strong specificity, and good precision, the instruments used are expensive and the reagents used are imported reagents, which are difficult to carry out in general laboratories and are not suitable for emergency testing.

The current common methods for detecting MYO are:

1 Latex agglutination method:

Latex agglutination is a commonly used serological method in clinical practice. The latex reagent is sensitized with purified anti-human MYO antibody, which can specifically react with MYO in the patient’s serum. Clear agglutination particles appear within a few minutes, and those with agglutination are positive. Those without agglutination were 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.

2 Immunoturbidimetry:

The principle of immunoturbidimetry is to use the specific combination of antigen and antibody to form a complex, and to quantify the antigen or antibody by measuring the amount of complex formation. The method is easy to operate, suitable for common automatic biochemical analyzers and common spectrophotometers, and can be carried out in almost all laboratories. The disadvantage is that the sensitivity and precision are not ideal, the amount of antiserum required is large, and the detection period is long.

3 Radioimmunoassay:

Radioimmunoassay is the most sensitive method for measuring serum MYO. MYO is used to compete with 125I-labeled MYO for limited antibodies, and the minimum measurement range is 2ng/ml. Due to the short half-life of radioactive isotopes in radioimmunoassay, radioactive contamination is not easy to preserve and has poor stability. And other shortcomings, there are many inconveniences in use, especially the wide application of enzyme immunoassay, so that this method is rarely used clinically.

Biotin-avidin system (biotinavidin system, BAS) is a new type of biological reaction amplification system, which is 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.

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 10 ¹⁵ M ^-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 of 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 myoglobin detection test using biotin-avidin system.

Utility model content

The purpose of this utility model is to provide a myoglobin immunochromatography quantitative detection test strip, which is based on double-antibody sandwich method, fluorescence immunological lateral chromatography and biotin-streptavidin amplification system, providing high sensitivity Myoglobin 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 its technical problem is: myoglobin 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, a nitrocellulose membrane and a water-absorbing pad. 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, Biotin-labeled myoglobin monoclonal antibody and streptavidin-labeled fluorescent protein are immobilized on the fluorescent marker binding pad; another surface for recognizing myoglobin 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.

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-conjugated 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 accompanying drawings described are only a part of the embodiments of the utility model, rather than all embodiments. Those skilled in the art can also obtain other design schemes and accompanying drawings according to these drawings without paying 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

The idea, specific structure and technical effects of the present utility model will be clearly and completely described below in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, characteristics and effects 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.

With reference to embodiment one shown in Fig. 1, myoglobin immunochromatography quantitative detection test strip, it comprises base plate 5, and described base plate 5 is provided with sample pad 1, fluorescent marker binding pad 2, nitrocellulose membrane successively 3 and a water-absorbing pad 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 It is a glass fiber membrane, and the sample pad 1 is overlapped and pressed on the fluorescent marker binding pad 2, and the biotin-labeled myoglobin monoclonal antibody (concentration: 0.3-1.5 mg) is immobilized on the fluorescent marker binding pad 2. /mL) and streptavidin-labeled (or avidin) fluorescent protein (using excitation light wavelength 530nm, emission light wavelength 570nm, concentration 0.1 ~ 1.0mg/mL); nitrocellulose membrane in the detection area 3 is immobilized with a detection line T (concentration 0.5-3 mg/mL) composed of a monoclonal antibody that recognizes another epitope of myoglobin and a quality control line C (concentration 0.2-2.0 mg/mL) composed of a goat anti-mouse IgG polyclonal antibody. mL), the quality control line C is used to detect the validity of the test strip.

Immobilize biotin-labeled myoglobin monoclonal antibody and streptavidin (or avidin)-labeled fluorescent protein on the fluorescent label binding pad 2, and there will be a monoclonal antibody that recognizes another epitope of myoglobin 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 myoglobin in the sample is combined with the fluorescent marker on the pad 2. The myoglobin antibody (Mab-MYO) bound to the fluorescent marker (fluorescent protein) *Fluoro) reacts to form a complex MYO-Mab-MYO*Fluoro, and when the reaction complex continues to move forward through the MYO antibody (detection line) coated on the nitrocellulose membrane under the action of chromatography, the reaction complex is coated MYO antibody captures and forms a complex (Mab-MYO-MYO-Mab-MYO*Fluoro) (detection line), and reads the reaction signal of the detection line through a fluorescent immunoassay analyzer. Under the action of an excitation light source, the fluorescent substance emits a specific wavelength Fluorescent signal, the fluorescent 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 myoglobin in the sample, and obtains the detection result of myoglobin.

As a further improvement of the above-mentioned technical solution, referring to the third embodiment shown in FIG. One end of a fluorescent marker binding 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 technical solution, referring to Embodiment 2 and Embodiment 4 shown in FIG. 2 and FIG. 4 , it also includes a cartridge, the bottom plate 5, the sample pad 1, the fluorescent marker binding pad 2, and the nitrocellulose membrane 3 and the water-absorbing pad 4 are all placed in the clamping shell 6, and the position corresponding to the nitrocellulose membrane 3 is provided with an observation window 8 on the shell surface of the clamping shell 6, and the sample injection hole 7 is provided on the shell surface of the clamping shell 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. myoglobins immunochromatographiassay assay quantitative detection test paper, it is characterized in that: it comprises base plate, described base plate is provided with successively sample pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads, described adsorptive pads and fluorescent marker pad are overlapping to be respectively pressed in behind the two ends of nitrocellulose filter in the formation detection zone, surface of nitrocellulose filter, described sample pad is overlapping to be pressed on fluorescent marker pad, described fluorescent marker pad is fixed with the fluorescin of biotin labeled myoglobins monoclonal antibody and marked by streptavidin; Nitrocellulose filter in described detection zone is fixed with the nature controlling line of detection line and the sheep anti-mouse igg polyclonal antibody formation identifying that the monoclonal antibody of myoglobins another one epi-position is formed.

2. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 1, it is characterized in that: described fluorescent marker pad comprises the first stacked fluorescent marker pad and the second fluorescent marker pad, one end pad of described first fluorescent marker pad in the below of sample pad, described second overlapping one end being pressed in nitrocellulose filter of fluorescent marker pad.

3. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 2, it is characterized in that: in described first fluorescent marker pad, the one end of inserting below sample pad is provided with positioning strip, and the lower surface of described sample pad is provided with the locating slot that can hold positioning strip.

4. the myoglobins immunochromatographiassay assay quantitative detection test paper according to any one of claims 1 to 3, it is characterized in that: also comprise and getting stuck, in described base plate, sample pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads are all placed in and get stuck, the position described shell face of getting stuck corresponding to nitrocellulose membrane is provided with view window, and position shell face of getting stuck corresponding to sample pad is provided with well.

5. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described view window is rectangular opening.

6. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described fluorescin is the one in green fluorescent protein, phycobniliprotein.

7. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described sample pad is glass fibre component dry after surfactant damping fluid immersion treatment.

8. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described base plate is polystyrene component or tygon component.

9. myoglobins immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described in get stuck and comprise plastics upper casing and plastics lower casing, described plastics upper casing fastens to be formed after on plastics lower casing and gets stuck.