CN111610335B - Time-resolved fluorescence immunochromatography test strip, kit containing same and application thereof - Google Patents

Time-resolved fluorescence immunochromatography test strip, kit containing same and application thereof Download PDF

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CN111610335B
CN111610335B CN202010472803.2A CN202010472803A CN111610335B CN 111610335 B CN111610335 B CN 111610335B CN 202010472803 A CN202010472803 A CN 202010472803A CN 111610335 B CN111610335 B CN 111610335B
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resolved fluorescence
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CN111610335A (en
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朱传增
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Shanghai Aopu Biomedical Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/585—Calcitonins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Abstract

The invention discloses a time-resolved fluorescence immunochromatography test strip, which comprises a bottom plate (1), a sample pad (2), a nitrocellulose membrane (3) and a water absorption pad (4), wherein the sample pad (2), the nitrocellulose membrane (3) and the water absorption pad (4) are sequentially connected and fixed on the bottom plate (1) in the horizontal direction, a fluorescent wire (5), a detection wire and a quality control wire (9) are coated on the nitrocellulose membrane (3), and the fluorescent wire (5) comprises one or more time-resolved fluorescent microspheres marked by antigens or antibodies. The invention also discloses a reagent and/or a kit containing the same and application thereof. The time-resolved fluorescence immunochromatography test strip and the like can rapidly quantify one antigen or simultaneously rapidly quantify multiple antigens in a short detection time, not only can improve the sensitivity and the linear range of detection, but also can realize the one-step sample loading operation, and can greatly improve the precision and the accuracy of the operation.

Description

Time-resolved fluorescence immunochromatography test strip, kit containing same and application thereof
Technical Field
The invention relates to a time-resolved fluorescence immunochromatography test strip, a reagent containing the same, a kit and application thereof.
Background
Procalcitonin (PCT) is a prohormone of calcitonin, consisting of 116 amino acids, and has a molecular weight of about 12.8kD, which is a hormone-free glycoprotein and is also an endogenous non-steroidal anti-inflammatory substance, produced by the thyroid gland in the case of non-infection. When systemic infection occurs, many different cell types of organs secrete procalcitonin after being stimulated by a pro-inflammatory reaction, and particularly when being infected by bacteria, the metabolism is little or not dependent on kidney function, the clearance rate of the patients with renal failure is not affected, and PCT metabolism is not affected by steroid hormones. As early as 1993, studies were carried out to find that the higher the PCT level is when the organism is severely infected, the heavier the infection is, and the worse the prognosis is, and the relationship between the PCT level and the severe degree of sepsis is demonstrated for the first time.
In clinical diagnosis, procalcitonin concentration of patients with sepsis rises earlier, and early diagnosis and monitoring of doctors are facilitated. The literature reports that PCT in serum begins to rise in the case of systemic infections in 2-4 hours, peaking for 8-24 hours, lasting days or weeks, and above a certain value taking into account the risk that patients may develop severe sepsis and septic shock. In addition, procalcitonin is an important marker for specifically distinguishing inflammatory reactions caused by bacterial infections and other causes, and viral infections, allergies, autoimmune diseases and graft rejection do not cause significant elevation of procalcitonin, whereas local bacterial infections can cause moderate elevation of procalcitonin concentrations. In some cases (neonates, multiple injuries, burns, major surgery, prolonged or severe cardiogenic shock), the elevation of procalcitonin may be irrelevant to the infection, usually returning to normal values soon. The current expert consensus of Procalcitonin (PCT) emergency clinical applications derives from the cut-off value of BRAHMS PCT: PCT concentrations <0.5ng/mL indicate no or low systemic inflammatory response, possibly local inflammation or local infection; PCT concentrations ranging from 0.5ng/mL to 2.0ng/mL indicate a moderate systemic inflammatory response, with the possible presence of infection; PCT concentrations ranging from 2ng/mL to 10ng/mL are likely to be sepsis, severe sepsis or septic shock, with a high risk of organ dysfunction; PCT concentrations of > 10ng/mL indicate almost all severe bacterial sepsis or septic shock, often accompanied by organ failure, with a high risk of mortality. ROC curve research shows that the area under the curve PCT > white blood cell count > C reactive protein > neutrophil percentage, and the sensitivity and specificity of PCT are superior to the indexes of white blood cell count, C reactive protein, neutrophil percentage, etc., and are related to the severity of disease. Therefore, PCT is an ideal index for auxiliary diagnosis of serious bacterial infection, sepsis, septicemia and other diseases, and has high sensitivity and specificity for systemic bacterial infection, sepsis, septicemia and the like.
Shafer in 1984 found and successfully isolated Heparin Binding Protein (HBP) from polymorphonuclear leukocyte granules, a member of the trypsin-like serine protein family. Because HBP has relative molecular weight of 37000, has bactericidal activity and positive charge, and is also called CAP37 and azulene.
HBP has biological effects of killing bacteria, causing inflammation, resisting apoptosis, improving cell penetration and the like, is mainly stored in the azurin granules of neutrophils, and is a part of the innate defense system of human neutrophils. The HBP level in healthy human blood is very low and generally not more than 10 mug/L, when infection occurs, part of bacteria is immersed into blood vessels, and substances such as bacteria and toxins released by the bacteria stimulate neutrophils to release HBP, so that the HBP content in blood is increased. When SIRS (systemic inflammatory response syndrome, systemic inflammatory syndrome) is caused, systemic inflammatory reaction and coagulation dysfunction are caused, polymorphonuclear neutrophils are activated by thalli, toxins, coagulation factor complex and the like, HBP is provided with positive charges, neutrophils are adhered to vascular endothelial cells to release HBP, the HBP can induce vascular leakage and tissue injury, vascular permeability is increased, and uncontrolled vascular permeability is a characteristic of serious SIRS. It can be seen that HBP plays an important role in the development of SIRS.
Interleukin 6 (IL-6) is produced by fibroblasts, monocytes/macrophages, T lymphocytes, B lymphocytes, epithelial cells, keratinocytes, and a variety of tumor cells, has the ability to regulate immune responses, acute phase responses, and hematopoiesis, and plays an important role in the anti-infective immune response of the body. Interleukin-6 (IL-6) is elevated earlier in the inflammatory response than CRP, PCT, etc., and has a long duration, and thus can be used to aid in early diagnosis of acute infections. IL-6 increased rapidly after bacterial infection, PCT increased after 2h, and CRP increased rapidly after 6 h.
Florence C.Riche et al found that the elevated levels of IL-6 were significantly different from inflammatory diseases, bacterial infection resulted in elevated levels of IL-6 significantly higher than non-bacterial infection, and the levels of IL-6 were progressively elevated with increasing inflammation and degree of infection. IL-6 levels are important for assessing disease severity and for judging prognosis (Surgery, 2003, 133 (3): 257-262).
Dong WookJekarl et al found, through studies of the inflammatory index of sepsis patients, that the surviving group IL-6 concentration decreased rapidly after treatment, whereas the dead group IL-6 concentration decreased with a delay, and the kinetics of IL-6 decrease was used to evaluate the prognosis of patients over PCT and CRP; PCT is a better indicator of sepsis than another indicator of sepsis, whereas IL-6 can better assess the prognosis of sepsis patients, reflecting the effect of antibiotic therapy faster (Diagnostic Microbiology and Infectious Disease,2013, 75:342-347).
The time-resolved fluorescence (TRF) analysis technology is a novel non-radioactive micro-analysis technology established by taking lanthanide with unique fluorescence characteristics and a chelating agent thereof as a tracer. Since Pettersson et al in 1983, time-resolved fluorescence immunoassay (TRFIA) was adopted to quantitatively measure hCG (human chorionic gonadotrophin), the development of TRFIA methodology research and clinical application was rapid, and a new milestone for the development of marker immunoassay following RIA was developed, and various TRFIA instruments and matched commercial kits were developed successively at home and abroad. The TRFIA technology has the advantages of high sensitivity, wide standard curve range, simple operation, no radioactive pollution, multiple marks and the like, is widely applied to clinical laboratory diagnosis of tumors, infectious diseases, endocrine diseases, autoimmune diseases and hereditary diseases, and becomes one of common analysis means for biomedical research and clinical ultra-trace biochemical detection. The TRF, the polystyrene microsphere and the nitrocellulose membrane are combined, and can be used in the field of rapid in-vitro diagnosis.
At present, the combined detection method of PCT, IL-6 and HBP is mainly fluorescence immunochromatography. Most of the fluorescence immunochromatography methods use fluorescein or other substances as a light-emitting source, and have a high linear range, but cannot guarantee low-end sensitivity due to interference of background signals.
Although time-resolved fluoroimmunochromatography has been widely used for detection of various items, most of them use immobilization of time-resolved fluorogenic microspheres on a conjugate release pad, which results in great inaccuracy due to non-uniform release of microspheres, failing to meet the high sensitivity requirements. CN201610223300.5 and CN201610223337.8 disclose operations of placing time-resolved fluorescent microspheres in a fluorescent solution, and by adjusting the volume of the fluorescent solution, sensitivity and precision are effectively improved. However, this method requires a high demand for transportation and storage of the reagent, and is complicated to operate, requiring two steps.
In view of the above, there is a strong need for a time-resolved fluorescence immunochromatographic method with high precision, low detection background signal, high sensitivity, and wide linear range in one-step loading when detecting one protein or simultaneously detecting multiple proteins (e.g., simultaneously performing quantitative detection of PCT, IL-6 and HBP).
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of low sensitivity, low accuracy, complex operation, high reagent transportation and storage requirements and the like of the traditional time-resolved fluorescence immunochromatography, and improve a time-resolved fluorescence immunochromatography test strip, a reagent and a kit containing the same, and a preparation method and application thereof, and particularly to a time-resolved fluorescence immunochromatography test strip for rapidly and quantitatively simultaneously detecting PCT, IL-6 and HBP, a reagent and a kit containing the same, and a preparation method and application thereof. The time-resolved fluorescence immunochromatography test strip, the reagent and the kit containing the same and the preparation method thereof can rapidly quantify one antigen or simultaneously rapidly quantify a plurality of antigens (for example, simultaneously detect PCT, IL-6 and HBP), not only can improve the sensitivity and the linear range of detection, but also can realize the operation of one-step sample loading (for example, 80-100 mu l of samples), can greatly improve the precision and the accuracy of the operation, reduce the error of manual operation, and have simpler operation and are beneficial to industrialized production.
In the prior art, when time-resolved fluorescence immunochromatography is performed, time-resolved fluorescence microspheres are mostly fixed on a conjugate release pad, but because of uneven release of the microspheres, the time-resolved fluorescence microspheres have great inaccuracy and cannot meet the requirement of high sensitivity. In addition, the time-resolved fluorescent microspheres are put into the fluorescent solution (namely, the fluorescent microspheres are independently stored in a liquid form independent of the test strip), so that the sensitivity and the precision are effectively improved. However, this method requires a high demand for transportation and storage of the reagent, and is complicated to operate, requiring two steps. The inventor unexpectedly found that, instead of the conventional sample pad and conjugate release pad, a single sample pad is used to directly fix a solution containing time-resolved fluorescent microspheres on a nitrocellulose membrane, so that the one-step operation can be achieved without combining a release pad (also called a combination pad), and meanwhile, the phenomenon of uneven microsphere distribution and uneven release of fluorescent microspheres caused by improper treatment is avoided, and high precision, accuracy, sensitivity and linear range of detection can be ensured.
In order to solve the technical problems, the first aspect of the invention provides a time-resolved fluorescence immunochromatography test strip, which comprises a bottom plate (1), a sample pad (2), a nitrocellulose membrane (3) and a water absorption pad (4), wherein the sample pad (2), the nitrocellulose membrane (3) and the water absorption pad (4) are sequentially connected and fixed on the bottom plate (1) in the horizontal direction,
The nitrocellulose membrane (3) is coated with a fluorescent wire (5), a detection wire and a quality control wire (9), and the fluorescent wire (5) comprises one or more time-resolved fluorescent microspheres marked by antigens or antibodies.
Preferably, the test strip does not include a conjugate pad. The conjugate pad described herein generally refers to a conjugate pad conventionally used in the art to which time-resolved fluorescent microspheres are immobilized, and the time-resolved fluorescent microspheres are directly immobilized on a nitrocellulose membrane in the present invention, so that such a conjugate pad may not be included.
Preferably, the antigen is preferably a protein, such as PCT, IL-6 and/or HBP.
Preferably, the antibody is an antibody targeting a protein, such as PCT, IL-6 and/or HBP.
Preferably, the sample pad is Fusion5.
Preferably, the time-resolved fluorescent microsphere is a time-resolved fluorescent microsphere modified by polystyrene, carboxyl, hydroxyl and/or epoxy groups.
Preferably, the particle size of the time-resolved fluorescence microsphere is 100-500 nm. The current microspheres applied to fluorescence chromatography in the market are all 100-500 nm. If the particle size is higher than 500nm, the microsphere is unstable after being connected with the antibody, precipitation is easy to occur, and the detection linearity and stability of the project are seriously affected; if the particle size is less than 100nm, the antibody connection process becomes complex, the centrifugation time is greatly prolonged, and the detection linearity and stability of the project are seriously affected.
Preferably, the time-resolved fluorescence microsphere is internally filled with chelate of lanthanide; the lanthanide is preferably one or more of europium, samarium, zinc and dysprosium; the time-resolved fluorescence microsphere is preferably a europium particle chelate (europium particle chelate is a fluorescent substance, can detect signals and mainly can adjust each component of a system).
Preferably, the sample pad is a treated sample pad, preferably a sample pad treated with borax borate buffer, preferably at a concentration of 50mM (i.e. mmol/L), preferably at a pH of 7.9-8.1, preferably comprising cleaning antibodies and/or anti-erythrocyte antibodies, preferably at a concentration of 30 μg/ml, preferably at a concentration of 100 μg/ml, more preferably comprising 0.02% casein, 0.02% polyvinylpyrrolidone, 1% trehalose, 30 μg/ml cleaning antibodies (e.g. MARK33 model cleaning antibody available from Roche), 100 μg/ml anti-erythrocyte antibodies and a preservative; the treatment is preferably a step of oven drying at 45 ℃ for 24±1 hour. I.e. the sample pad is a sample pad on which a cleaning antibody and an anti-erythrocyte antibody are immobilized.
Preferably, the working concentration of the fluorescent wire (5) is as follows: 0.05 to 1mg/ml, for example 0.5mg/ml. The inventor finds that the sensitivity lower than 0.05 can not be achieved in the experimental process; above 1 microsphere release effect is poor, and precision is poor.
Preferably, the concentration of the antibody on the detection line and the quality control line (9) is 0.5-2 mg/ml, for example 1mg/ml. The inventor finds that the sensitivity lower than 0.5 is not achieved in the experimental process; higher than 2 does not change significantly, but the cost increases much.
Preferably, the scribing spray amount of the antibody on the detection line and the quality control line (9) is 0.5-2.0 mu l/cm. The inventor finds that the sensitivity lower than 0.5 is not achieved in the experimental process; higher than 2 does not change significantly, but the cost increases much.
Preferably, the fluorescent line (5) comprises time-resolved fluorescent microspheres marked by HBP monoclonal antibody, time-resolved fluorescent microspheres marked by PCT monoclonal antibody, time-resolved fluorescent microspheres marked by IL-6 monoclonal antibody and/or time-resolved fluorescent microspheres marked by antibody (when the antibody on the quality control line is a rabbit source, the antibody is preferably a goat anti-rabbit antibody).
Preferably, the detection line is an antibody detecting the one or more antigens, preferably a detection line made of detection line (6) HBP monoclonal antibody, detection line (7) PCT monoclonal antibody and/or detection line (8) IL-6 monoclonal antibody.
Preferably, the quality control line (9) is a quality control line composed of IgG antibodies (e.g., rabbit IgG antibodies).
Preferably, the monoclonal antibodies in the fluorescent line recognize the same site of antigen as the monoclonal antibodies directed against the same antigen in the detection line.
Preferably, the monoclonal antibodies in the fluorescent line recognize different sites of the antigen than the monoclonal antibodies directed against the same antigen in the detection line. The HBP monoclonal antibody on the fluorescent line and the HBP monoclonal antibody on the detection line preferably recognize different sites of the antigen; the PCT monoclonal antibody on the fluorescent line preferably recognizes a different site of antigen than the PCT monoclonal antibody on the detection line; the IL-6 monoclonal antibody on the fluorescent line preferably recognizes a different site of the antigen than the IL-6 monoclonal antibody on the detection line. The antibodies on the fluorescent lines and the antibodies on the detection lines can react to form a double-antibody sandwich complex. The two antibodies may be paired antibodies, typically based on examining a large number of clinical samples parallel to a reference system, and observing the correlation r. Typically, r values are about 0.95 or greater, indicating that the two antibodies selected perform well.
Preferably, the time-resolved fluorescence immunochromatography test strip is used for simultaneously detecting PCT, IL-6 and HBP.
In a preferred embodiment of the present invention, the step of labeling the time-resolved fluorescent microsphere may include: and (3) reacting the time-resolved fluorescent microsphere with the antigen or the antibody, and performing ultrasound after sealing. Wherein,
preferably, the time-resolved fluorescent microsphere is a time-resolved fluorescent microsphere dissolved in a buffer solution, and each 1mg of time-resolved fluorescent microsphere is preferably dissolved in 100-300 mu L of buffer solution, and the buffer solution is preferably MES buffer solution. The inventors also tried other buffer formulations during the experiment, and overall the signal values were not as good as those of MES.
Preferably, the time-resolved fluorogenic microsphere is an activated time-resolved fluorogenic microsphere, the activation time preferably being 15 minutes; the activator used for the activation is preferably NHS and/or EDC. Wherein the final concentration of EDC is preferably 0.05% to 0.1%. Wherein the final concentration of NHS is preferably 0.1% -0.2%.
Preferably, the time-resolved fluorescence microsphere is a time-resolved fluorescence microsphere connected with glycine; preferably 10-100. Mu.L of 50mM glycine is added per 1mg of time-resolved fluorogenic microspheres; the time-resolved fluorescence microsphere is connected with the glycine and then comprises a step of centrifugation and/or activation; the rotational speed of the centrifugation is preferably 20000-25000rpm; the time of activation is preferably 15 minutes; the activating agent used for the activation is preferably NHS and/or EDC, wherein the final concentration of EDC is preferably 0.05% -0.1%, and the final concentration of NHS is preferably 0.1% -0.2%; the activation preferably comprises a step of secondary centrifugation and reconstitution; the rotation speed of the secondary centrifugation is preferably 20000-25000rpm; the reconstituted reagent is preferably a boric acid buffer, preferably at a pH of 7-9, e.g. 8.5, and preferably at a concentration of 60-100mM. The inventors have found during the course of experiments that the long-term storage stability of fluorescent microspheres is better when using boric acid buffer.
Preferably, the mass ratio of the time-resolved fluorescent microsphere to the antigen or antibody is 10 (0.01-2.0), e.g. 10:0.5. In a preferred embodiment of the invention, the mass ratio of the time-resolved fluorescent microsphere to the antigen or antibody is 10 mg/0.5 mg.
Preferably, the reaction time is 1 to 2 hours.
Preferably, the blocking reagent is BSA.
Preferably, the final concentration of the blocking agent is 2-3%.
Preferably, the closing time is 2h.
Preferably, the sealing step further comprises the steps of re-centrifuging and re-dissolving, wherein the rotational speed of the re-centrifuging is preferably 20000-25000rpm; the reagent for reconstitution is preferably boric acid buffer solution and BSA, wherein the pH value of the boric acid buffer solution is preferably 7-9, and the concentration is preferably 60-100mM; the final concentration of BSA is preferably 2-3%.
Preferably, the final concentration of the time-resolved fluorogenic microspheres after sonication is 0.05-1.0 mg/ml, e.g., 2.5mg/ml stock solution is used.
In a preferred embodiment of the present invention, the step of labeling the time-resolved fluorescent microsphere comprises: 100mg of time-resolved fluorescence microsphere is dissolved in 30ml of MES (2- (N-morpholino) ethanesulfonic acid) buffer with pH of 6.0100mol/L, NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) are added for activation for 15 minutes; the final concentration of EDC was 0.05% (volume ratio), and the final concentration of NHS was 0.1%; 10ml of 50mM glycine was added to allow the surface of the time-resolved fluorogenic microspheres to attach to the arm, centrifuged at 25000rpm, and activated by the addition of NHS and EDC for 15 min; EDC at 0.05% and NHS at 0.1% and centrifuging at 25000rpm, re-dissolving time-resolved fluorescent microsphere with 50ml boric acid buffer with pH of 8.5 60mol/L, adding antigen or antibody, and reacting for 2 hours, wherein the mass ratio of microsphere to antigen or antibody is 10mg:0.5mg; adding BSA to carry out blocking for 2 hours after the reaction is finished; after blocking, the final concentration of BSA is 2%, and after centrifugation at 25000rpm, re-dissolving and ultrasonic treatment are carried out again by using boric acid buffer with pH of 8.5 and 60mol/L and BSA, so that the microspheres are uniformly dispersed in the buffer, and the final concentration of BSA is 2%; the final concentration of the obtained time-resolved fluorescence microsphere is preferably 0.05-1.0 mg/ml. The final microspheres can be uniformly dispersed in a buffer solution and can be stored at 2-8 ℃ in a dark place.
In order to solve the above technical problems, a second aspect of the present invention provides a reagent and/or a kit for time-resolved fluorescence immunochromatography, which comprises the time-resolved fluorescence immunochromatography test strip according to the first aspect of the present invention.
Preferably, the reagent and/or kit does not include a fluorescent liquid. The fluorescent solution described herein generally refers to a fluorescent solution used for storing time-resolved fluorescent microspheres in the prior art, and the time-resolved fluorescent microspheres are directly immobilized on a nitrocellulose membrane in the present invention, so that such a fluorescent solution may not be included.
Preferably, the reagent and/or kit further comprises a reagent for treating the sample pad, preferably a borax borate buffer solution, the borax borate buffer solution preferably has a concentration of 50mM, the pH is preferably 7.9-8.1, the borax borate buffer solution preferably comprises a cleaning antibody and/or an anti-erythrocyte antibody, the cleaning antibody preferably has a concentration of 30 μg/ml, the anti-erythrocyte antibody preferably has a concentration of 100 μg/ml, and the borax borate buffer solution more preferably comprises 0.02% casein, 0.02% polyvinylpyrrolidone, 1% trehalose, 30 μg/ml MARK33 cleaning antibody, 100 μg/ml anti-erythrocyte antibody and a preservative. I.e. the sample pad is a sample pad on which a cleaning antibody and an anti-erythrocyte antibody are immobilized.
In order to solve the technical problem, the third aspect of the present invention provides a method for preparing the time-resolved fluorescence immunochromatography test strip according to the first aspect of the present invention, which comprises the following steps: scribing the diluted fluorescent microspheres on the nitrocellulose membrane (3) to form the fluorescent line (5), scribing the diluted antibodies on the nitrocellulose membrane (3) to form the detection line (6), and scribing the diluted rabbit IgG antibodies on the nitrocellulose membrane (3) to form the quality control line (9); drying the obtained nitrocellulose membrane; the sample pad (2), the nitrocellulose membrane (3) and the absorbent paper (4) are adhered to the bottom plate (1) from left to right in sequence.
Preferably, the diluted diluent is citrate and/or sucrose diluent; the citrate or sucrose in the dilute citrate or sucrose solution preferably comprises 5-20%, for example 10%. The inventor finds that the diluent can ensure that the reagent has better stability and higher signal value in the experimental process; other buffers may reach comparable signal values, but have poor stability.
Preferably, the temperature of the drying is 37-45 ℃.
Preferably, the drying time is 24-48 hours.
Preferably, the drying is performed in a vacuum drying oven.
Preferably, when the detection line contains a plurality of antibodies, the plurality of antibodies are located at the same position or at different positions, and when the plurality of antibodies are located at different positions, the plurality of antibodies are located at the same side of the quality control line (9), preferably at the side where the sample to be detected is contacted first.
Preferably, the resulting test strip is cut into small strips.
In a preferred embodiment of the present invention, the preparation method of the inter-resolution fluorescence immunochromatographic test strip (for example, the preparation method of the time-resolution fluorescence immunochromatographic reagent for rapid quantitative simultaneous detection of PCT, IL-6 and HBP) is as follows:
(1) Preparation of a fluorescent line solution: respectively diluting HBP monoclonal antibody 1 (for example, 1E2 available from Zhuhai Bome Biotechnology Co., ltd.), PCT monoclonal antibody 1 (for example, MJG03 available from Hangzhou Kogyo Biotechnology Co., ltd., model B052), IL-6 monoclonal antibody 1 (for example, 2706 available from Medix, model c 35389) and goat anti-rabbit antibody to 0.5mg/ml with 10% (by volume) citrate solution;
(2) Preparation of detection line (T1, T2, T3) solutions: PCT monoclonal antibody 2 (e.g., MJG05, model B052, available from Kitag Biotechnology Co., ltd., hangzhou), IL-6 monoclonal antibody 2 (CSB-DA 436EmN 2, available from Wuhan Huamei Biotechnology Co., ltd.), HBP monoclonal antibody 2 (5B 6, available from Zhuhai Bomei Biotechnology Co., ltd.) were diluted to 1mg/ml, respectively;
(3) Preparation of a quality control line (C) solution: the rabbit IgG antibody was diluted to 1mg/ml with 10% citrate solution;
the base plate (1) with the back adhesive is lapped, the nitrocellulose membrane (3) is stuck first, and then the sample pad (2) and the absorbent paper (4) are stuck at two ends of the nitrocellulose membrane (3) respectively. A nitrocellulose membrane (3) and a Fusion5 membrane (2) were marked with a fluorescent line (5), a T1 (HBP capture line (6)), a T2 (PCT capture line (7)), a T3 (IL-6 capture line (8)), and a C (rabbit IgG) line was marked with a C (rabbit IgG) line at one end of the absorbent paper (4), and the pitches of T1, T2, T3 and C were all 3mm.
At the T line, T1, T2, T3 are marked, respectively, and at the C line, C line is marked. The final concentration of T1, T2 and T3 line antibodies was 1mg/ml, the final concentration of C line rabbit IgG was 1mg/ml, and the spray amount of C, T lines was 1.0. Mu.l/cm.
The sprayed big card is placed in a constant temperature oven at 37 ℃ to be baked for 24 hours. After baking, cutting the test paper strip with the width of 3.85mm by a slitter in a room with the humidity of less than 40%, putting the test paper strip into a plastic shell for compaction, putting a bag of drying agent into the plastic shell, sealing the plastic shell, and storing the plastic shell in a drying cabinet for later use.
In order to solve the above technical problems, a fourth aspect of the present invention provides a method for labeling time-resolved fluorescent microspheres, which comprises the steps of reacting time-resolved fluorescent microspheres with the antigen or antibody, and performing ultrasound after blocking.
Preferably, the time-resolved fluorescent microsphere is a time-resolved fluorescent microsphere dissolved in a buffer solution, and each 1mg of time-resolved fluorescent microsphere is preferably dissolved in 100-300 mu L of buffer solution, and the buffer solution is preferably MES buffer solution. The inventors also tried other buffer formulations during the experiment, and overall the signal values were not as good as those of MES.
Preferably, the time-resolved fluorogenic microsphere is an activated time-resolved fluorogenic microsphere, the activation time preferably being 15 minutes; the activator used for the activation is preferably NHS and/or EDC, wherein the final concentration of EDC is preferably 0.05% -0.1% and the final concentration of NHS is preferably 0.1% -0.2%.
Preferably, the time-resolved fluorescence microsphere is a time-resolved fluorescence microsphere connected with glycine; preferably 10-100. Mu.L of 50mM glycine is added per 1mg of time-resolved fluorogenic microspheres; the time-resolved fluorescence microsphere is connected with the glycine and then comprises a step of centrifugation and/or activation; the rotational speed of the centrifugation is preferably 20000-25000rpm; the time of activation is preferably 15 minutes; the activating agent used for the activation is preferably NHS and/or EDC, wherein the final concentration of EDC is preferably 0.05% -0.1%, and the final concentration of NHS is preferably 0.1% -0.2%; the activation preferably comprises a step of secondary centrifugation and reconstitution; the rotation speed of the secondary centrifugation is preferably 20000-25000rpm; the reconstituted reagent is preferably a boric acid buffer, preferably at a pH of 7-9, e.g. 8.5, and preferably at a concentration of 60-100mM.
Preferably, the mass ratio of the time-resolved fluorescent microsphere to the antigen or antibody is 10 (0.01-2.0), e.g. 10:0.5.
Preferably, the reaction time is 1 to 2 hours.
Preferably, the blocking reagent is BSA.
Preferably, the final concentration of the blocking agent is 2-3%.
Preferably, the closing time is 2h.
Preferably, the sealing step further comprises the steps of re-centrifuging and re-dissolving, wherein the rotational speed of the re-centrifuging is preferably 20000-25000rpm; the reagent for reconstitution is preferably boric acid buffer solution and BSA, wherein the pH value of the boric acid buffer solution is preferably 7-9, and the concentration is preferably 60-100mM; the final concentration of BSA is preferably 2-3%.
Preferably, the final concentration of the time-resolved fluorescent microsphere after sonication is 0.05-1.0 mg/ml (e.g. using a stock solution concentration of 2.5 mg/ml).
In a preferred embodiment of the present invention, the step of labeling the time-resolved fluorescent microsphere comprises: 100mg of time-resolved fluorescence microsphere is dissolved in 30ml of MES (2- (N-morpholino) ethanesulfonic acid) buffer with pH of 6.0100mol/L, NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) are added for activation for 15 minutes; the final concentration of EDC was 0.05% (volume ratio), and the final concentration of NHS was 0.1%; 10ml of 50mM glycine was added to allow the surface of the time-resolved fluorogenic microspheres to attach to the arm, centrifuged at 25000rpm, and activated by the addition of NHS and EDC for 15 min; EDC at 0.05% and NHS at 0.1% and centrifuging at 25000rpm, re-dissolving time-resolved fluorescent microsphere with 50ml boric acid buffer with pH of 8.5 60mol/L, adding antigen or antibody, and reacting for 2 hours, wherein the mass ratio of microsphere to antigen or antibody is 10mg:0.5mg; adding BSA to carry out blocking for 2 hours after the reaction is finished; after blocking, the final concentration of BSA is 2%, and after centrifugation at 25000rpm, re-dissolving and ultrasonic treatment are carried out again by using boric acid buffer with pH of 8.5 and 60mol/L and BSA, so that the microspheres are uniformly dispersed in the buffer, and the final concentration of BSA is 2%; the final concentration of the time-resolved fluorogenic microspheres obtained is preferably 0.05-1.0 mg/ml, enabling uniform dispersion of the microspheres in the buffer. The obtained microsphere can be stored at 2-8deg.C in dark place. And aminocaproic acid is added as an arm in the fluorescent particle activation process, so that the distance between the antibody and the microsphere is increased, the steric hindrance effect is effectively reduced, and the sensitivity of a detection system is improved.
Preferably, the time-resolved fluorescence microsphere filled with europium particle chelate has the particle size of: 100-500 nm.
Preferably, the mass ratio of the microsphere to the antibody is: 10mg to 0.01mg to 2.0mg.
Preferably, the working concentration of the fluorescent wire (5) is: 0.05-1 mg/ml.
In order to solve the above technical problems, a fifth aspect of the present invention provides a method for detecting an antigen or an antibody for non-diagnostic purposes, wherein the detection is performed using the time-resolved fluorescence immunochromatographic test strip according to the first aspect of the present invention, the reagent and/or the kit according to the second aspect of the present invention; the antigen is preferably a protein, for example PCT, IL-6 and/or HBP.
Preferably, the method (e.g. a detection method of a time-resolved fluorescence immunochromatographic strip for rapid quantitative simultaneous detection of PCT, IL-6, HBP) comprises the steps of:
(1) Drawing a calibration curve: taking 80 μl of PCT, IL-6 and HBP mixed calibrator, loading sample, reacting for 15-20 minutes, reading system detection signals 1, 2 and 3 by a time-resolved immunochromatography quantitative analyzer matched with a test strip, and fitting to prepare a calibrator curve by taking each concentration of the calibrator as an abscissa and the average value of the detection signals of each concentration system as an ordinate;
(2) And (3) detecting the sample to be detected, namely respectively taking 80 μl of the sample to be detected for loading, reacting for 15-20 minutes, and obtaining the concentration values of PCT, IL-6 and HBP in the sample to be detected through the value of the system detection signal.
In the above (1), the concentrations of PCT, IL-6, HBP mixed calibrator are preferably:
PCT：0.0、0.01、0.05、0.1、0.5、2.0、5.0、10.0、25.0、50.0、100.0ng/ml；
IL-6：0.0、1.0、5.0、10.0、50.0、100.0、500.0、1000.0、4000.0、5000.0pg/ml；
HBP：0.0、5.0、10.0、25.0、50.0、100.0、200.0、250.0ng/ml。
in order to solve the technical problem, the sixth aspect of the present invention provides a time-resolved fluorescence immunochromatography test strip according to the first aspect of the present invention, and an application of a reagent and/or a kit according to the second aspect of the present invention in detecting an antigen or an antibody. Wherein the detection is typically a detection for non-diagnostic purposes, e.g. for detection in a research and development process. Wherein the antigen is preferably a protein, such as PCT, IL-6 and/or HBP; the antibodies are antibodies targeting proteins such as PCT, IL-6 and/or HBP.
In the invention, the sample pad (also called as a loading pad) has the function of filtering impurities in a sample and ensuring uniform chromatography of the sample. The sample pad may typically be a sample pad treated with a treatment fluid.
In a preferred embodiment of the invention, the method for preparing the time-resolved fluorescence immunochromatographic reagent for rapid quantitative simultaneous detection of PCT, IL-6 and HBP comprises the following steps:
(1) Fluorescent line (5) is obtained by diluting fluorescent microspheres labeled with HBP monoclonal antibody 1 (for example, 1E2 available from Zhuhai Bome Biotechnology Co., ltd.), PCT monoclonal antibody 1 (for example, MJG03 available from Hangzhou Kotaki Biotechnology Co., ltd., model B052), IL-6 monoclonal antibody 1 (for example, 2706 available from Medix, model c 35389) and goat anti-rabbit antibody with a citrate and sucrose diluent, and streaking on the nitrocellulose membrane (3). The detection line (6) is formed by diluting an HBP monoclonal antibody 2 (5B 6 purchased from Zhuhai Bomei Biotechnology Co., ltd.) with a citrate and sucrose diluent and streaking on the nitrocellulose membrane (3); the detection line (7) is obtained by diluting PCT monoclonal antibody 2 (for example MJG05 available from the company, model B052, of the company, opening biotechnology, hangzhou) with citrate and sucrose diluent, and streaking on the nitrocellulose membrane (3); the detection line (8) is formed by diluting an IL-6 monoclonal antibody 2 (CSB-DA 436EmN from Wuhan Huamei organism) with a citric acid buffer solution and a sucrose solution, and streaking on the nitrocellulose membrane (3); the quality control line (9) is formed by diluting a rabbit IgG antibody by using citrate and sucrose diluent, and marking the line on the nitrocellulose membrane (3); placing the scratched nitrocellulose membrane in a vacuum drying oven, and drying for 24-48 hours at 37-45 ℃; the detection lines (6), (7) and (8) are positioned at the same position or at different positions, and if the detection lines are positioned at different positions, the detection lines (6), (7) and (8) are positioned at the same side of the quality control line (9), namely the position where liquid is contacted first;
(2) Assembling a test strip: a sample pad (2), a nitrocellulose membrane (3) and water absorbing paper (4) are adhered to a test strip bottom plate (1) from left to right in sequence; and cutting the assembled large plate into small strips to obtain the time-resolved fluorescence immunochromatographic test strip for rapidly quantifying and simultaneously detecting PCT, IL-6 and HBP.
Preferably, the citrate or sucrose in the citrate or sucrose diluent is 5-20%.
Preferentially, the working concentration of the fluorescent line (5) is as follows: 0.05-1 mg/ml.
Preferably, the scribing concentration of the detection line and the quality control line antibody is 0.5-2 mg/ml.
Preferably, the scribing spray amount of the detection line and the quality control line antibody is 0.5-2.0 mu l/cm.
In the present invention, the nitrocellulose membrane may be commercially available, for example, from Sidoris under the model CN 140.
In the present invention, the sample pad may be commercially available, for example, as sample pad model JY-Y125 available from Shanghai Jie-A organism.
In the present invention, the absorbent pad may be commercially available, for example, from Shanghai Jie-Biotechnology under the model H5076.
In the present invention, the base plate may be commercially available, for example, from Shanghai Jie-Biotechnology under the model DB-4.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the time-resolved fluorescence immunochromatography test strip, the reagent and the kit containing the same and the preparation method thereof can rapidly quantify one antigen or simultaneously rapidly quantify a plurality of antigens (for example, simultaneously detect PCT, IL-6 and HBP), not only can improve the sensitivity and the linear range of detection, but also can realize the operation of one-step sample loading (for example, 80-100 mu l of samples), can greatly improve the precision and the accuracy of the operation, reduce the error of manual operation, and have simpler operation and are beneficial to industrialized production. In a preferred embodiment of the invention, the precision is less than 10% when PCT, IL-6 and HBP are detected simultaneously. In a preferred embodiment of the invention, the sensitivity of detecting HBP can reach 5ng/ml, and the upper limit of detection can reach 250ng/ml; the sensitivity of detection PCT can reach 0.01ng/ml, and the upper limit of detection can reach 100ng/ml; the sensitivity of the I detection L-6 can reach 1.0pg/ml, and the upper limit of the detection can reach 500pg/ml.
Drawings
FIG. 1 is a schematic diagram of a time-resolved fluorescence immunochromatographic test strip for rapid quantitative simultaneous detection of PCT, IL-6 and HBP (1 is a bottom plate, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is a water absorbing pad, 5 is a fluorescence line, 6 is a HBP detection line, 7 is a PCT detection line, 8 is an IL-6 detection line, and 9 is a rabbit IgG quality control line).
Figure 2 is a calibrator curve for HBP of the present invention.
FIG. 3 is a calibration curve of PCT of the present invention.
FIG. 4 is a calibrator curve for IL-6 of the present invention.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 preparation of PCT, IL-6, HBP, goat anti-Rabbit time-resolved fluorescence microspheres
100mg of time-resolved fluorescence microsphere (particle size of about 200 nm) is dissolved in 30ml of MES (2- (N-morpholinoethanesulfonic acid) buffer solution with pH of 6.0100mol/L, NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) are added for activation for 15 minutes; the final concentration of EDC was 0.05% (volume ratio), and the final concentration of NHS was 0.1%; 10ml of 50mM glycine was added to allow the microspheres to attach to the arm, centrifuged at 25000rpm, and activated by adding NHS and EDC again for 15 minutes; EDC at 0.05% and NHS at 0.1% and centrifugation at 25000rpm, re-dissolving time-resolved fluorescent microsphere with 50ml boric acid buffer with pH of 8.5 60mol/L, and adding PCT monoclonal antibody 1 to react for 2 hours (the mass ratio of microsphere to antibody is 10mg:0.5 mg); adding BSA to carry out blocking for 2 hours after the reaction is finished; after blocking, the final concentration of BSA was 2%, centrifuged at 25000rpm, re-dissolved again with boric acid buffer at pH 8.5 at 60mol/L and BSA, sonicated to uniformly disperse the microspheres in the buffer, the final concentration of BSA was 2%, and stored at 4℃in the dark. The final concentration of the PCT monoclonal antibody 1 labeled time-resolved fluorescence microsphere is 0.05-1.0 mg/ml (the concentration of the stock solution used is 2.5 mg/ml), so that the microsphere is uniformly dispersed in a buffer solution, and the microsphere is preserved at 2-8 ℃ in a dark place.
The preparation process of the fluorescent microsphere marked by the HBP monoclonal antibody 1, the preparation of the fluorescent microsphere marked by the IL-6 monoclonal antibody 1 and the preparation process of the fluorescent microsphere marked by the goat anti-rabbit antibody are the same as the process of the PCT preparation of the microsphere.
Example 2 sample pad treatment
(1) The solution was prepared with 50mM borax borate buffer (pH 7.9-8.1) containing 0.02% casein, 0.02% polyvinylpyrrolidone, 1% trehalose, 30. Mu.g/ml MARK33 detergent antibody, 100. Mu.g/ml anti-erythrocyte antibody and preservative.
(2) The sample pad is soaked in the buffer solution in the step (1), and then is taken out and put into a 45 ℃ oven for drying for 24+/-1 hour. Then adding a drying agent, sealing and preserving for later use.
Example 3 preparation of PCT, IL-6 and HBP time-resolved fluorescence immunochromatographic test strip
(1) Preparation of a fluorescent line solution: respectively diluting the HBP monoclonal antibody 1-labeled fluorescent microsphere, the PCT monoclonal antibody 1-labeled fluorescent microsphere, the IL-6 monoclonal antibody 1-labeled fluorescent microsphere and the goat anti-rabbit antibody-labeled fluorescent microsphere to 0.5mg/ml by using 10% (volume ratio) citrate solution;
(2) Preparation of detection line (T1, T2, T3) solutions: PCT monoclonal antibody 2, IL-6 monoclonal antibody 2, HBP monoclonal antibody 2 to 1mg/ml were diluted with 10% citrate solution, respectively;
(3) Preparation of a quality control line (C) solution: the rabbit IgG antibody was diluted to 1mg/ml with 10% citrate solution;
and (3) sticking a nitrocellulose membrane (3) on a base plate (1) with back adhesive in a lap joint mode, and then sticking a sample pad (2) and absorbent paper (4) obtained in the embodiment 2 on two ends of the nitrocellulose membrane (3) respectively. The nitrocellulose membrane (3) and the sample pad (Fusion 5 membrane) (2) were marked with a fluorescent line (5), a T1 (HBP capture line (6)), a T2 (PCT capture line (7)), a T3 (IL-6 capture line (8)), and the absorbent paper (4) was marked with a C (rabbit IgG) line at one end thereof, and the pitches of T1, T2, T3 and C were all 3mm.
At the T line, T1, T2, T3 are marked, respectively, and at the C line, C line is marked. The final concentration of T1, T2 and T3 line antibodies was 1mg/ml, the final concentration of C line rabbit IgG was 1mg/ml, and the spray amount of C, T lines was 1.0. Mu.l/cm.
The sprayed big card is placed in a constant temperature oven at 37 ℃ to be baked for 24 hours. After baking, cutting the test paper strip with the width of 3.85mm by a slitter in a room with the humidity of less than 40%, putting the test paper strip into a plastic shell for compaction, putting a bag of drying agent into the plastic shell, sealing the plastic shell, and storing the plastic shell in a drying cabinet for later use. As particularly shown in fig. 1.
Example 4
(1) Drawing of calibration curves
PCT, IL-6, HBP calibrator were diluted with calf serum at the following concentrations:
PCT：0.0、0.01、0.05、0.1、0.5、2.0、5.0、10.0、25.0、50.0、100.0ng/ml；
IL-6：0.0、1.0、5.0、10.0、50.0、100.0、500.0、1000.0、4000.0、5000.0pg/ml；
HBP：0.0、5.0、10.0、25.0、50.0、100.0、200.0、250.0ng/ml。
And respectively taking 80 μl of calibrator, dripping onto the PCT, IL-6 and HBP time-resolved fluorescence immunochromatography test strips in the embodiment 1, reacting for 15 minutes, and respectively reading the signal values of the system detection signal 1, the system detection signal 2 and the system detection signal 3 by an immunochromatography reader matched with the PCT, IL-6 and HBP time-resolved fluorescence immunochromatography test strips, wherein the concentration detection of each calibrator is three times, and the specific values are shown in the table 1.
Table 1 calibrator test values
As can be seen from the data in Table 1, the sensitivity of HBP can reach 5ng/ml, and the upper limit of detection can reach 250ng/ml; the sensitivity of PCT can reach 0.01ng/ml, and the upper limit of detection can reach 100ng/ml; the sensitivity of IL-6 can reach 1.0pg/ml, and the upper limit of detection can reach 500pg/ml.
The combined detection of PCT, IL-6 and HBP has high sensitivity and wide linear range, and shows the advantages of time-resolved fluorescence immunochromatography.
(2) Manufacturing an ID card:
and (3) respectively taking the concentration value of the calibrator HBP, PCT, IL-6 as an abscissa and the T/C average value of each concentration as an ordinate, drawing a calibrator curve, specifically, burning an ID card as shown in figures 2, 3 and 4, and introducing the calibration curve into a matched instrument.
EXAMPLE 5 precision detection
Using the test method in example 2, mixed calibrators of HBP, PCT and IL-6 (HBP concentrations of 10 and 100ng/ml, PCT concentrations of 0.5 and 50ng/ml, IL-6 concentrations of 10 and 1000 pg/ml) were tested, respectively, and each calibrator was repeatedly tested ten times, with specific test values shown in Table 2 below.
Table 2 table of results of precision test
As can be seen from the data in Table 2, the precision of HBP, PCT, IL-6 by adopting the triple detection test strip of the invention is less than 10%, and the requirements of POCT products on precision less than 15% are completely met.

Claims (10)

1. A time-resolved fluorescence immunochromatography test strip is characterized by comprising a bottom plate (1), a sample pad (2), a nitrocellulose membrane (3) and a water absorption pad (4), wherein the sample pad (2), the nitrocellulose membrane (3) and the water absorption pad (4) are sequentially connected and fixed on the bottom plate (1) in the horizontal direction,
wherein, the nitrocellulose membrane (3) is coated with a fluorescent wire (5), a detection wire and a quality control wire (9),
the fluorescent line (5) comprises time-resolved fluorescent microspheres marked by HBP monoclonal antibodies, time-resolved fluorescent microspheres marked by PCT monoclonal antibodies and time-resolved fluorescent microspheres marked by IL-6 monoclonal antibodies;
the detection lines are three, and comprise an HBP monoclonal antibody detection line (6), a PCT monoclonal antibody detection line (7) and an IL-6 monoclonal antibody detection line (8);
the time-resolved fluorescence immunochromatography test strip does not comprise a binding pad;
The sample pad is a sample pad treated by borax borate buffer solution;
the concentration of the borax borate buffer solution is 50 mM, the pH is 7.9-8.1, and the borax borate buffer solution comprises 0.02% casein, 0.02% polyvinylpyrrolidone, 1% trehalose, 30 mug/ml MARK33 cleaning antibody, 100 mug/ml anti-erythrocyte antibody and preservative;
the particle size of the time-resolved fluorescence microsphere is 100-500 nm; the working concentration of the fluorescent wire (5) is as follows: 0.05-1 mg/ml; the film-dividing spraying amount of the antibody on the detection line and the quality control line (9) is 0.5-2.0 mu l/cm; the monoclonal antibody in the fluorescent line and the monoclonal antibody aiming at the same antigen in the detection line recognize different sites of the antigen; the time-resolved fluorescence microsphere is a time-resolved fluorescence microsphere dissolved in buffer solution, and each 1-mg time-resolved fluorescence microsphere is dissolved in 100-300 mu L MES buffer solution.
2. The time-resolved fluorescence immunochromatographic test strip as defined in claim 1, in which,
the sample pad is Fusion5; the time-resolved fluorescence microsphere is modified by polystyrene, carboxyl, hydroxyl and epoxy groups;
The time-resolved fluorescence microsphere is a europium particle chelate filled time-resolved fluorescence microsphere;
the treatment is a step of drying in a 45 ℃ oven for 24+/-1 hour;
the membrane dividing concentration of the antibody on the detection line and the quality control line (9) is 0.5-2 mg/ml.
3. The time-resolved fluoroimmunochromatographic test strip according to claim 1, wherein the quality control line (9) is a quality control line composed of rabbit IgG antibodies.
4. A time-resolved fluoroimmunochromatographic test strip as defined in any one of claims 1 to 3, in which the step of labeling the time-resolved fluorogenic microspheres comprises: reacting the time-resolved fluorescent microsphere with an antibody, and performing ultrasound after sealing;
the time-resolved fluorescence microsphere is connected with glycine; adding 10-100 mu L of glycine of 50-mM into each 1-mg time-resolved fluorescence microsphere; the time-resolved fluorescence microsphere is connected with the glycine and then comprises the steps of centrifugation and activation; the rotational speed of the centrifugation is 20000-25000 rpm; the activation time is 15 minutes; the activating agent used for activating is NHS and EDC, wherein the final concentration of EDC is 0.05% -0.1%, and the final concentration of NHS is 0.1% -0.2%; the activated method also comprises the steps of secondary centrifugation and redissolution;
The mass ratio of the time-resolved fluorescence microsphere to the antibody is 10 (0.01-2.0);
the reaction time is 1-2 hours;
the blocking reagent is BSA;
the final concentration of the blocking reagent is 2-3%;
the closing time is 2 h;
the sealing process also comprises the steps of centrifugation and redissolution.
5. The time-resolved fluoroimmunochromatographic strip according to claim 4, in which the step of labeling the time-resolved fluorogenic microsphere comprises: 100 mg time-resolved fluorescence microsphere is dissolved in 30 ml MES buffer with pH of 6.0 and 100 mol/L, and NHS and EDC are added for activation for 15 minutes; the final concentration of EDC was 0.05% and that of NHS was 0.1%; adding 10 ml of 50 mM glycine to enable the surface of the time-resolved fluorescence microsphere to be connected with an arm, centrifuging at 25000 rpm, adding NHS and EDC again to activate for 15 minutes; EDC at 0.05% and NHS at 0.1% and centrifuging at 25000 rpm, re-dissolving time-resolved fluorescent microsphere with 50 ml boric acid buffer with pH of 8.5 mol/L and adding the antibody for reaction for 2 hours, wherein the mass ratio of microsphere to antibody is 10 mg:0.5 mg; adding BSA to carry out blocking for 2 hours after the reaction is finished; after blocking, the final concentration of BSA is 2%, the BSA is centrifuged at 25000 rpm, and re-dissolved and sonicated by boric acid buffer with pH of 8.5 and 60 mol/L and BSA to uniformly disperse the microspheres in the buffer, wherein the final concentration of BSA is 2%; the final concentration of the obtained time-resolved fluorescence microsphere is 0.05-1.0 mg/ml.
6. A kit for time-resolved fluorescence immunochromatography, comprising the time-resolved fluorescence immunochromatography test strip according to any one of claims 1 to 5;
the kit does not comprise fluorescent liquid.
7. The method for preparing the time-resolved fluorescence immunochromatographic test strip according to any one of claims 1 to 5, which comprises the following steps: scribing the diluted fluorescent microspheres on the nitrocellulose membrane (3) to form the fluorescent line (5), scribing the diluted antibodies on the nitrocellulose membrane (3) to form the detection line, and scribing the diluted rabbit IgG antibodies on the nitrocellulose membrane (3) to form the quality control line (9); drying the obtained nitrocellulose membrane; the sample pad (2), the nitrocellulose membrane (3) and the water absorption pad (4) are adhered to the bottom plate (1) from left to right in sequence.
8. The method according to claim 7, wherein,
the diluted diluent is citrate diluent; citrate accounts for 5-20% in the diluted citrate diluent;
the temperature of the drying is 37-45 ℃;
The drying time is 24-48 hours;
the drying is carried out in a vacuum drying oven;
when the detection line contains a plurality of antibodies, the plurality of antibodies are positioned at the same position or at different positions, and when the antibodies are positioned at different positions, the plurality of antibodies are positioned at the same side of the quality control line (9) and at the side contacted with the sample to be detected first;
the resulting test strips were cut into small strips.
9. A method for detecting an antigen for non-diagnostic purposes, characterized in that the detection is performed using the time-resolved fluorescence immunochromatographic strip according to any one of claims 1 to 5 or the kit according to claim 6; the antigen is PCT, IL-6 and HBP.
10. Use of a time resolved fluorescence immunochromatographic test strip according to any one of claims 1 to 5 or a kit according to claim 6 for the preparation of a product for detecting an antigen, wherein the antigen is PCT, IL-6 or HBP.
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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112143484B (en) * 2020-09-24 2022-10-21 武汉生之源生物科技股份有限公司 Fluorescent microsphere activator redissolution and application thereof
CN112269024A (en) * 2020-10-15 2021-01-26 上海捷门生物技术有限公司 Detection reagent, test paper and kit for interleukin-6 and application thereof
CN112946259A (en) * 2021-02-02 2021-06-11 瑞莱生物科技江苏有限公司 Procalcitonin, interleukin 6 and heparin binding protein combined detection kit
CN113484525A (en) * 2021-07-05 2021-10-08 海南医学院 Heparin Binding Protein (HBP) time-resolved fluorescence immunochromatography semi-quantitative detection test strip
CN114062683A (en) * 2021-11-15 2022-02-18 湖南永和阳光生物科技股份有限公司 Test strip and kit for quantitatively detecting GDF-15
CN114324898B (en) * 2022-03-11 2022-05-31 南京岚煜生物科技有限公司 Binding pad treatment solution for heparin binding protein HBP detection

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106771253A (en) * 2017-01-17 2017-05-31 安徽同致生物工程股份有限公司 Heparin-binding protein determines kit
CN106872420A (en) * 2016-12-27 2017-06-20 厦门奥德生物科技有限公司 The kit and method of a kind of time-resolved fluorescence quantitative determination microdose urine protein
CN107167597A (en) * 2017-07-18 2017-09-15 深圳市惠安生物科技有限公司 Quantitatively detection SAA, CRP, PCT immunofluorescence chromatographs kit and preparation method thereof
CN108132347A (en) * 2018-02-09 2018-06-08 河南省生物工程技术研究中心有限公司 The time-resolved fluoroimmunoassay chromatograph test strip and kit of joint-detection CA19-9 and CEA
CN109975557A (en) * 2019-04-29 2019-07-05 厦门稀土材料研究所 IL-6/PCT joint-detection time resolution detection kit and method

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO337444B1 (en) * 2014-06-19 2016-04-11 Spinchip Diagnostics As Analysis Method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872420A (en) * 2016-12-27 2017-06-20 厦门奥德生物科技有限公司 The kit and method of a kind of time-resolved fluorescence quantitative determination microdose urine protein
CN106771253A (en) * 2017-01-17 2017-05-31 安徽同致生物工程股份有限公司 Heparin-binding protein determines kit
CN107167597A (en) * 2017-07-18 2017-09-15 深圳市惠安生物科技有限公司 Quantitatively detection SAA, CRP, PCT immunofluorescence chromatographs kit and preparation method thereof
CN108132347A (en) * 2018-02-09 2018-06-08 河南省生物工程技术研究中心有限公司 The time-resolved fluoroimmunoassay chromatograph test strip and kit of joint-detection CA19-9 and CEA
CN109975557A (en) * 2019-04-29 2019-07-05 厦门稀土材料研究所 IL-6/PCT joint-detection time resolution detection kit and method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Sustained increase in angiopoietin-2, heparin-binding protein, and procalcitonin is associated with severe sepsis;Xiao-Wei Liu 等;《Journal of Critical Care》;20180630;第45卷;第14-19页 *
外周血HBP,IL-6,CD64指数，PCT,CRP和SAA水平检测在血流感染诊断中的应用价值研究;任继欣 等;《现代检验医学杂志》;20200331;第35卷(第2期);第56-59页 *

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