CN115389760B

CN115389760B - Detection reagent for immunoassay test strip

Info

Publication number: CN115389760B
Application number: CN202211327642.3A
Authority: CN
Inventors: 郑敬仙; 李森渊; 毛丽敏
Original assignee: Acon Biotech Hangzhou Co Ltd
Current assignee: Acon Biotech Hangzhou Co Ltd
Priority date: 2022-10-27
Filing date: 2022-10-27
Publication date: 2023-04-18
Anticipated expiration: 2042-10-27
Also published as: CN115389760A

Abstract

The present invention provides a detection reagent for an immunoassay test strip, comprising a labeling reagent comprising a first signal label-labeled reagent and a second signal label-labeled third reagent, the first reagent being capable of specifically binding to an analyte, and a capture reagent capable of capturing the first signal label-labeled reagent, the capture reagent being capable of capturing the second signal label-labeled third reagent. The detection reagent can be applied to the immunodetection fields of chemiluminescence immunity and the like so as to qualitatively and quantitatively analyze physiological indexes such as inflammatory markers, tumor markers, bone metabolism series markers, sex hormone markers, alpha function markers, infectious disease markers, hepatic fibrosis markers, diabetes markers, intestinal tract health series markers, allergy markers, drug abuse and the like.

Description

Detection reagent for immunoassay test strip

Technical Field

The invention belongs to the technical field of biological detection, and relates to a detection reagent for an immunoassay test strip.

Background

In recent years, immunological rapid diagnostic techniques and detection methods have been rapidly developed and widely used in a series of fields such as medical examination, pharmaceutical analysis, environmental analysis, food analysis, and biomedicine. By means of the advantages of rapidness, simplicity, convenience, accuracy, stability and the like, the immunochromatography technology becomes one of the common technologies for clinical diagnosis at present. Based on the principle of immunochromatography, a signal substance for labeling an antibody or antigen (hereinafter, referred to as a signal label) is accumulated on a detection line (T line) of an immunoassay strip to generate a specific signal, the magnitude of which is correlated with the concentration of an analyte (antibody, antigen or hapten) to be measured, thereby qualitatively and/or quantitatively detecting the analyte.

In the quantitative immunochromatography based on optical detection, an immunoassay strip is irradiated with light emitted from a light source, and then light signals such as reflected light, transmitted light, fluorescence or phosphorescence generated by irradiation of a T-line and/or a control line (C-line) of the immunoassay strip are collected by a detector, and then the collected light signals are converted into electrical signals, the magnitude of which is related to the concentration of an analyte to be detected, thereby quantitatively detecting the concentration of the analyte. In addition, in calculating the concentration of the analyte, in order to eliminate or minimize the adverse effect of the variation in the manufacturing process of the immunoassay strip on the test result, the optical signal of the T line is usually corrected by the optical signal of the C line.

In the process of manufacturing an immunoassay strip, a streaking or hydrojet apparatus is generally used to add a T-line solution and a C-line solution to the T-line and C-line of an immunochromatographic strip, respectively. The former adds T-line solution and C-line solution on the surface of the detection pad of the immunochromatographic strip by means of spotting through the liquid-drawing head of the liquid-drawing instrument, and the latter sprays the T-line solution and C-line solution on the surface of the detection pad of the immunoassay strip respectively. However, whether the liquid is dispensed or sprayed, due to a certain failure rate of the apparatus itself in the manufacturing process, or due to manual error operation or improper setting of apparatus parameters, the solutions added to the T-line and the C-line of the same batch of immunochromatographic strips are not uniform, for example, the volumes of the solutions added to some T-lines and C-lines are too large, and the volumes of the solutions added to other T-lines and C-lines are too small, thereby causing the sizes of the T-lines and the C-lines of the same batch of immunochromatographic strips to be inconsistent; also for example, some of the solutions added on the T and C lines are not continuous, resulting in bands of T and C lines that are also not continuous. These all result in inconsistent signals generated on the T-line of the same batch of immunochromatographic strips, which are likely to lead to detection errors.

Furthermore, in the prior art, since the T-line solution and the C-line solution are not added to the same line on the surface of the detection pad, the amounts of the added T-line solution and C-line solution may not be synchronized, and the optical signal of the T-line may be corrected according to the optical signal passing through the C-line, i.e., the analyte concentration calculated by the T/C or T/(T + C) signal ratio may not be accurate.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an immunoassay test strip, a kit, a test card and a method for detecting an analyte in a sample, wherein after the sample is added, a detection signal T and a correction signal T 'are generated on a T line of the immunoassay test strip, the T signal is corrected by utilizing the T' signal to obtain a corrected T signal, and then the concentration of the analyte can be accurately obtained according to the corrected T signal.

To this end, the present invention provides a detection reagent for an immunoassay test strip, comprising a labeling reagent including a first signal marker-labeled reagent and a second signal marker-labeled third reagent, signals generated from the first signal marker and the second signal marker being distinguishable from each other, the first reagent being capable of specifically binding to an analyte, and a capture reagent capable of capturing the first signal marker-labeled reagent and the second signal marker-labeled third reagent at the same position of the immunoassay test strip (e.g., a T-line of the immunoassay test strip).

In some cases, the capture reagent includes only one substance (e.g., protein a, protein G), such as mouse IgG as the first reagent and mouse anti-goat IgG as the second reagent, and in this case, the capture reagent is protein G, and not only can capture mouse IgG but also can capture mouse anti-goat IgG.

Further, the capture reagent includes a second reagent that captures the first reagent labeled with the first signal label and a fourth reagent that captures the third reagent labeled with the second signal label. The second and fourth reagents may be combined together by a coupling reagent, or may be present independently.

Further, the signal generated by the captured second signaling marker may be used to correct the signal generated by the captured first signaling marker.

The present invention also provides a method for detecting an analyte in a sample, the method comprising the steps of: (1) Providing an immunoassay test strip, wherein the immunoassay test strip comprises an application region and a detection pad, the application region is positioned at the upstream of the detection pad, a T line is arranged on the detection pad, a marking reagent is positioned between the application region or the application region and the T line and moves towards the T line when in detection, the marking reagent comprises a first reagent marked by a first signal marker and a third reagent marked by a second signal marker, signals generated by the first signal marker and the second signal marker can be distinguished from each other, the T line is coated with a capture reagent, the first reagent can be specifically combined with an analyte, the capture reagent can capture the first reagent marked by the first signal marker on the T line, and the capture reagent can capture the third reagent marked by the second signal marker on the T line;

(2) Adding a sample to a sample adding area of the immunoassay test strip, and allowing a labeled reagent to move to a T line along with the sample and be captured by a capture reagent on the T line;

(3) Detecting a detection signal T generated by a first signal marker on the T line and a correction signal T 'generated by a second signal marker on the T line by using an analyzer, and correcting the T signal by using the T' signal to obtain a corrected T signal;

(4) The corrected T signal is used to calculate the concentration of the analyte.

Further, correcting the T signal using the T ' signal is achieved by calculating a T/T ' or T/(T + T ') ratio, the corrected T signal being the T/T ' or T/(T + T ') ratio.

The invention also provides an immunoassay test strip for detecting an analyte in a sample, the immunoassay test strip comprising a sample addition zone and a detection pad, wherein the sample addition zone is positioned at the upstream of the detection pad, the detection pad is provided with a T line, a marking reagent is positioned between the sample addition zone or the sample addition zone and the T line and moves towards the T line when in detection, the marking reagent comprises a first reagent marked by a first signal marker and a third reagent marked by a second signal marker, signals generated by the first signal marker and the second signal marker can be distinguished from each other, the T line is coated with a capture reagent, the first reagent can be specifically bound with the analyte, the capture reagent can capture the first reagent marked by the first signal marker on the T line, and the capture reagent can capture the third reagent marked by the second signal marker on the T line.

In some embodiments of the invention, the labeling reagent is coated on the immunoassay strip or added to the immunoassay strip at the time of detection.

In some embodiments of the invention, the capture reagent comprises a second reagent that captures a first reagent labeled with a first signal label on the T-line and a fourth reagent that captures a third reagent labeled with a second signal label on the T-line.

In some embodiments of the invention, the first signaling tag and the second signaling tag are selected from the group consisting of lanthanide and its chelates, platinum/palladium porphyrin compounds, time-resolved luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds.

In some embodiments of the invention, the first signal marker is a time-resolved fluorescent microsphere and the second signal marker is a green fluorescent microsphere.

In some embodiments of the invention, when the analyte is an antigen, the first reagent and the second reagent are antibodies that specifically bind to the analyte, or the first reagent is an antibody that specifically binds to the analyte (such as a mouse IgG antibody that specifically binds to the analyte), the second reagent is a secondary antibody that specifically binds to the first reagent (such as a rabbit anti-mouse IgG antibody that specifically binds to the mouse IgG antibody); when the analyte is an antibody, the first and second reagents are antigens that specifically bind to the analyte, or one of the first and second reagents is an antigen that specifically binds to the analyte and the other is a secondary antibody that specifically binds to the analyte, or the first reagent is an antigen that specifically binds to the analyte (such as the receptor binding domain of SARS-CoV-2 spike protein), the second reagent (such as human ACE protein) competes with the analyte (such as SARS-CoV-2 neutralizing antibody) for binding to the first reagent; when the analyte is a hapten, the first reagent is an antibody that specifically binds the analyte and the second reagent is a conjugate of the analyte or analog thereof and a carrier protein (e.g., BSA, KLH, etc.).

In some embodiments of the invention, the immunoassay strip includes a sample addition pad, and the sample addition region is located on the sample addition pad.

In some embodiments of the invention, the labeling reagents are coated on the sample addition pad.

In some embodiments of the invention, the immunoassay strip includes a label pad, the sample loading region is located on the label pad, and the labeled reagent is coated on the label pad.

In some embodiments of the invention, the immunoassay strip includes a sample addition pad and a label pad, the label pad is located between the sample addition pad and the detection pad, the sample addition pad is located on the sample addition pad, and the label reagent is coated on the label pad.

In some embodiments of the present invention, the T-line is further coated with a quality control label, and the signals generated by the quality control label, the first signaling label and the second signaling label are distinguishable from each other.

In some embodiments of the invention, the quality control label is selected from the group consisting of lanthanide and its chelates, platinum/palladium porphyrin compounds, up-conversion luminescent materials, time-resolved luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds.

In some embodiments of the invention, the first signal marker is a time-resolved fluorescent microsphere, the second signal marker is a green fluorescent microsphere, and the quality control marker is FITC.

In some embodiments of the present invention, the detection pad further comprises a C-line, the labeling reagent further comprises a fifth reagent labeled with a third signal marker, and a sixth reagent is coated on the C-line and can capture the fifth reagent labeled with the third signal marker on the C-line.

In some embodiments of the invention, at least one of the third/fourth and fifth/sixth agents is selected from the group consisting of a non-human antibody/anti-non-human antibody, biotin/streptomycin, DNP-BSA/anti-DNP antibody, and a receptor/ligand.

In some embodiments of the invention, the third/fourth agent is goat/chicken IgY antibody and the fifth/sixth agent is DNP-BSA/rabbit anti-DNP antibody.

In some embodiments of the invention, the third signaling moiety is selected from the group consisting of lanthanide and its chelates, platinum/palladium porphyrin compounds, time-resolved luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds.

In some embodiments of the invention, the first signal marker and the third signal marker are time-resolved fluorescent microspheres and the second signal marker is green fluorescent microspheres.

In some embodiments of the invention, the luminescent compound is selected from the group consisting of quantum dots, fluorescein and its derivatives, fluorescent proteins, and chemiluminescent labels.

In some embodiments of the invention, the third reagent and the fourth reagent do not specifically bind to the first reagent, the second reagent, and the analyte.

In some embodiments of the invention, the fifth reagent and the sixth reagent do not specifically bind to the first reagent, the second reagent, the third reagent, the fourth reagent, and the analyte.

As used herein, "capture" means that the capture reagent coated on the T-line or the sixth reagent on the C-line can capture the first signal marker-labeled first reagent, the second signal marker-labeled third reagent, or the third signal marker-labeled fifth reagent on the T-line or the C-line, directly or indirectly or through a bridging structure. Directly refers to capturing only the first signal-labeled first reagent, the second signal-labeled third reagent, or the third signal-labeled fifth reagent itself on the T-line or C-line, and indirectly refers to capturing the complex formed by the specific binding of the first signal-labeled first reagent to the analyte, or the complex formed by the specific binding of the second signal-labeled third reagent to the first non-analyte, or the complex formed by the binding of the third signal-labeled fifth reagent to the second non-analyte on the T-line or C-line. The bridging structure is formed by specific binding of compounds such as biotin and streptavidin, for example, the sixth reagent carries biotin and the fifth reagent carries streptavidin, and then the sixth reagent can capture the third signal labeled fifth reagent on the C line by the specific binding of streptavidin and biotin.

Has the advantages that: (1) The invention utilizes the correction signal (T 'signal) sent by the T line to correct the detection signal (T signal) sent by the same T line, namely the second reagent and the fourth reagent are coated on the T line at one time, the volume of the solution of the T line is too much or too little, and the quantity of the second reagent and the fourth reagent is too much or too little at the same time, and the T signal is corrected by the T' signal, so that the difference that the correction of the T signal by the contrast signal (C signal) generated by the C line cannot be eliminated due to the asynchronous change of the volumes of the solution of the T line and the solution of the C line added during the preparation of the test card can be effectively eliminated, and the concentration of the analyte in a sample can be accurately determined, therefore, the detection precision is obviously better than the conventional method for correcting the T signal by the contrast signal (C signal) generated by the C line; (2) In the invention, the quality control marker is carried by the second reagent coated on the T line, the T line can be directly irradiated by a laser flashlight without cutting the test strip plate and adding clinical samples, analyte calibration liquid and sample diluent in the process of preparing the test card, the quantity and the uniformity of the T line solution coated on the T line of the test strip plate are judged according to the optical signal generated by the second reagent coated on the T line and carrying the quality control marker, if the quality control requirements are obviously not met, the test strip plate is not required to be cut, further processing of the test strip plate obviously not meeting the quality control requirements can be avoided from the source to produce a large quantity of unqualified immunoassay test strips, so that the preparation time and the cost are reduced, meanwhile, when the test card or the immunoassay test strips are subjected to sampling inspection, the clinical samples, the analyte calibration liquid and the sample diluent are not required to be added, but the light emitted by a light source of an analyzer is directly used for irradiating the T line of the test card, the fluorescent signal generated by the T line can be used for judging whether the batch of test cards meet the quality control requirements or not, the production efficiency is improved, the production period is shortened, and the production cost is saved.

Drawings

FIG. 1 is an exploded view of a test card used in the present invention.

FIG. 2 is a schematic view of an immunoassay strip in a test card used in the present invention.

Fig. 3 is a schematic perspective view of an analyzer used in the present invention.

FIG. 4 is a schematic diagram of the internal circuitry of an analyzer used in the present invention.

Figure 5 is a test pad after addition of the T-line solution and before drying, and taking a picture obtained by taking a picture of the detection pad by using the photographing equipment.

Fig. 6 is a picture of the test pad after the T-line solution is added and dried, and the test pad is photographed by using a photographing apparatus.

Detailed Description

As shown in fig. 1 and 2, the test card 5 includes an immunoassay test strip 15, a card cover 14, and a card holder 16. The immunoassay strip 15 used is an immunochromatographic strip comprising a sample addition pad 51, a label pad 52, a detection pad 53 and a sample absorption pad 54, which are sequentially overlapped together. The detection pad 53 is made of a material such as nitrocellulose, glass fiber, polyethersulfone, or nylon, and is preferably a nitrocellulose membrane. The detection pad 53 is provided with a detection line (T line) 56 and a control line (C line) 57. The sample addition member 51 is made of a water-absorbing material, and can be made of glass fiber or nonwoven fabric. The marking pad 52 is made of a water-absorbent material, and may be selected from a polyester film, a glass fiber, or a non-woven fabric. The sample absorption pad 54 is disposed on the bottom support layer 55 and is made of a hydrophilic material, preferably filter paper.

The number of detection lines provided on the detection pad 53 can be adjusted according to actual needs, for example, when detecting one analyte, only one detection line needs to be provided, and when detecting two or more analytes, a corresponding number of detection lines should be provided.

Immunoassay strip 15 further comprises a bottom support layer 55, wherein bottom support layer 55 is made of a hydrophobic material such as polyvinyl chloride, which is commonly used, to ensure that the sample cannot permeate through bottom support layer 55. The sample addition pad 51, the label pad 52, the detection pad 53 and the sample absorption pad 54 are disposed on the bottom support layer 55. One end of the label pad 52 is partially overlapped with the sample application pad 51, and the other end of the label pad 52 is partially overlapped with the detection pad 53; one end of the sample absorption pad 54 partially overlaps the detection pad 53. In some cases, it is possible to use, the overlapping area between any two adjacent pads is 0.5 to 5 mm long.

The immunoassay test strip 15 is located in a housing formed by combining the card cover 14 and the card holder 16 by ultrasonic welding, snap fitting or glue bonding. In some cases, the card cover 14 and the card holder 16 are made of plastic. A strip groove 60 is provided in the middle of the cartridge 16 for receiving the immunoassay strip 15. Preferably, a plurality of downwardly extending hooks (not shown) are disposed on the cap 14, a plurality of upwardly extending slots 61 are disposed on the cartridge 16, and the hooks disposed on the cap 14 and the slots 61 disposed on the cartridge 16 correspond to each other one by one, so that the cap 14 and the cartridge can fix the immunoassay test strip 15 in the test strip slot 60 when the cap 14, the immunoassay test strip 15 and the cartridge 16 are assembled together.

The card cover 14 is also provided with a sample introduction port 58 and a viewing window 59. The sample enters the sample addition port 51 below the sample addition port 58 through the sample addition port 58, and the sample migrates toward the sample absorption pad 54 along the length direction of the immunoassay strip 15 by capillary action. The observation window 59 is provided above the detection line 56 and the control line 57 of the detection pad 53. Light from the light source of the analyzer can be directed through a transparent or translucent viewing window 59 onto the test line 56 and the control line 57 of the immunoassay strip 15. Upon illumination with light from the light source, the light signals generated by the detection line 56 and/or the control line 57 can be output to the detector of the analyzer.

Depending on the analyte to be detected (e.g., antigen, antibody or hapten) and the immunoassay principle (double antigen sandwich, double antibody sandwich, competition, indirect, capture), there will be variations in the substance coated on the label pad 52 and the detection line 56. The test analyte is calprotectin, and the immunoassay principle is a double-antibody sandwich method, wherein the calprotectin in the excrement can be used as a biomarker for evaluating inflammatory bowel disease and colorectal cancer. The label pad 52 is coated with a first reagent labeled with a first signal marker (e.g., the first signal marker is a time-resolved fluorescent microsphere, the first reagent is a calprotectin detection antibody), a third reagent labeled with a second signal marker (e.g., the second signal marker is a green fluorescent microsphere, the third reagent is goat anti-chicken IgY antibody), a fifth reagent labeled with a third signal marker (e.g., the third signal marker is a time-resolved fluorescent microsphere, the fifth reagent is DNP-BSA), the detection line 56 is coated with a second reagent (e.g., a calprotectin capture antibody) and a fourth reagent (e.g., chicken IgY antibody), the first reagent and the second reagent specifically bind to calprotectin in the clinical sample, so that the clinical sample flows along the length of the immunoassay test strip 15 after the clinical sample is applied to the sample pad 51. When the clinical sample reaches the label pad 52, the first signal marker-labeled first reagent specifically binds to calprotectin (if present) in the clinical sample, and the first signal marker-first reagent-calprotectin complex formed continues to flow and specifically binds to the second reagent on the test line 56 to capture the first signal marker-first reagent-calprotectin complex on the test line 56, while the fourth reagent on the test line 56 captures the second signal marker-labeled third reagent or the second signal marker-labeled third reagent in a complex formed by specific binding of the second signal marker to the first non-analyte on the test line 56, the first signal marker captured on the test line 56 upon irradiation with light from a light source producing a detection signal T related to the concentration of calprotectin and the second signal marker captured on the test line 56 producing a calibration signal T'. At the same time, the fifth reagent labeled with the third signal marker continues to flow, and when flowing onto the control line 57, the sixth reagent (e.g., rabbit anti-DNP antibody) coated on the control line 57 can capture the fifth reagent labeled with the third signal marker or a complex formed by the specific binding of the fifth reagent labeled with the third signal marker and the second non-analyte on the control line 57, and the third signal marker captured on the control line 57 generates a control signal after being irradiated by the light from the light source. The detection signal T, the correction signal T', and the control signal may be generated by light signals such as reflected light, transmitted light, fluorescence, phosphorescence, etc. generated after the T-line and the C-line are irradiated with light from the light source. The third signaling tag may be the same as or different from the first signaling tag. The first signal marker and the second signal marker are different, and optical signals generated by the first signal marker and the second signal marker can be distinguished through an analyzer and cannot interfere with each other.

The sample applied through the sample-applying port 58 first enters the application region of the immunoassay strip 15, which is located upstream of the detection pad 53; the sample loading region is located on the sample loading pad 51. Although the immunoassay strip 15 of fig. 1 and 2 includes the sample addition member 51 and the label member 52, the sample addition member 51 may be absent if necessary, and the sample addition region may be located on the label member 52; alternatively, the label pad 52 may be absent, in which case the reagent originally coated on the label pad 52 is stored in a test tube in a liquid or solid form, and in the detection, a sample is added to the test tube to form a mixture, and then the formed mixture is applied to the application region of the immunoassay strip 15 through the sample inlet 58, and moves toward the T-line and the C-line along the length direction of the immunoassay strip 15.

Preferably, the fourth reagent on the detecting line 56 may capture the second signal label-labeled third reagent directly on the detecting line 56; the sixth reagent coated on the control line 57 directly captures the fifth reagent labeled with the third signal marker on the control line 57. At this time, the third and fourth reagents and the fifth and sixth reagents may be selected from any one of the following combinations: non-human/anti-non-human antibodies (e.g., rabbit/goat anti-rabbit IgG, chicken/goat anti-chicken IgY), biotin/streptomycin, DNP-BSA/anti-DNP antibodies, receptors/ligands, and the like. For example, the third and fourth agents are selected from chicken IgY/goat anti-chicken IgY and the fifth and sixth agents are selected from DNP-BSA/anti-DNP.

The first non-analyte and the second non-analyte referred to in the present invention refer to substances that do not affect the detection of the analyte to be detected and do not affect each other at the time of detection, and may or may not be present in the sample, and if not present, may be added to the sample or the immunoassay strip in advance. The first non-analyte and the second non-analyte may be substances that are not present or are present in negligible amounts in the sample, such that the calibration and control signals are not affected by the clinical sample. The first non-analyte and the second non-analyte may also be substances present at higher levels in the sample, for example, when the analyte to be tested is calprotectin, the first non-analyte and the second non-analyte in the present invention may be human IgG and human IgM, respectively, present in a clinical sample, in this case, the third reagent and the fifth reagent may be a rabbit anti-human IgG antibody specifically binding to human IgG and a rabbit anti-human IgM antibody specifically binding to human IgM, respectively, while the fourth reagent coated on the T-line is a goat anti-rabbit IgG antibody specifically binding to rabbit anti-human IgG, and the sixth reagent coated on the control line 57 is a goat anti-rabbit IgM antibody specifically binding to rabbit anti-human IgM.

First, second and third signaling tagsThe substance may be selected from the group consisting of a time-resolved luminescent label, a colored luminescent microsphere, a colored colloidal particle (e.g., latex, colloidal gold, colloidal carbon, colloidal selenium), a magnetic nanoparticle, and a luminescent compound. The first signaling tag and the third signaling tag are preferably time-resolved luminescent tags. The time-resolved luminescent marker has the characteristic of luminescence delay, namely, when the exciting light emitted by the light source is turned off, the time-resolved luminescent marker can still continuously emit light within a certain time. The time-resolved luminescent labels may be present in the form of molecules, called time-resolved luminescent molecules, which may be selected from lanthanides such as samarium (Sm (III)), dysprosium (Dy (III)), europium (Eu (III)) and terbium (Tb (III)) and chelates thereof; a platinum/palladium porphyrin compound capable of emitting phosphorescence after excitation; an up-converting luminescent material. A suitable lanthanide chelate is N- (p-isothiocyanatobenzene) -diethylenetriamine tetraacetic acid-Eu ⁺³ . The time-resolved luminescent marker may also be present in another form: the time-resolved luminescent microsphere is prepared by wrapping time-resolved luminescent molecules in the interior or on the surface of a natural or artificially synthesized microsphere or microbead. Since each time-resolved luminescent microsphere can wrap thousands of time-resolved luminescent molecules, the detection sensitivity is effectively improved, and therefore, the time-resolved luminescent label is preferably a time-resolved luminescent microsphere. The time-resolved luminescent microspheres that produce fluorescence after excitation are called time-resolved fluorescent microspheres.

The colored luminescent microspheres refer to microspheres or microbeads which are coated with luminescent compounds such as quantum dots, fluorescent dyes and the like on the surface or in the interior, and can generate optical signals without luminescence delay characteristic by irradiation of exciting light with proper wavelength. The colored luminescent microspheres can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres and colored fluorescent microspheres (emitting fluorescence of a plurality of specific colors). The colored colloidal particles refer to colloidal particles that generate colored aggregates after aggregating on the T line and/or C line during the immunochromatographic reaction, and may be selected from latex, colloidal gold, colloidal carbon, and colloidal selenium.

Whereas the control signal generated by the C-line in the present invention can be used to indicate the effect of the added clinical sample flowing to the sample absorption pad 54, the control signal can be a color signal, such as a colored aggregate generated after colored colloidal particles aggregate on the control line 57, besides the light signal, and when the color signal is observed on the control line 57, the determination of whether the added clinical sample flows to the sample absorption pad 54 can be made, so that the reflected light signal generated by the C-line can be collected without the light source.

The luminescent compound in the invention refers to a light ray which can generate no luminescence delay characteristic by being irradiated by exciting light with proper wavelength and can be selected from quantum dots; fluorescein and its derivatives, such as Fluorescein Isothiocyanate (FITC); fluorescent proteins capable of emitting fluorescence after excitation and modified variants thereof, such as green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, and the like; chemiluminescent label selected from luminol, isoluminol and its derivatives, 1, 2-dioxetane derivatives (AMPPD, CSPD, CDP and CDP-Star and PPD, lumi-Phos and Lumi-Plus from Lumigen, etc. are common) and acridinium esters or acridine sulfonamides. The luminescent compound may be coupled to the antibody or antigen or hapten-carrier protein conjugate by conventional chemical coupling reagents.

The polymer forming the microspheres or microbeads, whether in time-resolved luminescent microspheres or colored luminescent microspheres, may be selected from polystyrene, butadiene styrene, styrene acrylic acid-vinyl terpolymer, polymethyl methacrylate, polyethyl methyl acrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinyl benzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, and the like, or aldehyde, carboxyl, amino, hydroxyl, hydrazide derivatives thereof, or mixtures thereof. In addition, the surface of the microsphere or microbead usually carries hydroxyl, carboxyl, amino, aldehyde, sulfo, etc. groups, and can be coupled with the antibody or antigen or hapten-carrier protein conjugate by conventional chemical coupling reagents, or coupled with the antibody or antigen or hapten-carrier protein conjugate by a bridging structure, such as coupling the groups on the surface of the microsphere or microbead with streptavidin, and then coupling the biotin-labeled antibody or antigen or hapten-carrier protein conjugate to the microsphere or microbead by the specific binding of streptavidin and biotin. In some cases, the time-resolved luminescent microspheres have a particle size of 20nm to 100 μm; the particle size of the colored luminescent microsphere is 100 nm-100 μm.

When detecting one or more analytes in a sample, the intensity of the detection signal generated by the first signal label on the T-line is positively or negatively correlated with the concentration of the analyte, depending on the immunoassay principle, wherein the intensity of the detection signal generated by the first signal label on the T-line is positively correlated with the concentration of the analyte, in addition to the competition method. However, regardless of the immunoassay principle employed, the control line 57 should produce a control signal, which if not, indicates a problem or failure of the immunoassay strip.

The clinical sample in the present invention may be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, nasopharyngeal swab, sputum, feces, skin lesion sample (including swabs of rash/pock exudate; pock fluid; pock, etc.), and the like. Depending on the type of clinical sample and the analyte, some clinical samples are pre-treated before detection (e.g., lysed with a lysis solution to release the analyte to be detected) before being added to the test card 5 for detection.

The detectable analytes of the present invention include antigens or antibodies, and even haptens, such as inflammatory markers, e.g., CRP, IL-6, procalcitonin (PCT), SAA, and the like; heart failure markers such as BNP, NT-proBNP, cTnI, CK-MB, myoglobin, D-dimer, and the like; tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, prostate Specific Antigen (PSA), neuron Specific Enolase (NSE), CYFRA21-1, CA19-9, CA50, CA125, CA153, CA724, and the like; bone metabolism markers such as 25-hydroxyvitamin D, beta-CrossLaps, bone alkaline phosphatase, calcitonin, parathyroid hormone, human N-terminal mid-osteocalcin, and total type I procollagen amino-terminal extension peptide; sex hormone markers such as Follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), prolactin, progesterone, testosterone, estradiol, estriol, beta-chorionic gonadotropin (beta-HCG); a functional marker such as total triiodothyronine (TT 3), total thyroxine (TT 4), free triiodothyronine (FT 3), free thyroxine (FT 4), thyroid Stimulating Hormone (TSH), thyroglobulin antibody, and thyroid peroxidase antibody; infectious disease markers, such as monkeypox virus antigen/antibody, gonococcus antigen/antibody, chlamydia antigen/antibody, mycoplasma antigen/antibody, hepatitis b quintuple (HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe), HCV antigen/antibody, treponema pallidum antigen/antibody, TORCH (human cytomegalovirus, rubella virus, toxoplasma, HSV-1 and HSV-2) IgG/IgM, HIV antigen/antibody, and the like; markers of diabetes, such as insulin, C-peptide, insulin autoantibodies, islet cell antibodies, glutamate decarboxylase antibodies; markers of liver fibrosis, such as laminin, hyaluronic acid, type IV collagen, type iii procollagen N-terminal peptide, chitinase 3-like protein 1, and the like; allergy markers such as allergen, allergen-specific IgE and total IgE, etc.; intestinal health series markers, such as intestinal adenovirus antigen, rotavirus antigen, enterovirus 71 type IgE/IgG, coxsackie virus A group 16 type IgG/IgM, coxsackie virus B group IgG/IgM, clostridium difficile toxin A, clostridium difficile toxin B, clostridium difficile glutamate dehydrogenase and the like; cocaine and morphine for evaluating drug abuse, etc.

At the time of detection, the test card 5 is detected by an analyzer. As shown in fig. 3 and 4, the analyzer 100 includes an analyzer housing 1, a display 2, and a test card insertion port 3. The test card 5 is connected to the analyzer 100 through the test card insertion port 3. The clinical sample is inserted into the analyzer 100 after reacting for a certain time outside the analyzer 100 after being added to the test card 5, or is inserted into the analyzer 100 immediately after being added to the test card 5 and then reacting for a certain time in the analyzer 100. The analyzer 100 further includes an optical system, an amplifying circuit 8, an analog-to-digital conversion chip (ADC) 9, a main control unit 10, and a digital-to-analog conversion chip (DAC) 11 inside the housing 1. The optical system comprises a light source 4, an optical path structure 6, a detector 7, a light source drive circuit 12 and a feedback circuit 13. The optical system can be provided with some optical devices such as narrow-band filters or gratings according to requirements. The light source 4 may be selected from a Light Emitting Diode (LED), a flashlight, and other suitable light sources, preferably an LED. The illumination of the light source 4 may be continuous or pulsed. The detector 7 may be selected from a photomultiplier, a photodiode, a charge coupled device, a charge injection detector, a CMOS photosensitive element, or the like, preferably a Photodiode (PD), such as a silicon photodiode. The detector 7 may be provided in plurality as necessary. The analyzer may be a commercially available analyzer.

The main control unit 10 includes a microprocessor, and the control signal applied by the microprocessor is subjected to digital-to-analog conversion by a digital-to-analog conversion chip 11, and then provides a light source current control signal for the light source driving circuit 12, and the light source 4 is made to operate in a constant current state after being fed back by a feedback circuit 13. During the scanning process of the optical system on the test card 5, the light emitted from the light source 4 is irradiated on the test card 5 to which the sample to be tested has been added through the light path structure (such as an optical fiber) 6. After a certain time of reaction by adding the sample, the detection line (also referred to as T line) 56 and the control line (also referred to as C line) 57 of the test card 5 are irradiated to generate optical signals. The generated optical signal is output to the detector 7 through an optical path structure 6 (such as an optical fiber, preferably, different from the optical fiber through which the light emitted from the light source 4 passes), and is converted into an electric current signal by the detector 7. The generated current signal is modulated and converted into a suitable voltage interval by the amplifying circuit 8, and then is subjected to analog-to-digital conversion by the analog-to-digital conversion chip 9 and then is transmitted to the main control unit 10. The microprocessor in the main control unit 10 performs calculations on the received analog-to-digital converted electrical signal to determine the presence or concentration of the analyte in the sample to be tested.

Example 1: preparation of the first type of immunoassay strip and test card of the present invention

An immunoassay test strip and a test card for quantitatively detecting calprotectin in excrement are prepared by the following steps:

A. preparing an antibody: the antigen can be used to immunize animals such as mice, rats, rabbits and the like or hybridoma cell counts to screen out paired monoclonal antibodies or polyclonal antibodies for detecting calprotectin, or commercial calprotectin paired antibodies (one is a calprotectin capture antibody, and the other is a calprotectin detection antibody) can be selected, and in this example, commercial calprotectin paired antibodies (Medix Biochemica, cat nos. 100460 and 100618) are taken as an example for explanation. The chicken IgY antibody, the goat anti-chicken IgY antibody and the rabbit anti-DNP antibody are prepared by self or purchased from the market.

B. Detecting a pad spotting liquid:

adding calprotectin capture antibody and chicken IgY antibody into 0.02M phosphate buffer (pH 7.2) to obtain T line solution, wherein the concentration of calprotectin capture antibody in the T line solution is 0.8mg/ml and the concentration of chicken IgY antibody is 0.4mg/ml, adding rabbit anti-DNP antibody into 0.02M phosphate buffer (pH 7.2) to obtain C line solution, wherein the concentration of rabbit anti-DNP antibody in the C line solution is 0.lmg/ml, coating the T line solution and the C line solution on a 2.5 cm-wide nitrocellulose membrane (used as a detection pad) at an interval of 0.8cm by using a quantitative spotting device (AUTOKUN continuous membrane-scribing machine spotting machine HGS101 of Hangzhou mountain navigation technology Co., ltd.) so as to form a T line and a C line respectively, drying at 45 ℃ for 18 hours, and adding a drying agent for sealing for later use.

C. Preparing fluorescent microspheres:

selecting fluorescent microspheres: selecting a time-resolved fluorescent microsphere (Suzhou Kongku biological technology Co., ltd., product number FT0200 CA) with the diameter of 100 nm-400 nm, wherein the lanthanide ion marker is embedded inside the time-resolved fluorescent microsphere and the surface of the time-resolved fluorescent microsphere carries carboxyl, and the excitation wavelength of the time-resolved fluorescent microsphere is 360nm and the emission wavelength of the time-resolved fluorescent microsphere is 615nm; green fluorescent microspheres (product number FG0300CA, suzhou Kongku Bio-technology Co., ltd.) with the diameter of 100 nm-400 nm, which are embedded with fluorescent dye and carry carboxyl on the surface are selected, and the excitation wavelength of the microspheres is 488nm and the emission wavelength of the microspheres is 520nm.

Preparing MES buffer solution: adding morpholine ethanesulfonic acid sodium salt into pure water, mixing uniformly to make the concentration of morpholine ethanesulfonic acid sodium salt be 1.066% (w/v), filtering with 0.22um microporous membrane for sterilization, and storing at 4 deg.C for use.

Preparation of a preservation buffer: 50mM (pH8.0) Tris-HCl buffer containing 20% sucrose (w/v), 5% trehalose (w/v), 2% Tween-20 (v/v), 0.5% PVP (w/v), 0.5% Casein-Na (w/v) was used, and the buffer was sterilized by filtration through a 0.22um Millipore filter and stored at 4 ℃ until use.

Preparing a time-resolved fluorescent microsphere coupled with a calprotectin detection antibody (hereinafter referred to as a detection time-resolved fluorescent microsphere): washing the fluorescent microspheres with MES buffer, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make the final concentrations of the microspheres 0.4mg/ml and 0.1mg/ml respectively, reacting at room temperature for 20 minutes to activate the time-resolved fluorescent microspheres, washing the activated microspheres with MES buffer sufficiently, adding calprotectin detection antibody at a ratio of 1mg; respectively preparing the time-resolved fluorescent microspheres coupled and combined with DNP-BSA and the green fluorescent microspheres coupled and combined with the goat anti-chicken IgY antibody in the same way of preparing and detecting the time-resolved fluorescent microspheres, and the final concentrations of the time-resolved fluorescent microspheres coupled and combined with DNP-BSA and the green fluorescent microspheres coupled and combined with the goat anti-chicken IgY antibody are both 0.2% g/ml by using a storage buffer solution, and the microspheres are stored at 4 ℃ for later use. And finally, mixing the time-resolved fluorescent microspheres coupled with the calprotectin detection antibody, the time-resolved fluorescent microspheres coupled with DNP-BSA, the green fluorescent microspheres coupled with the goat anti-chicken IgY antibody and a storage buffer solution in a ratio of 5:2:5:8 to obtain the microsphere mixture.

D. Spraying and drying the fluorescent microspheres:

the prepared microsphere mixture was uniformly sprayed on a 1.0cm wide marking pad (glass fiber) in an amount of 4 μ 1/cm using a dedicated liquid-spreading head of AUTOKUN continuous film-spreading machine pointing device HGS101, dried at 45 ℃ for 18 hours, and sealed with a desiccant for use.

E. Treatment of the sample pad:

a sample pad with the width of 2.5cm is placed into the sample pad treatment solution for soaking treatment for 1 hour, and then taken out and dried overnight (12-24 hours) at 37 ℃. The sample pad treatment solution was a buffer solution containing 0.05% sodium tetraborate (w/v), 0.01% BSA (w/v), 0.5% disodium ethylenediaminetetraacetate (w/v) and 0.5% Tween-20 (v/v) at pH 8.6.

F. Assembling and cutting a test strip plate:

assembling the trial strip plate: a nitrocellulose membrane 2.5cm wide, a marker pad 1.0cm wide, a sample pad 2.5cm wide, and a water absorbent paper 2.8cm wide (as a sample absorbent pad) were assembled manually or by machine on a plastic base plate 8cm long (as a bottom support layer) so that the sample pad 2.5cm wide, the marker pad 1.0cm wide, the nitrocellulose membrane 2.5cm wide, and the water absorbent paper 2.8cm wide were overlapped alternately with each other by 2mm in this order to assemble a strip board.

Cutting a test strip plate: the assembled strip was cut into single-portion immunoassay strips of 0.4cm width using an AUTOKUN HGS201 type slitter.

G. Assembling the test card:

the cut single-person immunoassay test strip is placed in a clamping groove on a plastic clamping seat, a clamping cover is covered, and the clamping seat and the clamping cover are tightly pressed by using a card pressing machine or manually, so that the whole immunoassay test strip is ensured to be in a tight state. Adding a drying agent, and sealing at room temperature for later use.

After the test card is assembled, it can be loaded into a kit containing a desiccant and instructions for use. In addition, according to the requirement, a sampling tube, a test tube containing lysate, a test tube containing sample diluent, a sampling swab and the like can be also arranged in the kit.

Example 2: preparation of the second type of immunoassay strip, test card, and detection method of the present invention

The immunoassay test strip and test card prepared in the first embodiment of this example differ from example 1 only in that the calprotectin capture antibody of example 1 is replaced by a FITC-calprotectin capture antibody conjugate for quality control of the dot volume and homogeneity of the T-line. FITC-calprotectin capture antibody conjugate is prepared by activating FITC with N-hydroxysuccinimide (NHS) and then conjugating it to calprotectin capture antibody, the resulting FITC-calprotectin capture antibody conjugate being diluted with the storage buffer of example 1 to a final concentration of 0.8mg/mL. In the process of preparing a batch of test cards, before the test strip plate is cut, a clinical sample, calprotectin calibration solution or sample diluent is not required to be added, a laser flashlight is directly used for irradiating a T line, the quantity and the uniformity of the T line solution coated on the T line of the test strip plate are qualitatively judged according to fluorescence emitted by the FITC-calprotectin capture antibody conjugate coated on the T line, and if the T line solution does not meet the quality control requirement, the test strip plate is not required to be cut, so that the preparation time and the preparation cost are saved.

The single immunoassay test strips generated after the test strip plate is cut are assembled to obtain a batch of test cards. When the test card is used for sampling inspection, a clinical sample, calprotectin calibration solution or sample diluent is not needed, excitation light with the wavelength of 494nm emitted by a light source of a dry fluorescence immunoassay instrument FIC-Q100N (Suzhou and Meyer precision instruments Co., ltd.) is used for directly irradiating a T line of the test card, emitted light with the wavelength of 520nm emitted by an FITC-calprotectin capture antibody conjugate coated on the T line is counted, a fluorescence signal of the T line is obtained and is used for quantitatively judging the quantity and the uniformity of a T line solution coated on the T line of a single-person immunoassay test strip, and then whether the batch of test cards meet the quality control requirement is judged.

The second embodiment of this example differs from the first embodiment of this example in that the labeling pad is coated with only time-resolved fluorescent microspheres coupled with calprotectin detection antibody, time-resolved fluorescent microspheres coupled with DNP-BSA, and storage buffer, and is not coated with green fluorescent microspheres of goat anti-chicken IgY antibody; and when the spotting fluid is detected, only the FITC-calprotectin capture antibody conjugate is coated on the T line, and the chicken IgY antibody is not coated on the T line. After a period of time of reaction by adding the clinical sample, the time-resolved fluorescence signal (T signal) emitted by the time-resolved fluorescent microspheres conjugated to the calprotectin detection antibody captured on the T-line can be corrected by using the fluorescence signal (T ' signal) emitted by the FITC-calprotectin capture antibody conjugate coated on the T-line after excitation, for example, T/T ' or T/(T + T ') can be calculated. The T signal and the T' signal can be distinguished by the detector without interfering with each other. The T signal and the T' signal come from the same T line, so that the collinear correction can be realized. Furthermore, FITC in FITC-calprotectin capture antibody conjugates may also be replaced by other signal labels, such as lanthanides and chelates thereof, platinum/palladium porphyrin compounds, time-resolved luminescent microspheres, colored colloidal particles, magnetic nanoparticles and luminescent compounds other than FITC.

Example 3: preparing conventional immunoassay test strip and test card

The conventional immunoassay test strip and test card prepared in example 3 are different from those of example 1 only in that the labeling pad is coated with only time-resolved fluorescent microspheres coupled with calprotectin detection antibody, time-resolved fluorescent microspheres coupled with DNP-BSA and a preservation buffer, and green fluorescent microspheres not coated with goat anti-chicken IgY antibody; when the spotting fluid is detected, only the calprotectin capture antibody is coated on the T line, and the chicken IgY antibody is not coated on the T line; coupling the time-resolved fluorescent microspheres coupled with calprotectin detection antibodies, coupling the time-resolved fluorescent microspheres coupled with DNP-BSA and storing buffer solution in a ratio of 5:2:13, and coating on the marking pad after being fully mixed. At this time, after the sample is added and reacts for a period of time, only a detection signal (T signal) emitted by the time-resolved fluorescent microspheres is generated on the T line, and a correction signal emitted by the green fluorescent microspheres is not generated; the time resolved fluorescent microspheres on line C emit a control signal (C signal). Thus, the concentration of calprotectin in the sample can be calculated from the ratio of T/C or T/(T + C) signals.

Example 4: detection method

The test card prepared in example 1 was used to detect calprotectin in a faecal sample by the following procedure: the obtained 10mg fecal sample was added to 4ml sample diluent (borax, BSA, tween-20, EDTA-2Na, preservative, pH 8.6) and shaken well to generate a turbid solution, then 10. Mu.l of the turbid solution was added to the sample addition port of the test card, and then 80. Mu.l of the sample diluent was added to the sample addition port of the test card. After 15min of incubation, the test cards were inserted into a dry fluoroimmunoassay FIC-Q100N (Suzhou and Mimeji instruments, inc.) for detection.

During detection, the light source emits exciting light with the wavelength of 360nm to excite the time-resolved fluorescent microspheres captured on the T line and the C line, then the light source is turned off, and the time-resolved fluorescent microspheres captured on the T line and the C line emit time-resolved fluorescent signals with the wavelength of 615 nm. After the light source is turned off for 300 mus, the count is carried out after the capture by the detector of the immunity analyzer, the time-resolved fluorescence signal (T signal) emitted on the T line and the time-resolved fluorescence signal (C signal) emitted on the C line were obtained, respectively. Then, the light source emits 488nm excitation light to excite the green fluorescent microspheres captured on the T line, after excitation, the green fluorescent microspheres captured on the T line emit green fluorescent signals with the wavelength of 520nm, and after capture, the green fluorescent signals (T' signals) emitted on the T line are obtained by counting through a detector of an immunoassay analyzer. In order to correct the adverse effect caused by the manufacturing difference in the manufacturing process of the test cards in the same batch, the T signal can be corrected in various ways. In the prior art, the T signal is often corrected by using the C signal, and the ratio of the T signal to the C signal is obtained and recorded as T/C, or T/(T + C) is obtained; this way of correction is also called out-of-line correction, given that the C and T signals come from different lines. In this embodiment, the T signal is corrected by using the T 'signal, and a ratio of the T signal to the T' signal is obtained and recorded as T/T ', or a ratio of T/(T + T') is obtained; this type of correction is also called inline correction, since the T signal and the T' signal come from the same T line. The T/T 'or T/(T + T') obtained after addition of the clinical sample can be used to determine the concentration of calprotectin according to a previously determined calibration curve.

Firstly, preparing a series of calibration solutions with different calprotectin concentrations, then respectively adding the calibration solutions with different concentrations into the same batch of test cards, incubating for 15min, detecting a T signal, a T ' signal and a C signal of each test card by using a dry type fluorescence immunoassay analyzer, and calculating a T/T ' or T/(T + T ') ratio. And drawing a calibration curve according to each T/T 'or T/(T + T') ratio and the corresponding calprotectin concentration value, and using the calibration curve to calculate the calprotectin concentration in the clinical sample during the collinear correction. For comparison, a calibration curve is also drawn from each T/C or T/(T + C) ratio and its corresponding calprotectin concentration value, for use in calculating the calprotectin concentration in the clinical sample at the time of the off-line correction.

Example 5: judging the liquid uniformity of the T line point

Three test card lots were prepared according to the preparation method of the first protocol of example 2, with the difference that in preparing the first test card lot (1), the spotting speed of the dedicated pipetting head was maintained at 1 μ l/cm; in the process of preparing the second batch of test cards (2), the liquid dropping speed of the special liquid-drawing head is increased to 2 mu l/cm; during the preparation of the third batch of test cards (3), the dot liquid speed of the dedicated liquid-scribing head was maintained at 1 μ l/cm, but the dot film buffer assembly of the continuous film-scribing machine dot film gauge HGS101 was removed so that the T-line solution was discontinuously added on the 2.5cm wide detection pad 62, thereby forming the T-line 63 on the 2.5cm wide detection pad 62. After the addition of the T-line solution, a photograph was taken with a photographing apparatus before drying the 2.5cm wide test pad 62, and the result of the photograph is shown in fig. 5, in which: fig. 5 (a) is a picture obtained by photographing a test pad with a photographing apparatus before drying the test pad after adding a T-line solution to the test pad having a width of 2.5cm during the process of preparing a first batch of test cards; fig. 5 (b) is a picture obtained by photographing the test pad using a photographing apparatus before drying the test pad after adding the T-line solution to the test pad having a width of 2.5cm in the process of preparing the second batch of test cards; fig. 5 (c) is a picture obtained by photographing the test pad using a photographing apparatus before drying the test pad after adding the T-line solution to the test pad having a width of 2.5cm during the preparation of the third batch of test cards. After drying the 2.5cm wide test pad 62, before cutting the strip, the strip was irradiated with a laser flashlight so that T-line 63 fluoresces, and a photograph was taken with a photographing apparatus, the result of which is shown in fig. 6, where: fig. 6 (a) is a picture obtained by drying a test pad 2.5cm wide after adding a T-line solution, irradiating the strip with a laser torch before cutting the strip, so that the T-line fluoresces, and photographing with a photographing device during the preparation of the first batch of test cards; fig. 6 (b) is a drawing obtained by drying a test pad 2.5cm wide after adding a T-line solution, irradiating the strip with a laser torch before cutting the strip so that the T-line fluoresces, and photographing with a photographing device during the process of preparing the second batch of test cards; fig. 6 (c) shows a picture obtained by drying a 2.5cm wide test pad after adding a T-line solution, irradiating the strip with a laser torch before cutting the strip, and photographing the T-line with a photographing device during the process of preparing the third batch of test cards. After the addition of the T-line solution, the 2.5cm wide test pad 62 was dried, and three test cards obtained after cutting the strip plate (10 test cards per batch) were tested by the method of example 2 to obtain the fluorescent signal on the T-line 56, and the results are shown in table 1.

TABLE 1

As can be seen from Table 1, FIG. 5 and FIG. 6, the addition of a greater volume of T-line solution resulted in a wider T-line for the strip strips used to prepare the second batch of test cards, and thus a wider T-line for the second batch of test cards; the continuous addition of the T-line solution also results in the T-lines of the strip sheet used to prepare the first batch of test cards and the T-lines of the strip sheet used to prepare the second batch of test cards being continuous, and thus the T-lines of the first and second batches of test cards being continuous, so that the coefficient of variation between the 10 test cards selected from the two batches of test cards is relatively small, 3.01% for the former and 1.93% for the latter. The discontinuous addition of the T-line solution results in the T-line of the strip sheet used to prepare the third batch of test cards also being discontinuous, i.e., many discontinuities 64 occur in the T-line of the strip sheet. Breakpoint 64 need not be completely free of T-line solution, but the fluorescence signal at breakpoint 64 is significantly weaker than the fluorescence signal at the non-breakpoint on the T-line of the strip plate.

Since the position of the T-line for cutting the strip board is uncertain, the T-line of the third lot of test cards prepared therefrom may be continuous or discontinuous, and therefore, when 10 test cards are randomly selected from the third lot of test cards for testing, the coefficient of variation between the 10 test cards is relatively large, and is 15.94%.

Considering that the spotting speed of the second batch of test cards is twice that of the first batch of test cards, and the volume of the solution of the T-line added to the second batch of test cards is twice that of the first batch of test cards, theoretically, the fluorescence signal emitted by the T-line of the second batch of test cards should be twice that of the first batch of test cards, but actually limited by the constraints of factors such as the sensitivity of the analyzer, there are always some deviations, but should be close to 2. For the selected 10 test cards in each lot, the fluorescence signal from the T-line of the second lot was 1.785 to 2.016 times that of the first lot, indeed close to 2, regardless of their average or per test card intensity. Therefore, the difference between the dot solution quantity and the dot solution uniformity of the T-line solution coated on the T-line of the test strip plate under different dot solution conditions can be easily judged according to the fluorescent signal on the T-line of the test card.

Example 6: detecting the influence of collinear correction on the detection result

Fecal samples of calprotectin at low (L), medium (M) and high (H) concentrations were tested using the same batch of test cards prepared in example 1 according to the test method described in example 4, and the test was repeated 10 times for each sample, the results of which are shown in table 2. The samples are subjected to fixed value detection by utilizing calprotectin detection kit (enzyme linked immunosorbent assay) of Boerman laboratories, inc., and the obtained detection values are as follows: low concentration samples: 20.57 mu g/g; medium concentration samples: 211.45 mu g/g; high concentration samples: 513.25 μ g/g.

TABLE 2

As can be seen from table 2, the T/T' based on-line calibration accurately detected calprotectin concentrations, and the Coefficient of Variation (CV) of calprotectin concentration values detected over multiple iterations was significantly better than the T/C based off-line calibration for stool samples of either low, medium or high concentration calprotectin. This is because the calprotectin capture antibody and the chicken IgY antibody are coated on the T-line at one time when the test card is prepared, and therefore, even if the volume of the T-line solution coated on the T-line is too large or too small, the number of the calprotectin capture antibody and the chicken IgY antibody is too large or too small at the same time, and the difference in detection caused by too large or too small of the volume of the added T-line solution can be effectively eliminated by calculating the ratio of T/T'. In contrast, calprotectin capture antibody and rabbit anti-DNP antibody were not coated on the same line on the surface of the test pad: the former is added on the T-line and the latter on the C-line, so that it is difficult to simultaneously increase or decrease the amounts of calprotectin capture antibody and rabbit anti-DNP antibody added at the time of spotting. Furthermore, because the T-line and C-line are separated by a distance, the time for the sample to flow to the T-line and C-line is not the same after the sample is added, so the time for the time-resolved fluorescent microsphere accumulation on the T-line is not synchronized with the time for the time-resolved fluorescent microsphere accumulation on the C-line, in contrast to the time for the time-resolved fluorescent microsphere accumulation on the T-line and the time for the green fluorescent microsphere accumulation on the T-line when the sample flows to the T-line. The two factors make the collinear correction based on T/T' obviously better than the collinear correction based on T/C in detection precision.

Example 7: detecting the influence of collinear correction on the detection result

Stool samples of moderate concentration (M) calprotectin from example 6 were tested according to the T/T' based on-line calibration method of example 4 using the same batch of test cards of the present invention prepared in example 1, and the test was repeated 10 times for each sample, while stool samples of moderate concentration (M) calprotectin from example 6 were also tested according to the T/C based on-line calibration method of example 4 using the same batch of conventional test cards prepared in example 3, and the test results are shown in table 3 for each sample, with the results being repeated 10 times.

TABLE 3

In this example, the T/T' based inline correction and the T/C based offline correction were performed on different test cards, not on the same test card as in example 6. As can be seen from the data in table 3, for stool samples with moderate calprotectin concentrations, the T/T' based on-line correction accurately detected calprotectin concentrations, and the Coefficient of Variation (CV) of the calprotectin concentration values detected over multiple repetitions was significantly better than the T/C based on-line correction. In addition, in the conventional test card, the green fluorescent microspheres which are not coated with the goat anti-chicken IgY antibody are arranged on the label pad, and the chicken IgY antibody is not coated on the T-line, so that when the test card is added into a sample, a detection signal emitted by the time-resolved fluorescent microspheres captured on the T-line and a control signal emitted by the time-resolved fluorescent microspheres captured on the C-line are not interfered by a fluorescent signal (T ') emitted by the green fluorescent microspheres, but the detection precision of the T/T ' based collinear correction method is still better than that of the T/C based collinear correction method, which shows that the advantage is not caused by the interference of the T ' signal generated by the green fluorescent microspheres on the T signal and the C signal, but is due to the following two factors: (1) The T signal and the T 'signal are generated on the T line, and the detection difference caused by too much or too little solution volume of the T line added during the preparation of the test card can be effectively eliminated by calculating the ratio of T/T'; (2) In contrast to conventional test cards in which the time of accumulation of time-resolved fluorescent microspheres on line T and the time of accumulation of green fluorescent microspheres on line T are synchronized in the present invention, because line T and line C are spaced apart, the time of flow of the sample to line T and line C is not the same when the sample is added.

Claims

1. A detection reagent for an immunoassay strip, comprising a labeling reagent and a capture reagent, wherein the labeling reagent comprises a first signal label-labeled reagent and a second signal label-labeled third reagent, signals generated by the first signal label and the second signal label are distinguishable from each other, the first reagent specifically binds to an analyte, the capture reagent comprises a second reagent and a fourth reagent, both the second reagent and the fourth reagent are located on a T-line of the immunoassay strip, and the second reagent and the fourth reagent capture the first signal label-labeled reagent and the second signal label-labeled third reagent on the T-line, respectively; and correcting the detection signal from the first signal label captured on the same T-line by the correction signal from the second signal label captured on the T-line, wherein the second reagent carries a quality control label, and the signals generated by the quality control label, the first signal label and the second signal label are distinguishable from each other.

2. The detection reagent of claim 1, wherein the second reagent and the fourth reagent are independently present on the T-line.

3. The detection reagent of claim 1, wherein the second signal label is selected from the group consisting of lanthanides and chelates thereof, and platinum/palladium porphyrin compounds.

4. The detection reagent of claim 1, wherein the second signal label is selected from the group consisting of time-resolved luminescent microspheres.

5. The detection reagent of claim 1, wherein the second signal label is selected from the group consisting of a colored luminescent microsphere.

6. The detection reagent of claim 1, wherein the second signal label is selected from the group consisting of colored colloidal particles.

7. The detection reagent of claim 1, wherein the second signal label is selected from the group consisting of magnetic nanoparticles.

8. The detection reagent of claim 1, wherein the second signaling label is selected from the group consisting of luminescent compounds.

9. The detection reagent of claim 1, wherein the first signal label is selected from the group consisting of lanthanides and chelates thereof, and platinum/palladium porphyrin compounds.

10. The detection reagent of claim 1, wherein the first signal label is selected from the group consisting of time-resolved luminescent microspheres.

11. The detection reagent of claim 1, wherein the first signal label is selected from the group consisting of a colored luminescent microsphere.

12. The detection reagent of claim 1, wherein the first signal label is selected from the group consisting of colored colloidal particles.

13. The detection reagent of claim 1, wherein the first signal label is selected from the group consisting of magnetic nanoparticles.

14. The detection reagent of claim 1, wherein the first signaling label is selected from the group consisting of luminescent compounds.

15. The detection reagent according to claim 1, wherein the quality control label is selected from the group consisting of lanthanides and chelates thereof, and platinum/palladium porphyrin compounds.

16. The detection reagent of claim 1, wherein the quality control marker is selected from the group consisting of time-resolved fluorogenic microspheres.

17. The detection reagent of claim 1, wherein the quality control marker is selected from the group consisting of a colored luminescent microsphere.

18. The detection reagent according to claim 1, wherein the quality control label is selected from the group consisting of colored colloidal particles.

19. The detection reagent of claim 1, wherein the quality control label is selected from the group consisting of magnetic nanoparticles.

20. The detection reagent of claim 1, wherein the quality control marker is selected from luminescent compounds.

21. The detection reagent of claim 2, wherein when the analyte is an antigen, the first reagent and the second reagent are antibodies that specifically bind to the analyte, or the first reagent is an antibody that specifically binds to the analyte and the second reagent is a secondary antibody that specifically binds to the first reagent; when the analyte is an antibody, the first reagent and the second reagent are antigens specifically binding to the analyte, or one of the first reagent and the second reagent is an antigen specifically binding to the analyte and the other is a secondary antibody specifically binding to the analyte, or the first reagent is an antigen specifically binding to the analyte and the second reagent competes with the analyte for binding to the first reagent; when the analyte is a hapten, the first reagent is an antibody that specifically binds the analyte and the second reagent is a conjugate of the analyte or analog thereof and a carrier protein.

22. The test reagent of any of claims 1,2, or 21, wherein the immunoassay strip comprises a sample addition zone and a detection pad, the sample addition zone is located upstream of the detection pad, the detection pad has a T-wire, the labeling reagent is located between the sample addition zone or the sample addition zone and the T-wire and moves toward the T-wire during the test, and the capture reagent is coated on the T-wire, and the capture reagent captures a first reagent labeled with the first signal label and a third reagent labeled with the second signal label on the T-wire.

23. The detection reagent of claim 22, wherein the detection pad further comprises a C-line, the labeling reagent further comprises a fifth reagent labeled with a third signal label, and the sixth reagent is coated on the C-line and captures the fifth reagent labeled with the third signal label on the C-line.

24. The test reagent of claim 23, wherein the third/fourth reagent is selected from the group consisting of a non-human antibody/an anti-non-human antibody.

25. The detection reagent of claim 23, wherein the third/fourth reagent is selected from the group consisting of biotin/streptomycin.

26. The detection reagent of claim 23, wherein the third/fourth reagent is selected from DNP-BSA/anti-DNP antibody.

27. The detection reagent of claim 23, wherein the third/fourth reagent is selected from the group consisting of receptors/ligands.

28. The test reagent of claim 23, wherein the fifth/sixth reagent is selected from the group consisting of a non-human antibody/an anti-non-human antibody.

29. The detection reagent of claim 23, wherein the fifth/sixth reagent is selected from the group consisting of biotin/streptomycin.

30. The detection reagent of claim 23, wherein the fifth/sixth reagent is selected from DNP-BSA/anti-DNP antibody.

31. The detection reagent of claim 23, wherein the fifth/sixth reagent is selected from the group consisting of receptors/ligands.

32. The detection reagent of claim 23, wherein the third/fourth reagent is goat/chicken IgY antibody and the fifth/sixth reagent is DNP-BSA/rabbit anti-DNP antibody.

33. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of lanthanides and chelates thereof, and platinum/palladium porphyrin compounds.

34. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of time-resolved luminescent microspheres.

35. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of a colored luminescent microsphere.

36. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of colored colloidal particles.

37. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of magnetic nanoparticles.

38. The detection reagent of claim 23, wherein the third signal label is selected from the group consisting of luminescent compounds.

39. The detection reagent of claim 23, wherein the first signal label and the third signal label are time-resolved fluorescent microspheres and the second signal label is green fluorescent microspheres.