阅读说明：本技术 多联检免疫层析试剂卡及其应用和多联检免疫层析试剂盒 (Multi-joint detection immunochromatography reagent card, application thereof and multi-joint detection immunochromatography kit ) 是由 刘丽萍 李文静 于鸫 迟晨 于 2019-09-16 设计创作，主要内容包括：本发明提供了一种多联检免疫层析试剂卡及其应用和多联检免疫层析试剂盒,涉及生物检测技术领域,本发明提供的多联检免疫层析试剂卡包括依次设置的样品垫、结合垫、NC膜和吸水垫,能够避免多条T线引起的各个待检测项目的检测性能差异,能够保证每个待检测项目的性能保持一致性。(The invention provides a multi-detection immunochromatography reagent card, application thereof and a multi-detection immunochromatography kit, and relates to the technical field of biological detection.)

1. The multi-joint detection immunochromatography reagent card is characterized by comprising a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially arranged;

The combination pad is coated with at least one label-antibody complex, and the NC membrane comprises at least one T line and one C line.

2. The reagent card of claim 1, wherein at least 2 binding antibodies or competitive antigens are coated on the T line in a mixed manner;

The bound antibody is capable of binding to the label-antibody-sample complex from the conjugate pad;

The competing antigen is capable of competitively binding to the sample from the label-antibody complex in the conjugate pad, thereby immobilizing the labeling group on the T-line.

3. The reagent card of claim 1, wherein the C-line is coated with a capture antibody capable of capturing free label-antibody complexes.

4. The multi-joint detection immunochromatography reagent card according to claim 1, wherein the label in the label-antibody complex comprises a fluorescent label, a quantum dot label or a biotin label, and preferably the fluorescent label.

5. The reagent card of claim 2, wherein the T line is coated with one or more labeled binding antibodies of CK-MB, cTnI and Myo.

6. The reagent card of claim 2, wherein the T line is coated with PCT antibody and CRP antigen.

7. The multi-test immunochromatographic reagent card of claim 1, further comprising a support layer for bonding the sample pad, the conjugate pad, the NC membrane and the absorbent pad.

8. Use of a multi-test immunochromatographic reagent card according to any one of claims 1 to 7 for non-diagnostic/therapeutic purposes for the combined detection of different types of antigens.

9. A multiple-combination immunochromatography kit, which comprises the multiple-combination immunochromatography reagent card of any one of claims 1 to 7.

Technical Field

the invention relates to the technical field of biological detection, in particular to a multi-joint detection immunochromatography reagent card, application thereof and a multi-joint detection immunochromatography kit.

Background

At present, the multi-combination detection immunofluorescence chromatography kit which is on the market is mainly classified according to diseases such as cardiac muscle (cTnI/NT-proBNP), inflammation (PCT/CRP/SAA/IL-6), gastric function (PGI/PGII) and the like, and combined to form a two-combination detection or three-combination detection kit aiming at specific diseases. The multi-examination reagent box in the prior art generally has the following defects:

1. when the current multi-detection fluorescence immunochromatography reagent card mainly carries out signal capture by single fluorescein, and determines that an NC membrane coats antibodies, each item must coat a separate T-line antibody.

2. in the prior art, the coating condition of the multi-connected detection reagent card is mainly formed by combining a control line C and a plurality of detection lines T, and each T line is coated with an antibody capable of combining a single-item antigen. The NC film coating condition is complex, the special requirement is required for a coating machine (for example, the number of coating pump heads needs at least 3 or 4), the coating cost is high, and the coating process is complex.

3. The characteristic that a plurality of T lines are coated determines that the NC film of the reagent card to be detected needs a certain length, certain material selection limitation exists, due to the limitation of the length, at most 3-4T lines can be coated, and joint inspection of more items cannot be realized.

4. When a plurality of T lines are coated by the multi-joint inspection reagent card, the requirement that the performances of all projects to be inspected are consistent cannot be met at the same time, the performances of all the projects are different, and the performances of the projects which are generally coated behind the multi-joint inspection reagent card have certain loss.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is a first object of the present invention to provide a multi-test immunochromatography reagent card to alleviate at least one of the technical problems of the prior art.

The second purpose of the invention is to provide the application of the multi-joint detection immunochromatography reagent card for the non-diagnosis/treatment purpose of joint detection of different types of antigens.

The third purpose of the invention is to provide a multi-detection immunochromatography kit, which can jointly detect various antigens with different types, realize joint detection of different types of diseases, and has low cost and easy popularization and application.

The invention provides a multi-joint detection immunochromatography reagent card, which comprises a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially arranged;

The combination pad is coated with at least one label-antibody complex, and the NC membrane is provided with at least one T line and one C line.

furthermore, the multi-joint detection immunochromatography reagent card is provided with a T line.

Further, a plurality of binding antibodies or competitive antigens are coated on the T line in a mixed mode;

The bound antibody is capable of binding to the label-antibody-sample complex from the conjugate pad;

The competing antigen is capable of competitively binding to the sample from the label-antibody complex in the conjugate pad, thereby immobilizing the labeling group on the T-line.

Further, a capture antibody capable of capturing the free label-antibody complex is coated on the C-line.

Further, the label in the label-antibody complex comprises a fluorescent label, a quantum dot label or a biotin label, preferably a fluorescent label.

Further, the T-line is coated with a labeled binding antibody of one or more of CK-MB, cTnI, and Myo.

Further, the T-line was coated with PCT antibody and CRP antigen.

Further, the multi-joint detection immunochromatography reagent card also comprises a bearing layer for bonding the sample pad, the combination pad, the NC membrane and the water absorption pad.

The invention also provides the application of the multi-joint detection immunochromatography reagent card in the joint detection of different types of antigens for non-diagnosis/treatment purposes.

In addition, the invention also provides a multi-joint detection immunochromatography kit, which comprises the multi-joint detection immunochromatography reagent card.

The invention provides a multi-joint detection immunochromatography reagent card which comprises a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially arranged, wherein at least one marker-antibody compound is coated on the combination pad, and the NC membrane is provided with a T line and a C line. The multi-joint detection immunochromatography reagent card provided by the invention only comprises one T line and one C line, and can realize the joint detection of different types of diseases by utilizing different detection results of different markers. And the NC membrane only needs to be coated with two lines (a T line and a C line), so that compared with the existing multi-joint inspection product, the coating process is simplified, the coating time is saved, only 2 pump heads of a coating machine are required, the machine cost is saved, and no specific requirement is imposed on the length of the NC membrane. In addition, the multi-joint detection immunochromatography reagent card only coats one T line, so that the detection performance difference of each item to be detected caused by a plurality of T lines can be avoided, and the consistency of the performance of each item to be detected can be ensured.

Based on the same principle as the multi-joint detection immunochromatography reagent card provided by the invention, the multi-joint detection immunochromatography kit provided by the invention has all the beneficial effects of the multi-joint detection immunochromatography reagent card provided by the invention, and the details are not repeated herein.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1A is a diagram showing the result of CK-MB single-test provided in Experimental example 1 of the present invention;

FIG. 1B is a diagram showing the result of the CK-MB multi-test provided in Experimental example 1 of the present invention;

FIG. 2A is a chart showing the results of a single test of cTnI provided in Experimental example 1 of the present invention;

FIG. 2B is a diagram showing the result of cTnI multi-plex assay provided in Experimental example 1 of the present invention;

FIG. 3A is a Myo single test result chart provided in Experimental example 1 of the present invention;

FIG. 3B is a chart showing the results of Myo multi-plex assay provided in Experimental example 1 of the present invention;

FIG. 4A is a diagram showing the results of PCT single test provided in Experimental example 2 of the present invention;

FIG. 4B is a diagram showing the results of PCT multi-plex assay provided in Experimental example 2 of the present invention;

FIG. 5A is a graph showing the results of a CRP single test provided in Experimental example 2 of the present invention;

Fig. 5B is a graph showing the results of CRP multi-combination test provided in experimental example 2 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. Furthermore, the use of the term "including" and other forms is not limiting.

generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to one aspect of the invention, the multi-joint detection immunochromatography reagent card comprises a sample pad, a combination pad, an NC membrane and a water absorption pad which are arranged in sequence;

The combination pad is coated with at least one label-antibody complex, and the NC membrane is provided with a T line and a C line.

The multi-joint detection immunochromatography reagent card provided by the invention only comprises one T line and one C line, and can realize the joint detection of different types of diseases by utilizing different detection results of different markers. And the NC membrane only needs to be coated with two lines (a T line and a C line), so that compared with the existing multi-joint inspection product, the coating process is simplified, the coating time is saved, only 2 pump heads of a coating machine are required, the machine cost is saved, and no specific requirement is imposed on the length of the NC membrane. In addition, the multi-joint detection immunochromatography reagent card only coats one T line, so that the detection performance difference of each item to be detected caused by a plurality of T lines can be avoided, and the consistency of the performance of each item to be detected can be ensured.

It is to be understood that in the present invention, "coating" is an immunological term, having the meaning of immobilization and/or adsorption.

In the present invention, the "T line" is a test detection line, and the "C line" is a control line.

In the present invention, the "label-antibody complex" refers to a substance in which a label is complexed with an antibody, wherein the label and the antibody may be bound by a peptide bond or other means conventional in the art, and the present invention is not limited thereto.

The water absorption layer comprises a strong water absorption substance, and can provide directional power for a sample to be detected through the water absorption of the strong water absorption substance, so that the sample to be detected sequentially passes through the combination pad and the NC membrane. The material of the water absorbing layer is not limited, and any material with strong water absorbing property can be used as the water absorbing layer of the embodiment. The sample pad is used for contacting with a sample to be detected, so that the sample to be detected can be more uniformly combined with the marker-antibody compound on the combination pad when moving to the combination pad through the directional power, and the accuracy of a detection result is ensured. In this embodiment, the material of the sample pad is not limited, and a typical sample pad may be glass fiber wool or a nylon film.

It should be noted that, in the present invention, the sample pad, the bonding pad, the NC membrane, and the absorbent pad are sequentially disposed, that is, the sample pad is connected to one end of the bonding pad away from the NC membrane, so as to ensure that the sample to be measured can move to the bonding pad by directional power. One end of the conjugate pad and one end of the NC membrane are tightly connected to ensure that free label-antibody complexes and label-antibody-sample complexes can transition from the conjugate pad to the NC membrane. The water absorption pad is tightly connected with one end of the NC membrane far away from the combination pad, so that the directional power can be provided for the sample to be detected through the strong water absorption property of the water absorption pad.

In the present invention, the "marker-antibody complex" may refer to a marker-antibody complex, and may also refer to a type of marker-antibody complex. When referring to a class of label-antibody complexes, wherein the detection principle for each label-antibody complex is the same, e.g. both using a double antibody sandwich or both using a competition method; wherein the label of each antibody complex is different, e.g., the label-antibody complex may comprise an organofluorescein a-antibody a complex, an organofluorescein B-antibody B complex, and the like.

in some preferred embodiments, the T-line is coated with a plurality of binding or competing antibodies in a mixed fashion;

The bound antibody is capable of binding to the label-antibody-sample complex from the conjugate pad;

the competing antigen is capable of competitively binding to the sample from the label-antibody complex in the conjugate pad, thereby immobilizing the labeling group on the T-line.

In the present invention, the sample refers to a test sample, i.e., a test antigen.

the label-antibody-sample complex refers to a substance formed by complexing a sample (antigen to be detected) and a label-antibody complex, wherein the sample (antigen to be detected) and the label-antibody complex can be bound by peptide bond or other means conventional in the art, and the present invention is not limited thereto.

in the present invention, the "bound antibody" is capable of capturing the label-antibody-sample complex, thereby immobilizing the label-antibody-sample complex on the T-line to form a double antibody sandwich complex. By using a double-antibody sandwich method, the requirement of joint detection can be met by different detection results of different markers. The "binding antibody" is not particularly limited as long as it can specifically bind to the label-antibody-sample complex, and may be, for example, but not limited to, a PCT antibody, a cTNI antibody, or the like.

In the present invention, the "competitive antigen" is capable of competitively binding to the sample from the label-antibody complex in the conjugate pad, thereby immobilizing the label group on the T-line. By utilizing a competition method, the requirement of joint detection can be met through different detection results of different markers. The "competing antigen" is not particularly limited as long as it can specifically bind to the free label-antibody complex.

it should be noted that, although the T-line in the multi-test immunochromatography reagent card provided by the present invention includes both the binding antibody and the competitive antigen, in practical applications, only the binding antibody may be selected according to the property of the sample to be detected, and detection may be performed by the double antibody sandwich method, or only the competitive antigen may be used, and detection may be performed by the competitive method, or both the binding antibody and the competitive antigen may be used, and detection may be performed by the double antibody sandwich method and the competitive method.

In some preferred embodiments, the antigen to be tested is one or more, preferably more than one.

When the antigen to be detected is a plurality of types, the detection principle of each of the antigens to be detected is the same, for example, the detection is performed by using a double antibody sandwich method or a competition method. When the antigens to be detected are various, the multi-joint detection immunochromatography reagent card provided by the invention can realize simultaneous detection of a plurality of items, and the detection efficiency is higher.

in some preferred embodiments, the C-line is coated with a capture antibody capable of capturing free label-antibody complexes.

In the present embodiment, the "capture antibody" is capable of capturing the free label-antibody complex, thereby immobilizing the free label-antibody complex on the C-line.

The "capture antibody" is not particularly limited as long as it can specifically bind to a free label-antibody complex, and examples thereof include, but are not limited to, goat anti-mouse antibody, rabbit anti-mouse antibody, and goat anti-rabbit antibody.

In some preferred embodiments, the label in the label-antibody complex comprises a fluorescent label, a quantum dot label, or a biotin label, preferably a fluorescent label. When the fluorescent marker is selected as the marker, different fluorescent group-antibody complexes can be formed by respectively coating the multiple examination items by utilizing different excitation wavelengths and emission wavelengths of different fluorescent markers, and the multiple examination items are mixed and coated on the same T line, so that the joint examination is realized.

the fluorescent label may be, for example, but not limited to Cy3, Cy5, Fitc, AMC, CMK, FMK, or the like.

In some preferred embodiments, the T-line is coated with a labeled binding antibody of one or more of CK-MB, cTnI, and Myo.

It is understood that the detection principles of CK-MB (creatine phosphokinase-MB isozyme), cTnI (cardiac troponin i) and Myo (myoglobin) are the same, e.g.both using a double antibody sandwich method or both using a competition method.

Wherein, "one or more" means that the T-line may be coated with a CK-MB labeled binding antibody, a cTnI labeled binding antibody, or a Myo labeled binding antibody, or a combination comprising a CK-MB labeled binding antibody and a cTnI labeled binding antibody, or a combination comprising a CK-MB labeled binding antibody and a Myo labeled binding antibody, or a combination comprising a cTnI labeled binding antibody and a Myo labeled binding antibody, or a combination comprising a CK-MB labeled binding antibody, a cTnI labeled binding antibody, and a Myo labeled binding antibody.

In some preferred embodiments, the T-line is coated with a PCT antibody and a CRP antigen.

PCT (procalcitonin) and CRP (C reactive protein ) are joint inspection items with different sample addition amounts and sample addition modes, and specifically, in the double-antibody sandwich single inspection, a PCT sample does not need to be diluted, while a CRP sample needs to be diluted, so that the joint inspection is difficult.

in some preferred embodiments, the multi-test immunochromatography reagent card further comprises a support layer for binding the adsorption layer.

The bearing layer can bear the adsorption layer, and the detection is more convenient to carry out.

In the present embodiment, the material of the support layer is not limited, and any material having a solid phase form and not affecting the antigen-antibody reaction may be used, and examples thereof include, but are not limited to, a plastic plate and a glass plate.

In addition, the invention also provides a multi-joint detection immunochromatography kit which comprises the multi-joint detection immunochromatography reagent card.

Based on the same principle as the multi-joint detection immunochromatography reagent card provided by the invention, the multi-joint detection immunochromatography kit provided by the invention has all the beneficial effects of the multi-joint detection immunochromatography reagent card provided by the invention, and the details are not repeated herein.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagents and instrument information used in the embodiment of the invention are as follows:

Fluorescein was purchased from ANASPEC under the model numbers HiLyte Fluor 405, HiLyte Fluor532 and HiLyte Fluor 750. The sample pad was purchased from Shanghai Jie-I under model XQ-Y1, the conjugate pad was purchased from Shanghai Jie-I under model GL0194, the absorbent pad was purchased from Shanghai Jie-I under model H5072, and the nitrocellulose membrane was purchased from Millipore under model Hi-FlowPlusHF 135.

The spraying film system adopts an XYZ3050 spraying film system of BioDot, and the adopted antigens/antibodies are self-produced antigens and antibodies of the Fipeng biological corporation. The cTnI antibody model is cTnI-MCAb-9#, cTnI-MCAb-11#, the MYO antibody model is MYO-Ab2, MYO-Ab2, the CK-MB antibody model is CK-MB-3, CK-MB-4, the CRP antigen model is CRP-Ag, the CRP antibody model is CRP-Ab7#, the PCT antibody model is PCT-Ab4#, and the PCT-Ab7 #.