CN211505565U - Quantum dot-labeled rapid immunochromatographic test strip for detecting type I Shiga toxin - Google Patents

Abstract

The utility model discloses a quantum dot mark rapid immunochromatographic test strip for detecting Shiga toxin I, which comprises a test strip main body, wherein the test strip main body comprises a rubber plate, and a sample pad, a quantum dot immune pad, a nitrocellulose membrane and a water absorption pad which are arranged on the rubber plate and are sequentially connected; the nitrocellulose membrane is provided with a detection T line and a quality control C line, wherein the detection T line is arranged close to the quantum dot immune pad, and the quality control C line is arranged close to the water absorption pad; the quantum dot immune pad is embedded with a quantum dot marked Shiga toxin I monoclonal antibody, a detection T line is coated with the Shiga toxin I monoclonal antibody, and a quality control C line is coated with a goat anti-mouse polyclonal antibody (goat anti-mouse IgG). The test strip provided by the utility model can detect the type I Shiga toxin, and the using method is simple, quick and low in cost, and does not need to add bacteria, separate strains, carry out biochemical identification and other lengthy steps.

Description

Quantum dot-labeled rapid immunochromatographic test strip for detecting type I Shiga toxin

Technical Field

The utility model relates to a biochemical analysis detects technical field, concretely relates to quick immunochromatography test paper strip of quantum dot mark for detecting type I shiga toxin.

Background

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen, and causes diarrhea, Hemorrhagic enteritis (HC), Thrombotic Thrombocytopenic Purpura (TTP), and lethal Hemolytic Uremic Syndrome (HUS) Syndrome after human infection. The STEC epidemic situation is outbreaked in 1999 + 2000 in the three provinces border areas of Jiangsu, Anhui and Shandong, which causes great personnel property loss; the epidemic has been developed many times in countries such as the United states, Europe, Japan, etc., and the main serotypes thereof are O157, O104, etc. Studies on the pathogenic mechanism of STEC have shown that Shiga toxin (Stx) is one of the main pathogenic factors causing STEC infection, and it can penetrate into the blood circulation through the damaged intestinal epithelial cells, bind to the epithelial cell receptor of the target organ, triosephylsphingosine (Globotriosylceramide, Gb3) or butosephylsphingosine (Gb 4), cause damage to organs such as intestinal tract and central nervous system, cause HUS in severe cases, endanger the life of patients, and have the characteristic of fulminant epidemic infection, so STEC infection has become a public health problem of global concern. Clinically, there are no effective treatment or prevention of STEC infection, and antibiotic use can aggravate the condition and increase the incidence of HUS, especially in infants and elderly patients. As a material basis for eliciting HUS, Stx is recognized as a targeting molecule for diagnosis and treatment of STEC infection, and is divided into StxI and StxII 2 subtypes, wherein StxI has three varieties (stxla, stxlc, stxld) and StxII has 7 varieties. Since cases caused by StxI are still common, the detection of StxI is a very important task and should be paid enough attention. In the traditional detection means, the StxI detection is generally realized by carrying out pathogen isolation culture on a patient excrement sample, but the method has the defects of complicated operation steps, long time consumption and incapability of meeting the requirement of on-site rapid diagnosis. Therefore, the development of a test strip capable of rapidly detecting shiga toxin type I is a technical problem to be solved urgently.

SUMMERY OF THE UTILITY MODEL

The method aims to solve the problems of time and labor waste, serotype dependence, low sensitivity and the like of the traditional detection method of the type I Shiga toxin. The utility model provides a short, the easy and simple to handle quick immunochromatography test paper strip of quantum dot mark for detecting type I shiga toxin consuming time.

The utility model discloses a quantum dot mark rapid immunochromatographic test strip for detecting Shiga toxin type I, which comprises a test strip main body, wherein the test strip main body comprises a rubber plate and a sample pad, a quantum dot immune pad, a nitrocellulose membrane and a water absorption pad which are arranged on the rubber plate and are sequentially connected; a detection T line and a quality control C line are arranged on the nitrocellulose membrane, wherein the detection T line is arranged close to the quantum dot immune pad, and the quality control C line is arranged close to the water absorption pad; the quantum dot immune pad is embedded with a quantum dot marked Shiga toxin I monoclonal antibody, the detection T line is coated with the Shiga toxin I monoclonal antibody, and the quality control C line is coated with a goat anti-mouse polyclonal antibody (goat anti-mouse IgG).

Further, the sample pad is a glass fiber membrane layer, the quantum dot immune pad is a nitrocellulose membrane layer, and the rubber plate is a polyvinyl chloride membrane layer (PVC). In the arrangement, the materials of the sample pad, the quantum dot immune pad and the rubber plate are specifically limited, and the detection result is accurate and reliable in the arrangement.

Further, still include the protection casing, the protection casing is including dismantling last casing and the lower casing of connection, go up the casing with lower casing form can hold the cavity of test paper strip main part, the offset plate place one side in the test paper strip main part is close to the casing setting down. In the arrangement, the protective shell is arranged outside the test strip main body, so that the test strip is protected, and the influence of the external environment on the test strip is reduced to the maximum extent.

Furthermore, the upper shell and the lower shell are connected through a buckle. In the arrangement, the upper shell and the lower shell are more convenient to disassemble and use by adopting the buckle structure.

Furthermore, the upper shell is provided with a track groove along the length direction of the upper shell, the lower shell is provided with a slide bar structure along the length direction of the lower shell, and the slide bar structure corresponds to the track groove. In the arrangement, the combination of the slide bar structure and the track groove structure is adopted, the structure is simple, and the use is convenient.

The test strip further comprises a spacer, the spacer is arranged in the cavity, and the spacer is arranged in the lower shell and used for shielding the test strip main body; set up on the spacer in having application of sample hole and visual window, application of sample hole with the sample pad corresponds the setting, the visual window with detect the T line with quality control C line corresponds the setting, application of sample hole with the visual window is for running through the through-hole of spacer. In this kind of setting, set up the spacer that is used for sheltering from the test paper strip main part to set up application of sample hole and visual window on the spacer, thereby can realize the application of sample under the condition of not opening the spacer, and can look over the testing result through the visual window.

Further, still include the application of sample hole extension, the application of sample hole extension is in the spacer is close to towards on test paper strip main part one side the test paper strip direction sets up, application of sample hole extension is provided with the poroid structure with application of sample hole intercommunication, application of sample hole extension offsets with test paper strip main part. In the arrangement, the extended part of the sample adding hole is designed, so that a sample added in the detection process can flow onto the sample pad more smoothly, and the accuracy of the detection result is ensured.

Furthermore, the sample adding hole and the porous structure are integrally a flat-top conical hole, wherein the top of the flat-top conical hole is abutted to the test strip main body. In the arrangement, the whole of the sample adding hole and the porous structure is a flat-top conical hole, so that the sample can flow onto the sample pad more smoothly.

Furthermore, the whole spacer is made of elastic material, and the spacer is connected with the lower shell in an interference fit manner. In the arrangement, the isolating sheet is made of elastic materials and is connected with the lower shell in an interference fit mode, so that the isolating sheet can better play a role of comprising the test paper strip main body.

Furthermore, a plurality of fixture block structures are arranged in the lower shell, and at least two fixture block structures are respectively arranged close to two side walls in the length direction of the lower shell; the end faces, close to the spacers, of the fixture block structures are abutted to the spacers, and the side faces of the fixture block structures are abutted to the sample test strip and used for fixing the test strip main body and supporting the spacers. In the arrangement, the fixture block structure can fix the test strip main body to prevent the test strip main body from shaking, so that the stability of the whole structure and performance is ensured; meanwhile, the fixture block structure can also support the spacer, so that the spacer is not contacted with the test strip main body, and the spacer is prevented from rubbing the test strip main body to influence the overall property of the test strip.

Furthermore, a transparent part is arranged on the upper shell, and the transparent part and the visual window are correspondingly arranged. In the arrangement, the transparent part is arranged, and the detection structure can be observed under the condition that the upper shell is covered, so that the upper shell can be covered in the detection process, the detection environment is not influenced by the external environment, and the accuracy of the result is ensured.

Compare with the detection technology based on immunology of tradition, the utility model provides a test paper strip has following advantage: based on the quantum dot labeled rapid immunochromatographic test strip for detecting the type I Shiga toxin, a rapid, simple and accurate detection method can be established, so that the limitation of the traditional method for identifying the thalli through biochemical experiments and serotype classification can be broken through; solves the problems of time and labor waste, serotype dependence, low sensitivity and the like of the traditional and conventional detection methods. Based on the utility model provides a test paper strip detects type I shiga toxin, and the method is simple quick, with low costs, need not to increase the tedious steps such as fungus, isolate bacterial strain, biochemical identification, can directly or indirectly detect toxin protein from patient's sample to effectively shorten the check-out time from traditional approach's 2 ~ 3 weeks to within 15min, can win the valuable time for patient's treatment, control epidemic situation. To sum up the utility model provides a test paper strip has extensive market prospect and very big economic effect.

Drawings

Fig. 1 is a schematic diagram of an overall structure of a test strip main body in a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in an embodiment of the present invention;

fig. 2 is a schematic perspective view of the overall structure of a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in an embodiment of the present invention;

FIG. 3 is a schematic cross-sectional structure view of the quantum dot labeled rapid immunochromatographic test strip for detecting Shiga toxin type I in FIG. 2;

fig. 4 is a schematic view of the overall structure of the open state of the casing on the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

fig. 5 is a schematic diagram of the overall structure of the upper case of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

fig. 6 is a schematic view of the overall structure of the lower case of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

fig. 7-9 are schematic diagrams of the overall structure of three kinds of spacers in the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin I in the embodiment of the present invention;

fig. 10 is a schematic perspective view of the overall structure of a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in an embodiment of the present invention;

FIG. 11 is a schematic cross-sectional structure view of the quantum dot labeled rapid immunochromatographic test strip for detecting Shiga toxin type I in FIG. 10;

fig. 12 is a schematic structural diagram of the open state of the casing on the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

fig. 13 is a schematic diagram of the overall structure of the upper case of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

fig. 14 is a schematic view of the overall structure of the lower case of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the embodiment of the present invention;

in the figure, 100, a test strip body; 110. a sample pad; 120. a quantum dot immune pad; 130. a nitrocellulose membrane; 131. detecting a T line; 132. a quality control line C; 140. a water absorbent pad; 150. a rubber plate; 200. a protective housing; 210. an upper housing; 211. an upper housing floor; 212. the short side plate of the upper shell; 213. a long side plate of the upper shell; 214. a protrusion; 215. an open structure; 216. a track groove; 217. a transparent portion; 220. a lower housing; 221. a lower housing floor; 222. the short side plate of the lower shell; 223. a lower shell long side plate; 224. a groove; 225. a slide bar structure; 300. a separator; 310. a sample application hole; 320. a visual window; 330. a sample application extension; 340. a handle; 400. a clamping block structure; 500. and (7) a plug.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to the accompanying drawings, examples and experimental examples. In the present invention, the antibody coating is a conventional technical means in the field, and is not particularly limited herein, and those skilled in the art can select and prepare the antibody according to actual needs. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.

The embodiment of the utility model provides an in used reagent and instrument and equipment's source: goat anti-mouse IgG antibody, PVC rubber plate, absorbent paper and glass fiber membrane were purchased from Shanghainej, Biotechnology GmbH; nitrocellulose membranes were purchased from PALL, usa; the three-dimensional plane film scribing instrument and the slitting machine are purchased from Shanghai gold-labeled Biotech Co.

Example 1

Fig. 1 shows an overall structure diagram of a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in embodiment 1 of the present invention, which is a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I, and includes a test strip main body 100, where the test strip main body 100 includes a rubber plate 150, and a sample pad 110, a quantum dot immune pad 120, a nitrocellulose membrane 130, and a water absorption pad 140, which are disposed on the rubber plate 150 and connected in sequence; a detection T line 131 and a quality control C line 132 are arranged on the nitrocellulose membrane 130, wherein the detection T line 131 is arranged close to the quantum dot immune pad 120, and the quality control C line 132 is arranged close to the absorbent pad 140; the quantum dot immune pad 120 is embedded with a quantum dot-labeled Shiga toxin I monoclonal antibody, the detection T line 131 is coated with a Shiga toxin I monoclonal antibody, and the quality control C line 132 is coated with a goat anti-mouse polyclonal antibody.

Preferably, as shown in fig. 1, the nitrocellulose membrane 130 is disposed close to the gel plate 150, two ends of the nitrocellulose membrane 130 in the length direction of the test strip are respectively connected to the quantum dot immune pad 120 and the absorbent pad 140, and one end of the quantum dot immune pad 120 away from the nitrocellulose membrane 130 is connected to the sample pad 110; the sample pad 110, the quantum dot immune pad 120, the nitrocellulose membrane 130, and the absorbent pad 140 are partially overlapped at the phase connection. In the present invention, the test strip length direction refers to the direction from the free end of the sample pad 110 to the free end of the absorbent pad 140.

In a preferred embodiment, the distance between the detection T line 131 and the quality control C line 132 is 5 mm.

In a preferred embodiment, the sample pad 110 is a glass fiber membrane layer, the quantum dot immune pad 120 is a nitrocellulose membrane 130, and the glue board 150 is a polyvinyl chloride (PVC) glue board 150. The materials of the sample pad 110, the quantum dot immune pad 120 and the rubber plate 150 are selected based on the types of the test samples, the shiga toxin I monoclonal antibody of the quantum dot markers embedded on the quantum dot immune pad 120, the shiga toxin I monoclonal antibody coated on the detection T line 131 on the nitrocellulose membrane 130, the goat anti-mouse secondary antibody coated on the quality control C line 132 and other factors, so that the sensitivity of the test strip and the accuracy of the detection structure are ensured.

Example 2

Fig. 2 is a schematic overall perspective view of a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in this embodiment, a protective casing 200 is added on the basis of embodiment 1, the protective casing 200 includes an upper casing 210 and a lower casing 220 that are detachably connected, the upper casing 210 and the lower casing 220 form a cavity that can accommodate the test strip main body 100, and one side of the test strip main body 100 where the rubber plate 150 is located is disposed near the lower casing 220. The protective casing 200 has a good supporting and protecting effect on the test strip main body 100, and the influence of the external environment on the test strip main body 100 is avoided.

As a further improvement of the housing, a snap connection is adopted between the upper housing 210 and the lower housing 220. Fig. 3 is a schematic sectional view of a quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in the present embodiment, and fig. 4 is a schematic overall structural view of an open state of the upper case 210 in the present embodiment; fig. 5 and 6 are schematic views of the overall structures of the upper and lower cases 210 and 220, respectively. As shown in fig. 2, the protective case 200 in the present embodiment has a cubic structure as a whole. As shown in fig. 5 and 6, the upper case 210 in the present embodiment includes an upper case bottom plate 211 and upper case 210 side plates, and the lower case 220 includes a lower case bottom plate 221 and lower case 220 side plates, wherein the upper case 210 side plates include two upper case short side plates 212 oppositely disposed and two upper case long side plates 213 oppositely disposed; wherein the lower case 220 side plates include two lower case short side plates 222 disposed opposite to each other and two lower case long side plates 223 disposed opposite to each other; wherein the upper housing 210 is sleeved on the lower housing 220. Further, the upper case short side plate 212 is provided with a projection 214 on the face facing the inside of the upper case 210 as shown in fig. 5, the projection 214 being provided in the width direction of the upper case 210; the lower casing short side plate 222 is provided with a groove 224 in a direction towards the outside of the lower casing 220 as shown in fig. 6, wherein the groove 224 is provided in correspondence with the protrusion 214; such an arrangement is simple in structure, and the detachable connection of the upper case 210 and the lower case 220 can be well achieved. Preferably, the opening structures 215 are arranged on the short side plate 212 of the upper shell at the positions corresponding to the two ends of the protrusion 214, so that the short side plate 212 of the upper shell at the position of the protrusion 214 is more easily deformed under the condition of applying an external force, thereby facilitating the detachment of the upper shell 210 and the lower shell 220.

In some preferred embodiments, as shown in fig. 3, the test strip in this embodiment further includes a spacer 300, as shown in fig. 7, the spacer 300 in this embodiment is schematically shown in the overall structure of the test strip in this embodiment, as shown in fig. 3, the spacer 300 in this embodiment is disposed in the cavity, and the spacer 300 is disposed in the lower housing 220 for shielding the test strip main body 100; the spacer 300 is provided with a sample hole 310 and a visual window 320, the sample hole 310 corresponds to the sample pad 110, the visual window 320 corresponds to the detection T line 131 and the quality control C line 132, and the sample hole 310 and the visual window 320 are through holes penetrating through the spacer 300.

As shown in fig. 7, in this embodiment, an extension of the sample adding hole 310 is further provided, the extension of the sample adding hole 310 is disposed toward the test strip direction on the side of the spacer 300 close to the test strip main body 100, the extension of the sample adding hole 310 is provided with a hole-like structure communicated with the sample adding hole 310, and the extension of the sample adding hole 310 abuts against the test strip main body 100.

Fig. 8 and 9 show the overall structure of the spacer 300 according to another embodiment of the present invention, and fig. 8 and 9 show that the sample adding hole 310 and the hole structure are a flat-top taper hole, wherein the top of the flat-top taper hole is against the test strip main body 100.

Preferably, in this embodiment, the whole of the spacer 300 is made of an elastic material, and the spacer 300 is connected with the lower housing 220 in an interference fit manner. The spacer 300 may be made of silica gel or rubber, and other materials that can be used to manufacture the resilient spacer 300, and those skilled in the art can select the material as required, and the present invention is not limited to this. To facilitate removal of the spacer 300 from the lower housing 220, a handle 340 may be provided on the side of the spacer 300 remote from the test strip body 100 as shown in FIGS. 7-9. As shown in fig. 7, the handle 340 is connected to the spacer 300 by a hinge, so that the handle 340 can be folded onto the spacer 300.

Further preferably, in this embodiment, a plurality of fixture block structures 400 are disposed in the lower housing 220, and at least two fixture block structures 400 are disposed close to two side walls of the lower housing 220 in the length direction, respectively; the end surfaces of the fixture block structures 400 close to the spacer 300 are abutted against the spacer 300 for supporting the spacer 300. In this embodiment, four fixture block structures 400 are respectively disposed at four corners of the lower housing 220, that is, four fixture block structures 400 are provided in this embodiment, and each fixture block structure 400 is a cubic structure, wherein one surface close to the spacer 300 is a first surface of the fixture block structure 400, wherein one surface close to the test strip main body 100 is a second surface of the fixture block structure 400, wherein the first surface of the fixture block structure 400 abuts against the spacer 300, and the second surface of the fixture block structure 400 abuts against a side surface of the test strip main body 100. Therefore, the fixture block structure 400 in this embodiment can play a role of supporting the spacer 300 while fixing the test strip body 100. And because the arrangement of fixture block structure 400, there is a clearance between test paper main part 100 and lower casing long side board 223 to thereby be convenient for take out the test paper in lower casing 220, thereby be convenient for protect the reuse of casing 200, resources are saved reduce cost.

Example 3

The overall structure of the test strip in this embodiment is the same as that in embodiment 2, except for the structure and connection manner of the upper housing 210 and the lower housing 220. Fig. 10 is a perspective schematic view of the overall structure of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in this embodiment, and fig. 11 is a schematic sectional structure of the quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in this embodiment; fig. 12 is a schematic structural diagram illustrating the open state of the upper case 210 of the test strip in this embodiment. In the present embodiment, the upper housing 210 and the lower housing 220 are detachably connected by a sliding connection. Fig. 13 and 14 are schematic diagrams illustrating overall structures of the upper housing 210 and the lower housing 220 in this embodiment, respectively, in which the upper housing 210 in this embodiment is provided with a track groove 216 along a length direction of the upper housing 210, the lower housing 220 is provided with a slide bar structure 225 along a length direction of the lower housing 220, and the slide bar structure 225 is arranged corresponding to the track groove 216. As shown in fig. 13 and 14, in the present embodiment, the upper case 210 includes an upper case bottom plate 211 and upper case 210 side plates, and the lower case 220 includes a lower case bottom plate 221 and lower case 220 side plates, wherein the upper case 210 side plates include two oppositely disposed upper case short side plates 212 and two oppositely disposed upper case long side plates 213; wherein the lower case 220 side plates include two lower case short side plates 222 disposed opposite to each other and two lower case long side plates 223 disposed opposite to each other. In this embodiment, two rail grooves 216 are provided on the inner side surfaces of the long side plates 213 of the upper case, respectively; the two sliding bar structures 225 are respectively arranged on the outer side surface of the long side plate 223 of the lower shell, and the upper shell 210 and the lower shell 220 are closed and opened through the sliding between the track groove 216 and the sliding bar structures 225. Further, in this embodiment, a sliding inlet of the lower shell 220 is provided on one of the short side plates 212 of the upper shell, and in this embodiment, a plug 500 for plugging the sliding inlet is further provided, and the plug 500 is in interference fit with the sliding inlet.

Further, in the case of covering the upper case 210, in order to facilitate observation of the detection, the upper case 210 is provided with a transparent portion 217, and the transparent portion 217 is provided corresponding to the visual window 320.

The specific detection principle of the quantum dot labeled rapid immunochromatographic test strip in the utility model is as follows: in the double-antibody sandwich method adopted by the test strip in the embodiment, if a detection sample is positive, an antigen Shiga toxin I and a Shiga toxin I type monoclonal antibody marked by a quantum dot embedded on a quantum dot immune pad are combined to form a compound, the compound moves forwards along the test strip paper under the action of chromatography, and reacts with the Shiga toxin I type monoclonal antibody coated on a detection T line when passing through the detection T line to form an immune compound, so that a fluorescent strip is shown under the irradiation of an ultraviolet lamp, and the Shiga toxin I type monoclonal antibody marked by a free quantum dot is combined with a goat anti-mouse antibody at a quality control C line to show the fluorescent strip; if the detection sample is negative, the shiga toxin I is not contained, so that immune complexes cannot be formed, fluorescence cannot appear at the position of a detection T line, and only the quality control C line is used for developing color; the quality control C line should have strips when detecting positive samples and negative samples, and the fluorescence strips are the standard for judging whether the chromatography process is normal or not, and are also used as the internal control standard of the reagent.

According to the utility model discloses, it is preferred detect the T line with the distance of matter accuse C line is 5 mm.

As the preferred technical scheme of the utility model, the Shiga toxin I type monoclonal antibody on the quantum dots is a mouse source antibody, and the antibody as the quality control C line is goat anti-mouse IgG. The murine antibody is the most common antibody form in the current commercial antibodies, and has the advantages of wide source, low cost, good specificity and stability. The virus antibody may be a polyclonal antibody or a monoclonal antibody, and methods for preparing the polyclonal antibody or the monoclonal antibody are well known to those skilled in the art, and for example, an animal, such as a mouse, may be immunized with the corresponding virus and the polyclonal antibody may be purified from the serum of the animal, but the polyclonal antibody may have a problem of poor specificity compared to the monoclonal antibody, and may have a false positive result. In particular, the present invention relates to a Shiga toxin type I monoclonal antibody, which has more obvious advantages, and is preferably used for preparing the monoclonal antibody, such as corresponding antigen Shiga toxin type I immune animals, such as mice, by fusing animal spleen B cells and mouse myeloma cells to form hybridoma cells, and obtaining the monoclonal antibody with stronger specificity.

According to the present invention, the shiga toxin type I monoclonal antibody is a monoclonal antibody of shiga toxin type I antigen. Preferably, the preparation method of the Shiga toxin I monoclonal antibody comprises the following steps: the shiga toxin I antigen is used as an immunogen to immunize a mouse to obtain a specific cell strain, and ascites obtained by immunizing an animal with the specific cell strain is separated and purified to obtain the shiga toxin I antigen. The purification method is a saturated ammonium sulfate salting-out precipitation method and/or an affinity chromatography method.

The utility model discloses an on the other hand still includes the preparation method of the quick immunochromatography test paper strip of quantum dot mark, include: antibody coating, preparation of a quantum dot immune pad, assembly of a quantum dot mark rapid immunochromatography test strip and the like.

In the present invention, the antibody coating is a conventional technical means in the field, and is not particularly limited herein, and those skilled in the art can select and prepare the antibody according to actual needs.

Preferably, the antibody coating buffer for antibody coating is PBS buffer. Preferably, the PBS buffer has a pH of 6-8.5, e.g., can be 6, 6.1, 6.2, 6.3, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, or 8.5, preferably 7-8.

According to the utility model discloses, wherein the preparation of quantum dot immunity pad specifically includes following step:

(1) mixing the quantum dot particles with carboxyl, StxI antibody and 3-dimethylaminopropyl carbodiimide hydrochloride (EDC), stirring at room temperature in the dark for reaction for 3 hours, centrifuging at 1000r/min for 5 minutes, and taking the supernatant;

(2) adding a sealing liquid into the supernatant solution obtained in the step (1) for sealing;

(3) centrifugally separating and purifying the quantum dot solution sealed in the step (2) to obtain shiga toxin I type monoclonal antibody marking solution marked by the electronic dots;

(4) spraying the Shiga toxin I type monoclonal antibody marking solution obtained in the step (3) on a glass fiber membrane, wherein each milliliter of the solution is coated by 2.5cm²-3cm²Drying and sealing to obtain the quantum dot immune pad, and storing at 4 ℃ in a dark place for later use.

According to the utility model, the particle size of the quantum dot particles in step (1) is 5nm, the pH value in step (1) is 7.4, and the confining liquid in step (2) is 1% BSA solution.

The utility model discloses an on the other hand still provides a detect reagent box, the reagent box includes above the test paper strip.

The utility model discloses an on the other hand still discloses the adoption test paper strip detects method for detecting shiga toxin I, specifically as follows:

(1) sucking 80 mu L of sample to be detected, and slowly dripping the sample to be detected into a sample adding hole of the test strip;

(2) the results were observed under an ultraviolet lamp, timed, and read 10-20 minutes, since the results may be abnormal after 20 minutes.

And (4) analyzing results:

positive (+): two fluorescence events, one at the detection T line and the other at the quality control C line, were present, indicating the presence of Shiga toxin I in the sample.

Negative (-): only one fluorescence band appeared on the control C line and no fluorescence band appeared on the detection T line, indicating either no StxI in the sample or StxI below the detection level.

And (4) invalidation: the absence of fluorescence from the control C-line may be due to improper handling or reagent failure. In any case, it should be retested. If the problem still exists, the batch should be stopped immediately.

Compared with the prior art, the utility model provides a test paper strip has following beneficial effect:

(1) the quantum dot marked rapid immunochromatographic test strip of the utility model adopts a sandwich method to detect Shiga toxin I, has better sensitivity, specificity, repeatability and stability, has higher recovery rate to target compounds, and has accurate and reliable test results; the detection limit of the immunochromatographic test strip is further reduced, and the lowest detection limit can reach 10 ng/mL;

(2) the colloidal gold immunochromatographic test strip of the utility model has higher sensitivity and specificity to Shiga toxin I three-variant (StxIa, StxIC and StxId) strains.

The preparation and performance of the test strip of the present invention will be described and verified with reference to examples 1-2.

Experimental example 1 preparation of quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I

The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I in embodiments 1 to 3 of the present invention can be prepared as follows. The following preparation method that mainly is the test paper strip main part, the preparation of protection casing belongs to the conventional technology of mechanical manufacturing, and the skilled person in the art basis the utility model discloses a content can be prepared, the utility model discloses do not do the perusal. The specific structure of the test strip is shown in figure 1, and the specific preparation method is as follows:

one end pastes sample pad, quantum dot immunity pad, nitrocellulose membrane, absorbent filter paper (the pad that absorbs water) in order each other overlap joint ground on polyvinyl chloride (PVC) offset plate, then cuts into the test paper strip that the width is 4mm with PVC offset plate and attached material to in packing into the protective housing, can open application hole and visual window on the protective housing like conventional technique, certainly can be as in the utility model discloses the mode that adopts protective housing and spacing block to combine in embodiment 2-3.

In the utility model, the sample pad is a glass fiber membrane; wherein the quantum dot is immunologically embedded with a quantum dot-labeled Shiga toxin I monoclonal antibody with the concentration of 1.5-2 mg/mL; the nitrocellulose membrane comprises: the detection T coil is coated with Shiga toxin I monoclonal antibody, the quality control C coil is coated with goat anti-mouse IgG antibody, and the distance between the detection line and the quality control line is 5 mm.

(1) Preparation of Shiga toxin I monoclonal antibody:

the shiga toxin I antigen is used as an immunogen to immunize an animal to obtain a specific cell strain, and ascites obtained by immunizing the animal with the specific cell strain is separated and purified to obtain the shiga toxin I antigen;

(2) antibody coating:

diluting Shiga toxin I type monoclonal antibody and goat anti-mouse IgG antibody to 2mg/mL by using PBS (phosphate buffer solution) with 0.01-0.05mol/L of coating buffer solution and pH of 7.2-7.4 to obtain quality control C-line goat anti-mouse coating solution and detection T-line Shiga toxin I type monoclonal antibody diluent;

and secondly, spraying the diluent obtained in the step one onto an NC membrane (nitrocellulose membrane).

Thirdly, drying the NC membrane, adding a drying agent, and sealing and storing at 4 ℃;

(3) preparing a quantum dot immune pad:

adjusting the pH value to 6.5 by using quantum dot particles, connecting 2mg/mL shiga toxin I type monoclonal antibody with the quantum dot particles with carboxyl under the action of a connecting agent EDC, stirring and reacting for 3 hours at room temperature in a dark place, centrifuging for 5min at 1000r/min, and taking the supernatant after centrifugation.

Secondly, adding 5% BSA blocking solution into the quantum dot solution obtained in the step one to the final concentration of 1%, and blocking for 30 min; performing centrifugal separation and purification to obtain a quantum dot labeled Shiga toxin I monoclonal antibody;

thirdly, spraying the Shiga toxin I type monoclonal antibody marking liquid obtained in the second step on a glass fiber membrane;

fourthly, drying the glass fiber membrane in the third step, adding a drying agent, sealing and storing at 4 ℃ to obtain the quantum dot immune pad;

(4) assembling the quantum dot marked rapid immunochromatographic test strip:

firstly, assembling the coated NC membrane and quantum dot immune pad, absorbent paper and sample pad on a PVC plate;

cutting the assembled plate obtained in the step one into quantum dot marked fast immunochromatographic test strips with the size of 4mm by using a slitting device;

and thirdly, placing the test strip obtained in the step two into a sealed bag, and adding a drying agent for preservation.

The utility model discloses a detection principle of quantum dot mark quick immunochromatography test paper strip specifically as follows:

after the sample to be detected is added to the sample adding region (sample pad), the area for detecting the Shiga toxin I monoclonal antibody is detected through capillary action: the antigen-antibody-quantum dot conjugate in the sample moves towards one end of the absorbent paper. If the detected sample contains the antigen of the Shiga toxin I, when the sample moves to a detection T line, namely a coating line of a coating antibody of a quantum dot marked Shiga toxin I type monoclonal antibody, the antigen-antibody-quantum dot conjugate is captured, and an antibody-antigen-antibody-quantum dot conjugate is generated at the detection T line, so that a fluorescent strip is displayed; and (3) continuously flowing the sample, and generating a fluorescence strip when the sample flows to a quality control C line, namely the goat anti-mouse IgG antibody, so as to prove the effectiveness of the test strip. If no antigen of the Shiga toxin I exists in the detected actual sample, no immune complex can be formed, and a fluorescence band can not be displayed at the position of the detection T line, and only the quality control C line is used for color development. As long as the quality control C line does not develop color, the test strip is proved to be invalid, and the actual sample needs to be detected again.

Experimental example 2 the test strip obtained in Experimental example 1 was used for detection

The quantum dot labeled rapid immunochromatographic test strip in the embodiment 1-2 is used for detection, and the specific steps are as follows:

(1) sucking 80 mul of the bacterial liquid 3 group, and slowly dripping the bacterial liquid into a sample adding hole of a test strip;

(2) and (4) observing the result under an ultraviolet lamp, timing, reading the result after 15-20 minutes, and observing the result after 20 minutes.

In summary, the test strip of the utility model adopts the sandwich method to detect the shiga toxin I, so that the detection limit of the immunochromatography test strip is further reduced, and the lowest detection limit can reach 10 ng/mL; the quantum dot marked rapid immunochromatographic test strip has better sensitivity, specificity, repeatability and stability.

The above description of the embodiments is only intended to illustrate the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several modifications can be made to the present invention, and these modifications will fall within the protection scope of the claims of the present invention.

Claims (10)

1. A quantum dot-labeled rapid immunochromatographic test strip for detecting Shiga toxin I is characterized by comprising a test strip main body, wherein the test strip main body comprises a rubber plate, and a sample pad, a quantum dot immune pad, a nitrocellulose membrane and a water absorption pad which are arranged on the rubber plate and connected in sequence; a detection T line and a quality control C line are arranged on the nitrocellulose membrane, wherein the detection T line is arranged close to the quantum dot immune pad, and the quality control C line is arranged close to the water absorption pad; the quantum dot immune pad is embedded with a quantum dot marked Shiga toxin I monoclonal antibody, the detection T line is coated with the Shiga toxin I monoclonal antibody, and the quality control C line is coated with a goat anti-mouse polyclonal antibody.

2. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 1, further comprising a protective shell, wherein the protective shell comprises an upper shell and a lower shell which are detachably connected, the upper shell and the lower shell form a cavity capable of accommodating the test strip main body, and one side of the test strip main body where the rubber plate is located is arranged close to the lower shell.

3. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 2, wherein the upper case and the lower case are connected by a snap fit.

4. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 2, wherein the upper case is provided with a rail groove along a length direction of the upper case, the lower case is provided with a slide bar structure along a length direction of the lower case, and the slide bar structure is arranged corresponding to the rail groove.

5. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to any one of claims 2 to 4, further comprising a spacer disposed in the cavity, the spacer being disposed in the lower case for shielding the test strip main body; set up on the spacer in having application of sample hole and visual window, application of sample hole with the sample pad corresponds the setting, the visual window with detect the T line with quality control C line corresponds the setting, application of sample hole with the visual window is for running through the through-hole of spacer.

6. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 5, further comprising a sample application hole extension part, wherein the sample application hole extension part is arranged on one side of the spacer close to the test strip main body and faces the test strip direction, the sample application hole extension part is provided with a hole-like structure communicated with a sample application hole, and the sample application hole extension part abuts against the test strip main body.

7. The quantum dot labeled rapid immunochromatographic strip for detecting shiga toxin type I according to claim 6, wherein the sample application hole and the porous structure are a truncated conical hole as a whole, wherein the top of the truncated conical hole abuts against the strip body.

8. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 5, wherein the whole spacer is made of an elastic material, and the spacer is connected with the lower housing in an interference fit manner.

9. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 5, wherein a plurality of fixture block structures are arranged in the lower case, and at least two fixture block structures are respectively arranged close to two side walls in the length direction of the lower case; the fixture block structures are close to the end faces of the spacers and abut against the spacers, and are used for supporting the spacers.

10. The quantum dot labeled rapid immunochromatographic test strip for detecting shiga toxin type I according to claim 5, wherein a transparent portion is provided on the upper case, and the transparent portion is provided corresponding to the visible window.

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