CN112315511B

CN112315511B - Eye surface liquid collector and eye surface disease diagnosis device

Info

Publication number: CN112315511B
Application number: CN202110000688.3A
Authority: CN
Inventors: 陶勇; 陆成慧; 常海涛; 付欣怡
Original assignee: Zhidemingchuang Biotechnology Beijing Co ltd
Current assignee: Zhide mingchuang Biotechnology (Wuxi) Co.,Ltd.
Priority date: 2021-01-04
Filing date: 2021-01-04
Publication date: 2021-04-06
Anticipated expiration: 2041-01-04
Also published as: CN112315511A

Abstract

The invention provides an ocular surface liquid collector and an ocular surface disease diagnosis device, and relates to the technical field of medical diagnosis reagents.

Description

Eye surface liquid collector and eye surface disease diagnosis device

Technical Field

The invention relates to the technical field of medical diagnostic reagents, in particular to an ocular surface liquid collector and an ocular surface disease diagnosis device.

Background

The immunochromatography technology is a novel immunoassay developed in the last 80 th century. The main principle of the method is that a nitrocellulose membrane and the like are used as carriers for solid phase chromatography, a substance to be detected is combined with an antibody for marking a trace particle (colloidal gold, latex microspheres, fluorescent microspheres and the like) in advance, then the nitrocellulose membrane is subjected to chromatography through capillary action, and then the nitrocellulose membrane is combined with an antigen or an antibody which is coated in advance by a detection area of the chromatography membrane to develop color. Compared with other detection methods, the immunochromatography technology has the greatest characteristics of simplicity, rapidness, no need of instruments and equipment for the result, capability of directly observing the test result by naked eyes, simple treatment of the standard in the detection process, and particular suitability for large-area diagnosis of basic units.

The tears cover the ocular surface and have the functions of shielding, lubricating, sterilizing, regulating immunity and the like. Many eye diseases and even systemic diseases can affect the composition of tears, and changes in tear composition are often also important causes of ocular surface diseases. Quantitative studies of specific protein components in tears have been increasingly used for diagnosing ocular surface diseases, judging the severity of diseases, etc., and can be applied to scientific research and clinical as an indication for preventing or treating ocular surface diseases. Extensive application of tear samples in scientific research and clinic puts higher requirements on tear collection and preservation. For eye diseases, the detection requirement is higher due to the sensitivity and fragility of eye organs; in actual operation, tear, aqueous humor and eye surface moistening lotion which can be collected by eyes belong to trace samples, so that the detection difficulty is high; such as redness and swelling of the patient's eyes, conjunctival congestion, it is difficult for the clinician to determine whether the cause is an infection or an allergy without the aid of a detection technique. Therefore, accurate and rapid diagnosis of the etiology plays an important role in guiding medication. The current tear collection methods mainly comprise a capillary method, a filter paper adsorption method and the like, and have the defects of long collection time, difficult elution of adsorbed protein components and the like. Taking the capillary method as an example: the shape and the glass material of the glass capillary suction tube ensure that a patient is easy to be nervous, the operation of an inspector cannot be well matched, the edge of the tip of the glass capillary suction tube is sharp, the epithelium of the patient is easy to be damaged, and the sampling failure is easy to cause; meanwhile, the glass capillary pipette is too thin to be held and operated by an inspector easily.

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 an ocular surface fluid collector that alleviates at least one of the technical problems of the prior art.

A second object of the present invention is to provide an ocular surface disease diagnosis device.

The invention provides an ocular surface fluid collector, which comprises:

the sampling rod is arranged at one end of the sampling rod;

the sampling material comprises a sampling membrane, and the sampling membrane comprises a nanofiber membrane;

the water absorption of the sampling membrane is 100-1000%; the water absorption speed is more than or equal to 1 mm/s; the instant protein elution rate is more than or equal to 60 percent.

Further, the thickness of the sampling membrane is 100-500 μm;

the raw materials for preparing the nanofiber membrane comprise polyacrylonitrile and/or PVA.

Further, the collecting rod comprises a first collecting rod and a second collecting rod which are arranged in parallel, and a connecting rod which is obliquely arranged between the first collecting rod and the second collecting rod;

the connection angle between the first acquisition rod and the connecting rod is larger than 90 degrees and smaller than 180 degrees;

the bottom of the first collecting rod is provided with a sampling material, and the bottom of the second collecting rod is provided with a handle;

the connection angle of the first collecting rod and the connecting rod is 135 degrees.

In addition, the invention also provides a device for diagnosing eye surface diseases, which comprises the eye surface liquid collector and a detection card matched with the eye surface liquid collector.

Further, the detection card comprises a shell and a chromatography test strip fixed in the shell;

the shell is provided with a sample adding hole, a collector groove and an observation window;

and the shell is also provided with a collector groove positioning hole.

Further, along the flow direction of a sample to be detected, the chromatographic test strip comprises a sample pad, a marking pad, a cellulose membrane and absorbent paper which are sequentially connected on the bottom plate;

the sample pad is connected with a sampling material in the ocular surface fluid collector.

Further, the cellulose membrane comprises a cellulose acetate membrane or a cellulose nitrate membrane;

the cellulose membrane comprises a quality control line and at least one detection line;

the quality control line contains one or more of a goat anti-mouse antibody, a goat anti-rabbit antibody, a goat anti-chicken antibody or a goat anti-human antibody;

the detection line contains an antibody against the factor to be detected.

Further, the marking pad comprises a glass fiber film, a nylon film or a polyester film;

the label pad contains a conjugate of an antibody for resisting a factor to be detected and a tracer particle.

Further, the sample pad comprises a glass fiber film, a nylon film, or a polyester film;

the sample pad contains one or more of a protein protectant, a blocking agent, or a surfactant.

Furthermore, the bottom of the handle of the ocular surface liquid collector is provided with a positioning bolt which is matched with the groove positioning hole of the collector, and when the positioning bolt is combined with the groove positioning hole of the collector, the sampling material can be fixed on the sample pad through the groove of the collector.

Compared with the prior art, the invention has at least the following beneficial effects:

the sampling material in the ocular surface fluid collector provided by the invention is used for direct contact and adsorption of ocular surface fluid, and has the characteristics of high water absorption rate, high water absorption speed, high protein instant elution efficiency and the like by using the sampling material with specific water absorption rate, specific water absorption speed and specific instant protein elution rate, so that the ocular surface fluid collector can conveniently, simply and rapidly collect ocular surface fluid samples of patients, and the defects of psychological stress, difficult collection and the like in the collection process of the patients in the prior art are overcome.

The invention provides an eye surface disease diagnosis device, which comprises an eye surface liquid collector and a detection card matched with the eye surface liquid collector. After the ocular surface liquid collector is used for simply and quickly collecting samples such as tears of the ocular surface, the ocular surface disease diagnosis device can be directly applied to a matched detection card to quickly diagnose related protein factors and pathogenic microorganisms of the collected samples, has the advantages of convenience, quickness, high accuracy and sensitivity, low detection cost and the like, and is easy to realize the purpose of on-site diagnosis.

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. 1 is a schematic structural view of an ocular surface fluid collector provided in embodiment 1 of the present invention;

FIG. 2 is a topographical view of a prepared PAN-nano provided in example 1 of the present invention;

fig. 3 is a schematic structural view of an ocular surface disease diagnosis device provided in embodiment 2 of the present invention;

fig. 4 is a schematic structural diagram of an analytical test strip in a diagnostic device for ocular surface diseases according to embodiment 2 of the present invention;

fig. 5 is a graph showing the results of the linear regression equation and the regression coefficient of the standard curve provided in experimental example 1 of the present invention.

Icon: 1-a first acquisition rod; 2-a connecting rod; 3-a second acquisition rod; 4-sampling material; 5-a handle; 6-a housing; 7-chromatographic test paper; 8-a sample application hole; 9-collector groove; 10-an observation window; 11-a base plate; 12-sample pad; 13-a marker pad; 14-cellulose film; 15-absorbent paper.

Detailed Description

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. In this application, unless otherwise indicated, 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.

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.

According to one aspect of the present invention, there is provided an ocular surface fluid collector comprising:

the sampling rod is arranged at one end of the sampling rod;

the water absorption of the sampling material is 100-1000%; the water absorption speed is more than or equal to 1 mm/s; the instant protein elution rate is more than or equal to 60 percent.

In the present invention, the raw material for preparing the sampling material is some polymer material with water absorption property, such as but not limited to wool polyester, cotton fiber, biological cellulose, nylon, polyacrylonitrile or polyethylene oxide. Besides the water absorption performance, the material needs to have the characteristics of high water absorption rate, high water absorption speed, high instant protein elution efficiency and the like, and particularly the water absorption rate is 100-1000%; the water absorption speed is more than or equal to 1 mm/s; the instant protein elution rate is more than or equal to 60 percent.

In order to obtain a sampling material having a higher water absorption rate, a faster water absorption speed, and a higher instant protein elution efficiency, polyacrylonitrile or PVA is preferably used as a raw material of the sampling material.

Wherein, the sampling material can be connected with one end of the collecting rod through modes such as microwave welding, hot melting and the like.

When the ocular surface fluid collector provided by the invention is used for collecting samples, the collection modes are two types: firstly, the eye mask directly contacts the palpebral conjunctiva and is removed from the palpebral conjunctiva after a certain time; secondly, a drop of eye drop is dripped at the eye socket, and the eye drop is collected at the surface of the eyes after the eyeball of the patient rotates.

The sampling material in the ocular surface liquid collector provided by the invention is used for direct contact and adsorption of ocular surface liquid samples, and has the characteristics of high water absorption rate, high water absorption speed, high protein instant elution efficiency and the like by using the sampling material with specific water absorption rate, specific water absorption speed and specific instant protein elution rate, so that the ocular surface liquid collector can conveniently, simply and rapidly collect the ocular surface liquid samples of patients, and the defects of psychological stress, difficult collection and the like in the collection process of the patients in the prior art are overcome.

In some preferred embodiments, the sampling material comprises a sampling membrane.

The sampling membrane in this embodiment is a series of membrane materials, such as filter paper, absorbent paper, polyacrylonitrile nanofiber membrane (PAN-nano), cellulose membrane, etc., prepared from a polymer material with water absorption property by using known processes, such as an electrospinning technique and a mold pressing technique.

The thickness of the sampling membrane is preferably 100-500 μm, and may be, but is not limited to, 100 μm, 200 μm, 300 μm, 400 μm or 500 μm; the dimensions may be chosen to be 5 mm by 5 mm square.

The reason for adopting this thickness interval is that, when the thickness of the membrane is too low, the support performance of the sampling membrane is poor and the water absorption rate is low, which does not meet the requirements; when the thickness of the membrane is too high, the cost of the sampling membrane is increased, and more eluent is required in the elution process.

In some preferred embodiments, the harvesting rods comprise a first harvesting rod and a second harvesting rod arranged in parallel, and a connecting rod obliquely arranged between the first harvesting rod and the second harvesting rod;

the bottom of the first collecting rod is provided with a sampling material, and the bottom of the second collecting rod is provided with a handle.

Further adjustment and optimization are carried out through the structure to gathering the pole for when using this collection pole to carry out the collection of ocular surface liquid sample, when the person of gathering of being convenient for grips, can make the sample material contact ocular surface liquid sample more convenient and fast ground, alleviate patient's intense tension.

When the sampling device is used, a collector holds the handle at the bottom of the second collecting rod by hand, and brings the first collecting rod close to the eyes of a patient, so that the sampling material at the bottom of the first collecting rod is in contact with an ocular surface liquid sample, and the collection of the ocular surface liquid sample is completed.

The invention verifies that when the connection angle of the first acquisition rod and the connecting rod is 135 degrees, the acquisition rod is more convenient to use.

According to a second aspect of the invention, an ocular surface disease diagnosis device is provided, which comprises the ocular surface fluid collector provided by the invention and a detection card matched with the ocular surface fluid collector.

The ocular surface disease diagnosis device provided by the invention can be used for directly applying a matched detection card to rapidly diagnose related protein factors and pathogenic microorganisms of the collected sample after simply and rapidly collecting samples such as tears of the ocular surface by the ocular surface liquid collector, has the advantages of convenience, rapidness, high accuracy and sensitivity, low detection cost and the like, and is easy to realize the purpose of on-site diagnosis.

It is understood that the tear analytes to be detected by the detection card of the present invention include, but are not limited to, proteins, peptides, metabolites, electrolytes, small molecules, lipids, saccharides, nucleic acids, etc. in tears. In some embodiments, the analyte includes some protein biomarkers, including but not limited to: immunoglobulins (immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin E (IgE)), cytokines (tumor necrosis factor-

（TNF-

) Transforming growth factor-

（TGF-

) Interleukins), proteins (lactoferrin, lipocalins, cathepsins, mucins or other glycoproteins), and the like.

In some preferred embodiments, the test card comprises a housing and a chromatographic strip secured within the housing. For ease of inspection and observation, the housing may be an elongated flat shell-like housing.

Preferably, the shell is provided with a sample adding hole, a collector groove, an observation window and an optional collector groove positioning hole.

The sample adding hole is used for adding a sample to be detected to a test strip in the detection card; the observation window is used for observing the detection result of the test strip; the collector groove is used for placing a sampling material at one end of the collecting rod so as to realize the matched use function of the detection card and the ocular surface fluid collector.

Optionally, the bottom of the handle of the ocular surface fluid collector is provided with a positioning bolt adapted to the positioning hole of the collector groove. After collection is completed, when the positioning bolt is tightly combined with the positioning hole of the collector groove, the sampling material at one end of the collecting rod can be tightly fixed on the sample pad through the collector groove.

In some preferred embodiments, the chromatographic test strip comprises a sample pad, a label pad, a cellulose membrane and a water-absorbent paper, which are connected to a bottom plate in sequence along the flow direction of a sample to be tested.

Wherein, the bottom plate is made of plastic for providing support, and the material can be PVC and the like.

The cellulose membrane comprises a cellulose acetate membrane or a cellulose nitrate membrane;

specifically, the cellulose membrane comprises a quality control line and at least one detection line;

wherein, at least one means that one, two or three or more detection lines can be set according to the actual detection requirement, and is used for detecting a single pathogenic factor or a plurality of pathogenic factors.

Preferably, the quality control line contains one or more of a goat anti-mouse antibody, a goat anti-rabbit antibody, a goat anti-chicken antibody or a goat anti-human antibody;

preferably, the detection line contains an antibody against the factor to be detected.

In the present embodiment, the detection line is not limited to an antibody against the factor to be detected, and may contain a substance such as a receptor protein, a polypeptide, or an aptamer capable of specifically binding to the factor to be detected.

The marking pad comprises a fiberglass film, a nylon film or a polyester film;

preferably, the label pad contains a conjugate of an antibody against the factor to be detected and a tracer particle.

The tracer particles may be, for example, but not limited to, colloidal gold, colloidal selenium, colored latex microspheres, fluorescent microspheres, and the like.

The sample pad comprises a fiberglass film, a nylon film, or a polyester film;

preferably, the sample pad contains one or more of a protein protectant, a blocking agent, or a surfactant.

The absorbent paper is absorbent filter paper with strong water absorption capacity.

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.

Example 1

This embodiment provides an ocular surface fluid collector, the ocular surface fluid collector includes:

the sampling rod and the sampling material are arranged at one end of the sampling rod.

As shown in fig. 1, the collection rod of the ocular surface fluid collector provided by this embodiment includes a first collection rod 1 and a second collection rod 3 arranged in parallel, and a connection rod 2 obliquely arranged between the first collection rod 1 and the second collection rod 3.

Wherein, first collection pole 1 and connecting rod 2, connecting rod 2 and second collection pole 3 can fixed connection respectively independently, also can articulate as long as satisfy first collection pole 1 and connecting rod 2 the angle of connection be greater than 90 and be less than 180 can.

When the first acquisition rod 1 is fixedly connected with the connecting rod 2, the connection angle of the first acquisition rod 1 and the connecting rod 2 is preferably 135 degrees, so that the acquisition rod is more convenient for an acquirer to use; when first collection pole 1 is articulated with connecting rod 2, do not specifically limit connection angle, can adjust wantonly according to the in-service use demand.

The bottom of the first collecting rod 1 is fixedly connected with a sampling material 4, and the bottom of the second collecting rod 3 is fixedly connected with a handle 5.

The raw material for preparing the sampling material 4 is a polymer material with water absorption property, such as wool polyester, cotton fiber, biological cellulose, nylon, polyacrylonitrile or polyethylene oxide. Besides the water absorption performance, the water-absorbing agent also needs to have the characteristics of high water absorption rate, high water absorption speed, high instant protein elution efficiency and the like, and specifically, the water absorption rate is 100-1000%; the water absorption speed is more than or equal to 1 mm/s; the instant protein elution rate is more than or equal to 60 percent.

When the raw material of the sampling material 4 is polyacrylonitrile, the method for preparing the polyacrylonitrile nano fiber membrane (PAN-nano) by the electrospinning technique can be as follows:

12.0 g of Polyacrylonitrile (PAN) was added to 93 mL of N, N-Dimethylformamide (DMF) solvent, and magnetically stirred for 8 hours to obtain a 12 wt% PAN solution. The solution was transferred to a syringe and mounted on a micro syringe pump. The spinning parameters were set as: the flow rate is 1 mL/h; voltage +20.0 kV; the distance between the needle head and the receiver is 25 cm; humidity is 40%; the temperature is 25 ℃; the spinning time was 16 h. Obtaining the polyacrylonitrile nano-fiber membrane (PAN-nano) with the thickness of 500 mu m. Subsequently dried at 60 ℃ for 8 h to remove the non-volatile solvent. The topography of the PAN-nano is shown in FIG. 2. And rolling the PAN-nano at 60 ℃ by using a hot roller press to obtain the PAN-nano sampling membrane with the thickness of 100-500 mu m.

After the preparation is finished, the prepared PAN-nano membrane is adhered to the bottom of the first collecting rod 1 of the ocular surface fluid collector through an ultrasonic welding process, and the ocular surface fluid collector is used after being sterilized.

The shape of the handle 5 is not limited, and any shape can be used for holding.

When using the ocular surface liquid collector that this embodiment provided, the handheld handle 5 of second collection pole 3 bottom of collection person will gather the stick and be close to patient's eye, and the collection of ocular surface liquid is accomplished to the contact of the sampling material 4 of first collection pole 1 bottom and after absorbing patient's ocular surface liquid.

Example 2

The present embodiment provides an ocular surface disease diagnostic apparatus.

As shown in fig. 3 and 4, the diagnosis device for eye surface diseases provided by this embodiment includes an eye surface liquid collector and a detection card used in cooperation with the eye surface liquid collector.

Wherein, the ocular surface fluid collector may be the ocular surface fluid collector provided in embodiment 1 of the present invention.

The test card may comprise a housing 6 and a chromatographic strip 7 secured within the housing.

In this embodiment, the chromatographic test strip includes a sample pad 12, a label pad 13, a cellulose membrane 14 and a absorbent paper 15 connected to a bottom plate 11 in sequence along the flow direction of the sample to be tested.

The connection with the base plate 11 may be fixed connection, for example, the sample pad 12, the label pad 13, the cellulose membrane 14 and the absorbent paper 15 are fixed on the base plate 11 by adhesive. The adhesive substance is preferably a non-setting adhesive.

The sample pad 12, the label pad 13, the cellulose membrane 14, and the absorbent paper 15 may be joined in series or may be joined in series. When the sample pad 12, the label pad 13, the cellulose film 14 and the absorbent paper 15 are sequentially overlapped, the sampling material 4, the sample pad 12, the label pad 13 and the cellulose film 14, and the absorbent paper 15 overlapped on the cellulose film 14 are sequentially arranged from top to bottom in the sample flow direction.

The shell 6 is also provided with a sample adding hole 8, a collector groove 9 and an observation window 10.

The collector groove 9 is arranged on the sample pad 12 and used for receiving the sampling material 4 in the ocular surface fluid collector, so that a sample to be detected obtained from the sampling material 4 can be in contact with the sample pad 12 in the chromatographic test strip, and the sample to be detected is introduced into the chromatographic test strip for detection.

The observation window 10 is arranged on the upper part of the cellulose membrane 14, so that a user can directly observe the detection result of the chromatography conveniently.

In order to enable the sampling material 4 in the ocular surface liquid collector to be better combined with the detection card, a collector groove positioning hole is preferably additionally arranged on the shell 6, and a positioning bolt which can be matched with the collector groove positioning hole is additionally arranged at the bottom of the handle 5 of the ocular surface liquid collector. When the locating pin is engaged with the collector well locating hole, the sampling material 4 can be secured to the sample pad 12 through the collector well 9.

Example 3

The present embodiment provides a method for preparing a detection reagent used in the chromatographic test strip of embodiment 2, including:

1. preparation of tracer antibody

Firstly, diluting the fluorescent microspheres by 10 times by using ultrapure water, wherein the solid content is 0.1%; and (4) carrying out ultrasonic dispersion for 5 minutes by using an ultrasonic cleaner to obtain the fluorescent microsphere suspension.

Weighing 50mg of N-hydroxysuccinimide (NHS) and dissolving the NHS in 0.1M MES to prepare a solution (solution A) with the concentration of 50mg/mL, weighing 50mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and dissolving the solution in 0.1M MES to prepare a solution (solution B) with the concentration of 50mg/mL, adding 10mL of fluorescent microsphere suspension and uniformly mixing, then adding 0.4mL of the solution B into the fluorescent microsphere suspension and uniformly mixing, and reacting at normal temperature for 10 minutes; then, the mixture was centrifuged at 12000r/min for 30min, the supernatant was decanted, 0.01M PBS was added, and the suspension was resuspended. Then taking the activated fluorescent microsphere suspension, uniformly dispersing by ultrasonic, and then dropwise adding an antibody, wherein the preferable dosage of the anti-IgE rabbit polyclonal antibody is 0.10 mg; stirring at 2-8 deg.C for 1-2 hr, adding 10% BSA (final concentration of 0.5%), and blocking for 0.5 hr; centrifuging the sealed fluorescent microspheres at the speed of 12000r/min for 20min, and adding the preservation solution into the centrifuged fluorescent microspheres to uniformly disperse the fluorescent microspheres. And (3) respectively and uniformly spreading the marked antibodies on the marking pads 13, drying for 12 hours at the temperature of 20-40 ℃ and the humidity of 10-30%, and sealing and bagging for later use.

2. Coating of detection line (T) and quality control line (C)

0.05M Carbonate Buffer (CBS) with a dilution pH of 9.6 was prepared as a coating solution, an anti-IgE monoclonal antibody was used as a detection line (hereinafter referred to as "T line") and an anti-mouse IgG polyclonal antibody was used as a quality control line (hereinafter referred to as "C line"), and the solutions of different dilutions were coated on the cellulose film 14, respectively. The preferred concentrations are respectively: 2.0mg/ml and 1.0 mg/ml. Drying at 40 deg.C for 12 hr, sealing and bagging.

3. Preparation of sample pad 12

The sample pad 12 was uniformly coated with the treatment solution (PBST 0.01M, pH7.4, Tween-200.5%), dried in a drying room (temperature 20-40 ℃ C., humidity 10-30%) for 24 hours, and cut into a working specification by a paper cutter for use.

5. Assembly of test strips

The components are sequentially arranged on the bottom plate 11 in sequence, cut into test strips with the length of 4mm by a slitter and then placed in the shell 6 of the detection card for standby.

Example 4

This embodiment provides a method for preparing another detection reagent used in the chromatographic test strip of embodiment 2, which comprises the following steps:

1. preparation of gold-labeled antibody

Taking 8 15ml test tubes, and respectively adding 5ml of 30 nm colloidal gold; with 25 mM K₂CO₃Adjusting the pH value to 4, 5, 6, 6.5, 7, 7.5, 8, 9 and 10 respectively; adding 75 mu L of protein to be marked (adenovirus antibody or mouse IgG) with the concentration of 1 mg/ml into each test tube, mixing, and standing at room temperature for 10-15 min; adding 1ml of 10% NaCl solution into each tube, mixing, and standing at room temperature for 5 min; observing the color change of the colloidal gold, and recording the optimal pH value for keeping the purple-red color to the red color. Repeating the steps, wherein the pH gradient is X-0.1 and X-0.5; x-0.3; x; x + 0.3; x + 0.5; x +1, observing the color change of the colloidal gold until standing at room temperature for 2 hours, recording the lowest pH at which the red color remained.

The pH value of the colloidal gold solution is adjusted to the determined optimum pH value, and the colloidal gold solution is divided into 10 tubes with 1ml of each tube. After the protein to be labeled (adenovirus antibody or mouse IgG) is serially diluted to 5-45 mug/ml, 1ml is added into 1 tube of colloidal gold solution. The control tube was filled with 1ml of diluent. When the amount of the added protein reaches or exceeds the minimum stable amount of the colloidal gold, the colloidal gold solution keeps red; if the amount of protein added is less than the minimum stable amount of colloidal gold, the colloidal gold solution will undergo coagulation from red to blue. Then, of all tubes exhibiting red color, the tube with the lowest protein content was selected as the tube containing the amount of antigen required to stabilize 1ml of colloidal gold.

And uniformly spreading the labeled gold-labeled antibody (labeling pad) on the labeling pad 13, drying for 6 hours at the temperature of 20-40 ℃ and the humidity of 10-30%, and sealing for later use.

2. Coating of detection line (T) and quality control line (C)

0.05M Carbonate Buffer (CBS) with a pH of 9.6 as a diluent is prepared as a coating solution, an anti-adenovirus monoclonal antibody is used as a detection line (hereinafter, referred to as T line) and an anti-mouse IgG polyclonal antibody is used as a quality control line (hereinafter, referred to as C line), and the solutions with different dilutions are coated on the cellulose membrane 14 respectively. The preferred concentrations are respectively: 1.0mg/ml and 0.5 mg/ml. Drying at 40 deg.C for 12 hr, sealing and bagging.

3. Preparation of sample pad 12

The sample pad 12 treatment solution PBST and 0.1% BSA solution (0.01M, pH7.4, Tween-200.5%) are uniformly coated, dried in a drying room (temperature of the drying room is 20-40 ℃, humidity is 10-30%) for 24 hours, and the sample pad is cut into working specifications by a paper cutter for standby.

4. Assembly of test strips

Experimental example 1 implementation of quantitative determination of allergen-specific Total IgE antibodies in tear samples (fluorescent microsphere method)

1. Principle of reaction

The detection card contains an anti-IgE monoclonal antibody fixed on a detection area (T) on a membrane in advance and an anti-mouse IgG polyclonal antibody of a quality control area (C), meanwhile, the method provided by the embodiment 3 of the invention is used for preparing the anti-IgE polyclonal antibody and mouse IgG marked by fluorescent microspheres, and the anti-IgE polyclonal antibody and mouse IgG are matched with other test strip supports to prepare the detection card, so that the double-antibody sandwich method is used for detecting the total IgE content in the ocular surface fluid, and the detection card is suitable for auxiliary diagnosis of allergic conjunctivitis.

During testing, a drop of artificial tears is dripped into the eye socket of a patient to be discharged, and a sample secreted at the canthus is collected by using an ocular surface fluid collector; then buckling the sampling membrane on the detection card, and dropwise adding a diluent (normal saline) into the reagent adding hole; when the sample contains IgE, HIgE and the microsphere-labeled anti-IgE polyclonal antibody firstly form a reaction complex; under the action of chromatography, the reaction complex moves forwards along the nitrocellulose membrane, and when the reaction complex meets the situation that the detection area (T) is coated with the anti-IgE monoclonal antibody, the reaction complex is finally intercepted on the detection area and reacts and combines to form a fluorescent bright band; the quality control region (C) is coated with an anti-mouse IgG polyclonal antibody, and reacts with mouse IgG marked by microspheres to form a fluorescent bright band as quality control. The standard for judging whether the chromatography process is normal appears in the quality control area (C), and the standard is also used as the internal control standard of the reagent. The intensity of the fluorescent band is correlated with the reaction concentration.

2. Performance testing

2.1 Standard Curve construction

IgE standards were diluted to working concentrations using artificial tear fold ratios. A drop of the standard substance with the working concentration is added to a sampling diaphragm (40 mu L), the sampling diaphragm is buckled in a collector groove of the detection card, a drop of diluent (40 mu L) is added to a sample adding hole, the reaction is waited for 15 minutes, and then a fluorescence reader is used for reading the fluorescence brightness ratio of C, T lines. The mean value was taken after 3 replicates for each concentration (results are shown in the table below); curve fitting was then performed and the linear regression equation and regression coefficients were calculated (see fig. 5).

。

2.2 specificity test

And (3) diluting IgA, IgG and IgM standard products to working concentrations by using the multiple ratio of artificial tears, and testing according to the sampling detection requirements, wherein the test results are no cross under the detection limit concentration of 1000 times.

2.3 detection Limit test

Taking the artificial tears to test a blank sample according to the requirements of sampling detection, testing for 6 times, and calculating a standard difference value which is 3 times of the blank concentration value under a standard curve; the formula is as follows: y =3 SD; the detection limit result was 0.027. mu.g/kg.

2.4 precision measurement

Diluting IgE standard substance with artificial tear to working concentration (0.1 μ g/Kg, 1 μ g/Kg, 10 μ g/Kg), testing each concentration for 20 times according to detection method, and calculating Relative Standard Deviation (RSD) of blank concentration value; the 3 concentration detection results are respectively as follows: 12.1%, 5.4% and 7.9%.

2.5 accelerated stability test

Sealing each detection card and a drying agent in an aluminum foil bag, and placing for 1 month at 37 ℃; again, the detection limit and precision were tested. The detection limit result is 0.025 mu g/kg; the precision results were: 13.4%, 4.9%, 7.1%.

Experimental example 2 qualitative detection of adenovirus antigen in tear sample (colloidal gold method)

1. Principle of reaction

The detection card contains an anti-adenovirus surface antigen monoclonal antibody which is fixed on a detection area (T) on a membrane in advance and an anti-mouse IgG polyclonal antibody of a quality control area (C), meanwhile, the method provided by the embodiment 4 of the invention is used for preparing the anti-adenovirus surface antigen monoclonal antibody and the mouse IgG which are marked by colloidal gold, and the monoclonal antibody and the mouse IgG are matched with other test strip supports to prepare the detection card, and the double-antibody sandwich method is used for detecting adenovirus in ocular surface liquid, so that the detection card is suitable for auxiliary diagnosis of epidemic keratitis.

During testing, the eyelid of a patient is directly wiped, then the eyelid is sampled by using an ocular surface fluid collector and then is buckled on a test card, and 2 drops of diluent is dripped into a reagent sampling hole; when the sample contains adenovirus with the detection limit and the concentration above the detection limit, the adenovirus and the anti-adenovirus monoclonal antibody marked by the colloidal gold form a reaction complex Ag-Ab-Au reaction complex; under the action of chromatography, the reaction complex moves forwards along the nitrocellulose membrane, and when encountering the anti-adenovirus monoclonal antibody of another binding site coated by the detection area (T), the reaction complex is finally trapped and reacts and is combined on the detection area to form a colloidal gold red strip; the quality control region (C) is coated with anti-mouse IgG polyclonal antibody, and reacts with mouse IgG marked by microspheres to form a red strip as quality control. The standard for judging whether the chromatographic process is normal or not appears in the quality control area (C).

2. Performance testing

2.1 Positive coincidence

The samples of 20 positive patients were tested and the compliance rate was 100%.

2.2 negative match Rate

The samples of 20 positive patients were tested and the compliance rate was 90%.

2.3 repeatability

A positive patient is repeatedly sampled for 10 times, the result is positive, and the color development is uniform.

Accelerated destructive test at 2.437 deg.C

After the reagent is stored for 30 days at 37 ℃, the negative coincidence rate, the positive coincidence rate and the repeatability test result still accord with each other.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The device for diagnosing the eye surface diseases is characterized by comprising an eye surface liquid collector and a detection card matched with the eye surface liquid collector;

the ocular surface fluid collector comprises:

the sampling rod is arranged at one end of the sampling rod;

the water absorption of the sampling material is 100-1000%; the water absorption speed is more than or equal to 1 mm/s; the instant protein elution rate is more than or equal to 60 percent;

the collecting rod comprises a first collecting rod and a second collecting rod which are arranged in parallel, and a connecting rod which is obliquely arranged between the first collecting rod and the second collecting rod;

the detection card comprises a shell and a chromatography test strip fixed in the shell;

the shell is also provided with a collector groove positioning hole;

along the flow direction of a sample to be detected, the chromatographic test strip comprises a sample pad, a marking pad, a cellulose membrane and absorbent paper which are sequentially connected on a bottom plate;

the sample pad is connected with a sampling material in the ocular surface fluid collector;

the bottom of the handle of the ocular surface liquid collector is provided with a positioning bolt which is adaptive to the groove positioning hole of the collector, and when the positioning bolt is combined with the groove positioning hole of the collector, the sampling material can be fixed on the sample pad through the groove of the collector.

2. The ocular surface disease diagnostic device of claim 1, wherein the sampling material comprises a sampling membrane comprising a nanofiber membrane;

the thickness of the sampling membrane is 100-500 mu m;

3. The ocular surface disease diagnostic device of claim 1, wherein the cellulose membrane comprises a cellulose acetate membrane or a cellulose nitrate membrane;

the detection line contains an antibody against the factor to be detected.

4. The ocular surface disease diagnostic device of claim 1, wherein the marker pad comprises a fiberglass membrane, a nylon membrane, or a polyester membrane;

5. The ocular surface disease diagnostic device of claim 1, wherein the sample pad comprises a glass fiber membrane, a nylon membrane, or a polyester membrane;