CN102445536A - Gold-labeled immune test strip for detecting equine encephalitis virus antibody and preparation method and application thereof - Google Patents

Abstract

The invention discloses a gold-labeled immune test strip for detecting equine encephalitis virus antibodies, a preparation method and application thereof, which can simultaneously detect eastern equine encephalitis virus, western equine encephalitis virus and venezuelan equine encephalitis virus antibodies, and a preparation method and application of a gold-labeled immune chromatography system capable of respectively quantitatively detecting the antibodies, and comprises the preparation of the gold-labeled immune test strip and the application of quantitative detection of a gold-labeled analyzer. The invention combines the technical principles of gold-labeled marker and immunochromatography, ensures the sensitivity and specificity of the detection method, and has the advantages of simple operation, short detection time, no need of professional, suitability for rapid on-site screening, capability of achieving the aim of quantitative detection and the like.

Description

Gold-labeled immune test strip for detecting equine encephalitis virus antibody, preparation method and application thereof

technical field

The invention belongs to the field of biological detection, and specifically relates to a gold-labeled immune test strip capable of simultaneously detecting antibodies to Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus and quantitatively detecting them respectively, and its preparation methods and applications. the

Background technique

Eastern Equine Encephalitis Virus (EEEV) is a group A arbovirus Togaviridae Alphavirus genus, containing single-stranded positive-strand RNA, the virus is mainly transmitted by mosquitoes, and can be transmitted through the air containing the virus. Sol infects humans through the respiratory tract, and the meningoencephalitis it causes is an acute, viral, and zoonotic infectious disease. After infection, the morbidity rate is high, the infectivity is strong, the fatality rate is high, there is no effective preventive method, and sequelae are often left after recovery, which is very harmful to the crowd, so it is listed as one of the main diseases to prevent bioterrorism by the international community. It has attracted the attention of countries all over the world. the

my country's research on the detection and medical protection of Eastern Equine Encephalitis Virus biological warfare agents is still blank, and there is a lack of medium and long-term research and development plans. There are no supporting diagnostic reagents and effective immunotherapy and treatment for terrorist attacks against Eastern Equine Encephalitis Virus. Therefore, it is urgent to invent a fast, simple, and on-site detection method for detecting eastern equine encephalitis virus, which is of great significance to my country's image and detection status in the international community.

Western Equine Encephalitis (Western Equine Encephalitis, abbreviated as WEE) (hereinafter referred to as Western Equine Encephalitis) is an acute viral encephalitis caused by Western Equine Virus that can kill humans, horses and other animals. Simma virus can transmit infection through the respiratory tract, but it is mainly transmitted by mosquitoes. The antigenicity of Simma virus is determined by the envelope glycoprotein E1 and equine encephalitis virus. E1 contains a neutralizing antigenic site, and the corresponding antibody will be produced after infection with Simma virus, so the detection of West Ma virus E1C antibody can tell whether you have been infected with the virus. In 1990, He Haihuai and others isolated Simma virus from Anopheles mosquitoes collected in Wusu County, Xinjiang and Ixodes spp. from Bole County. Lv Xinjun and others found in the survey of human serum in my country that the positive rate of West Malaysia virus antibody was 2.71%. These circumstances all confirmed the existence of Simma virus in our country, which indicated that Simma virus may become one of the new pathogens of infectious diseases in our country. the

Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, abbreviated as VEEV) (hereinafter referred to as Venezuelan equine encephalitis virus) belongs to Togaviridae, Alphavirus genus, and its genome is a single-stranded positive-strand RNA that can cause Venezuelan equine encephalitis. Virus. The virus is mainly transmitted by mosquitoes sucking blood, which can cause diseases in horses, humans or other animals. Studies have shown that aerosol is also an important transmission route. The virus has a high morbidity and fatality rate, and sequelae will remain after recovery. It has been listed as a bioterrorist agent by the US Centers for Disease Control and Prevention. Although no reports of Venezuelan equine encephalitis virus have been found in my country, with the increasing frequency of international tourism and trade exchanges, the risk factor of carrying this virus into my country and the risk of potential bioterrorism attacks are increasing. Therefore, the ports of entry and exit should be more vigilant and strengthen the detection of the Weima virus to prevent the outbreak and prevalence of the virus in our country. the

Gold Standard Immunochromatography (GICA) is a new in vitro diagnostic technology developed on the basis of monoclonal antibody technology, gold standard technology and new material technology since the 1990s. It has developed rapidly in recent years and has been widely used in the field of biomedicine, especially in medical testing. This technology is mainly to immobilize the specific antigen or antibody on the NC membrane in a strip shape, and the gold labeling reagent is adsorbed on the binding pad. When the sample to be tested is added to the sample pad at one end of the test strip, it moves forward through the siphon principle. Move, dissolve the gold-labeled reagent on the binding pad and react with each other, and then move to the fixed antigen or antibody area, the complex of the analyte and the gold-labeled reagent will specifically bind to it and be trapped, and gather in the detection area. On the belt, the visual color development results can be obtained through the visual inspection of the gold-labeled markers. The free markers cross the detection zone to achieve the purpose of automatic separation from the bound markers. Because it is fast, simple, accurate, highly specific and sensitive, the results are intuitive and reliable, and the amount of reagents and samples is very small, each sample only needs 1-2μl, no equipment is needed, which simplifies the tedious routine operation process, and also The error caused by the operation is reduced, and the gold standard immunochromatography technology is a compound immunochromatography technology with the fastest development at present. The gold label itself is red, does not need to add color reagents, and is non-toxic to the human body. At present, this technology has been widely used in clinical medical testing, and is considered to be one of the most promising new technologies in the diagnosis of microorganisms, infectious diseases and parasitic diseases. the

Contents of the invention

The object of the present invention is to provide an immunochromatography that is easy to use, quick to detect, qualitative, specific and sensitive, and can simultaneously detect Eastern equine encephalitis virus antibodies, Western equine encephalitis virus antibodies, and Venezuelan equine encephalitis virus antibodies Another object of the present invention is to provide a preparation method and application of the test strip and the gold-labeled immunochromatography system capable of quantitative detection thereof. the

In order to achieve the above object, a gold standard immunochromatographic system for detecting equine encephalitis virus antibody of the present invention comprises a gold standard immunoanalyzer for quantitative detection and a gold standard immunological test paper for detecting Eastern equine encephalitis virus antibody Strip A, gold-labeled immune test strips for detecting Western equine encephalitis virus antibodies B, gold-labeled immunological test strips for detecting Venezuelan equine encephalitis virus antibodies, all three test strips include reaction supports, Closely connected to the nitrocellulose membrane in the middle of the upper surface of the reaction support, the upper surface of the initial end of the nitrocellulose membrane is partially overlapped and connected with the water-absorbing pad, the upper surface of the end of the nitrocellulose membrane is partially overlapped and connected with the gold-labeled antibody protective film, and the gold-labeled antibody is protected The upper surface of the membrane is partially overlapped and connected with a sample pad;

Among them, the gold-labeled antibody protective film in the gold-labeled immune test strip A includes the film body and the gold-labeled Staphylococcus aureus A protein or recombinant Eastern Equine Encephalitis Virus E1C antigen coating evenly coated on it, A quality control strip coated with goat anti-rabbit IgG or rabbit anti-EEEV IgG is set on the nitrocellulose membrane near its starting end, and a detection strip coated with recombinant Eastern Equine Encephalitis Antigen is set near its end ;

The gold-labeled antibody protective film in the gold-labeled immunoassay strip B includes the film body, and the gold-labeled Staphylococcus aureus protein A or recombinant western equine encephalitis virus E1C antigen coating evenly coated on it, nitrocellulose A quality control band coated with goat anti-rabbit IgG or rabbit anti-Western equine encephalitis virus IgG is set on the membrane near its starting end, and a detection band coated with recombinant Western equine encephalitis virus antigen is set near its end;

The gold-labeled antibody protective film in the gold-labeled immune test strip C includes the film body, and the gold-labeled Staphylococcus aureus protein A or recombinant Venezuelan equine encephalitis virus E antigen coating evenly coated on it, nitrocellulose A quality control band coated with goat anti-rabbit IgG or rabbit anti-Venezuelan equine encephalitis virus IgG is set on the membrane near its starting end, and a detection band coated with recombinant Venezuelan equine encephalitis virus antigen is set near its end.

Further, the gold-labeled immune test strip A, the gold-labeled immune test strip B and the gold-labeled immune test strip C are made side by side. the

Further, the reaction support is a PVC board, and the water-absorbing pad is oil filter paper; the gold-labeled antibody protective film is a glass fiber film, a polyester film or a filter paper fiber. the

Further, the sample pad is glass fiber membrane, polyester membrane or filter paper fiber. the

Further, the gold-labeled immune test strip A, the gold-labeled immune test strip B and the gold-labeled immune test strip C are respectively wrapped with a shell, and the shell is provided with a sample hole corresponding to the sample pad. , observation windows are arranged on the housing corresponding to the quality control strips and detection strips. the

A preparation method of the above-mentioned gold standard immune test strip A, gold standard immune test strip B or gold standard immune test strip C, specifically:

1) Preparation of colloidal gold;

2) Use the prepared colloidal gold to label the corresponding Staphylococcus aureus protein A or recombinant Eastern equine encephalitis virus E1C antigen, Staphylococcus aureus protein A or recombinant Western equine encephalitis virus E1C antigen, Staphylococcus aureus protein A Or recombinant Venezuelan equine encephalitis virus E antigen;

3) Spray goat anti-rabbit IgG or rabbit anti-Oriental equine encephalitis virus IgG on the beginning of the nitrocellulose membrane 1 to form a quality control band 1. Spray anti-rabbit IgG or rabbit anti-Western equine encephalitis virus IgG to the beginning of nitrocellulose membrane 2 to form a quality control band 2. Spray goat anti-rabbit IgG or rabbit anti-Venezuela equine encephalitis virus IgG to the end of nitrocellulose membrane 3 At the beginning end to form quality control band 3, spray recombinant equine encephalitis protein onto the ends of the three nitrocellulose membranes to form corresponding detection bands;

4) Apply the three colloidal gold-labeled proteins or antigens prepared in step 2) evenly on three glass fiber membranes, and dry or freeze-dry to make three corresponding gold-labeled antibody protective films;

5) Set the three nitrocellulose membranes in step 3) in the middle of the upper surface of the reaction support, the upper surfaces of the starting ends of the three nitrocellulose membranes overlap and connect with the corresponding absorbent pads respectively, and the upper surfaces of the ends of the three nitrocellulose membranes It is partially overlapped and connected with the corresponding gold-labeled antibody protective film, and the upper surface of the gold-labeled antibody protective film is partially overlapped and connected with the corresponding sample pad;

6) Cut the test paper containing three test strips formed in step 5) into the shape of the specified size, dry and pack the test paper to form the test strip.

Further, the specific steps for preparing colloidal gold in the step 1) are:

1) Prepare HAuCl ₄ aqueous solution: add HAuCl ₄ under magnetic stirring, and add trisodium citrate aqueous solution at the same time, continue heating after the color is stable, add pure water to make up to the set value after cooling to room temperature, and store in the dark at 4 °C;

2) Use K ₂ CO ₃ to adjust the pH value to 6.0-6.4;

3) Adjust the colloidal gold to pH 6.0-6.4, dilute SPA with PB to 0.2 mg/ml;

4) Keep colloidal gold stable.

Further, in step 3), the film spraying speed is 10-100 mm/s; the added amount of the recombinant equine encephalitis protein is 0.1-5 mg/ml, and the dilution of the recombinant antigen can be PBS; goat anti-rabbit IgG Or rabbit anti-eastern equine encephalitis virus IgG, goat anti-rabbit IgG or rabbit anti-western equine encephalitis virus IgG, goat anti-rabbit IgG or rabbit anti-Venezuela equine encephalitis virus IgG at a concentration of 0.1-5 mg/ml. the

Further, the pH of the colloidal gold-labeled protein or antigen is 6.0-6.4; the sample pad uses phosphate buffer containing NP 40 to pretreat the glass cellulose membrane. the

An application of the above-mentioned gold-labeled immune test strip, wherein the gold-labeled immune test strip A, the gold-labeled immune test strip B and the gold-labeled immune test strip C are organically integrated with a gold-labeled analyzer to form a gold-labeled immune chromatography Quantitative detection system, the reacted test strip is scanned by the gold standard immunoassay analyzer, and the corresponding reflected light intensity is recorded, represented by the optical density detection value T, quality control value C and T/C, so as to correctly interpret the test result of the test strip . the

The present invention is based on colloidal gold labeled with SPA protein or recombinant equine encephalitis protein, constructs an indirect method or a double-antigen sandwich method to detect antibodies, and evaluates the sensitivity, specificity and stability of the method. The feasibility of this method for detecting equine encephalitis virus antibodies in serum samples was evaluated by detecting the simulated environmental samples of healthy human serum artificially spiked with rabbit anti-recombinant equine encephalitis protein antibodies. The detection by using the test strip provided by the invention has the advantages of simple operation, short detection time, no need of professionals, and is suitable for fast on-site detection. Moreover, with the help of relevant equipment, qualitative and quantitative detection can be achieved, thereby greatly improving its promotion and application. the

Description of drawings

Fig. 1 is the front schematic view of the internal structure of the immunochromatographic test strip of the present invention;

Fig. 2 is the schematic diagram of the side structure of the internal structure of the immunochromatographic test strip of the present invention;

Fig. 3 is the positive detection result schematic diagram of immunochromatography test strip of the present invention;

Fig. 4 is the negative detection result schematic diagram of immunochromatography test strip of the present invention;

Fig. 5 is the invalid detection result schematic diagram of immunochromatography test strip of the present invention;

Fig. 6 is a structural schematic diagram of three kinds of gold-labeled immunological test strips arranged side by side in one body in the present invention.

Detailed ways

The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. the

For ease of description, spatially relative terms such as "upper," "lower," "left," and "right" may be used herein to describe the relationship of one element or feature relative to another element or feature shown in the figures. It will be understood that the spatial terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "lower" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative specifications used herein interpreted accordingly. the

In Fig. 1 and Fig. 2, 1: Absorbent pad; 2: Nitrocellulose membrane, T: detection band coated with recombinant equine encephalitis virus protein; C: coated with goat anti-rabbit IgG or goat anti-recombinant equine encephalitis virus protein IgG 3: Protective film of gold-labeled antibody containing gold-labeled SPA (Staphylococcal Protein A, SPA) or recombinant equine encephalitis protein; 4: Sample pad; 5: Reaction support . the

Fig. 3, Fig. 4, and Fig. 5 are schematic diagrams of detection results of the present invention; two lines of T and C are positive; one line of C is negative; the test strip is invalid if no lines appear. The overall outer package of the test strip is provided with a casing 6, and a sample hole 7 is arranged on the casing 6 corresponding to the sample pad, and an observation window 8 is arranged on the casing corresponding to the quality control strip and the detection strip. the

Fig. 6 is a schematic diagram of the structure of gold-labeled immune test strips that can simultaneously detect Eastern equine encephalitis virus, Western equine encephalitis virus, and Venezuelan equine encephalitis virus antibodies that are arranged side by side; Paper strip A, gold standard immune test strip B or gold standard immune test strip C, the three kinds of gold standard immunochromatographic test strips can be placed separately to achieve the purpose of simultaneous detection. the

In the present invention, it is used to detect the gold-labeled immune test strip A of the Eastern equine encephalitis virus antibody, the gold-labeled immune test strip B used to detect the Western equine encephalitis virus antibody, and the gold-labeled immune test strip B used to detect the Venezuelan equine encephalitis virus antibody. Standard immune test strip C, the basic structure of these three test strips is the same, the main difference is the gold-labeled antibody protective film: 

The gold-labeled antibody protective film in the gold-labeled immunoassay strip A includes the film body, and the gold-labeled Staphylococcus aureus protein A or recombinant Oriental equine encephalitis virus E1C antigen coating evenly coated on it, nitrocellulose A quality control strip coated with goat anti-rabbit IgG or rabbit anti-EEEV IgG is provided on the membrane near its starting end, and a detection strip coated with recombinant Eastern Equine Encephalitis Virus antigen is provided near its end;

The gold-labeled antibody protective film in the gold-labeled immunoassay strip B includes the film body, and the gold-labeled Staphylococcus aureus protein A or recombinant western equine encephalitis virus E1C antigen coating evenly coated on it, nitrocellulose A quality control band coated with goat anti-rabbit IgG or rabbit anti-Western equine encephalitis virus IgG is set on the membrane near its starting end, and a detection band coated with recombinant Western equine encephalitis virus antigen is set near its end;

The gold-labeled antibody protective film in the gold-labeled immune test strip C includes the film body, and the gold-labeled Staphylococcus aureus protein A or recombinant Venezuelan equine encephalitis virus E antigen coating evenly coated on it, nitrocellulose A quality control band coated with goat anti-rabbit IgG or rabbit anti-Venezuelan equine encephalitis virus IgG is set on the membrane near its starting end, and a detection band coated with recombinant Venezuelan equine encephalitis virus antigen is set near its end.

The invention is a gold-labeled immunochromatographic test strip for detecting equine encephalitis virus antibody, which has a sensitivity of 60 ng/mL for qualitative detection of eastern equine encephalitis virus antibody, a sensitivity of 120 ng/mL for detection of western equine encephalitis virus antibody, and a sensitivity of 120 ng/mL for detection of Venezuelan equine encephalitis virus antibody. Equine encephalitis virus antibody sensitivity reaches 1/20000 times serum dilution. the

The present invention is a gold-labeled immunochromatographic test strip for detecting equine encephalitis virus antibody, which includes mixing the sample to be tested with the sample diluent, and then adding the sample mixture to the sample hole of the test paper, and the liquid in the sample relies on siphon The effect is upward, and the result is interpreted in 10-15 minutes; the immunochromatographic test strip includes:

(1) Reaction support;

(2) Absorbent pad;

(3) Nitrocellulose membrane, which is coated with detection bands and quality control bands of recombinant equine encephalitis virus protein and quality control antibody;

(4) Gold-labeled antibody protective film, which contains gold-labeled SPA (Staphylococcal Protein A, SPA) or recombinant equine encephalitis virus protein;

(5) Sample pad;

Among them, the reaction support is made of PVC board; the absorbent pad is made of oil filter paper; the nitrocellulose membrane is provided with a quality control strip coated with goat anti-rabbit IgG or rabbit anti-recombinant equine encephalitis virus IgG near its starting end, and near its end. A detection band coated with recombinant equine encephalitis virus protein is set. The material of the gold-labeled antibody protective film is selected from polyester film, glass fiber or filter paper fiber, which contains gold-labeled SPA or recombinant equine encephalitis virus protein; the material of the sample pad is selected from polyester film, glass fiber or filter paper fiber.

The absorbent pad 1 in the test paper involved in the present invention is selected from filter paper, the reaction support 5 is selected from PVC board, and the material of the gold-labeled antibody protective film 3 is selected from polyester film, glass fiber or filter paper fiber, and the gold-labeled mark is evenly coated on it. antibodies. the

The gold-labeled antibody protective film in the test paper involved in the present invention is made by uniformly coating gold-labeled SPA or recombinant equine encephalitis virus protein on polyester film, glass fiber film or filter paper fiber film. the

The structure of a gold standard immunochromatographic test strip for detecting equine encephalitis virus antibody of the present invention is as follows: the reaction support 5 is located at the bottom layer, the nitrocellulose membrane 2 is located in the middle of the reaction support 5, and the T of the nitrocellulose membrane 2 It is the detection band coated with recombinant equine encephalitis virus protein, C is the quality control band coated with goat anti-rabbit IgG or rabbit anti-recombinant equine encephalitis virus protein IgG; the gold-labeled antibody protective film 3 is located on the nitrocellulose membrane 2 One side of the upper part and partly overlapped with it, the membrane contains the recombinant equine encephalitis virus antibody of the gold label; the absorbent pad 1 is located on the other side of the nitrocellulose membrane 2 upper part relative to the gold label antibody protective membrane 3, and It partially overlaps with the nitrocellulose membrane 2; the sample pad 4 is located on the opposite side of the nitrocellulose membrane 2 to the absorbent pad 1 and partially overlaps with the gold-labeled antibody protective membrane 3. the

In the above test paper, the side of the absorbent pad 1 is the starting end, the side of the gold-labeled antibody protective film 3 is the end, the detection strip is located near the end, and the quality control strip is close to the initial end. the

In the above test paper, the concentration of recombinant equine encephalitis virus protein is 0.1-5 mg/ml, and the concentration of quality control goat anti-rabbit IgG is 0.2-5 mg/ml. the

In the above test paper, the concentration of goat anti-rabbit IgG is preferably 0.5-2.5 mg/ml. the

In the above test paper, the amount of SPA labeled 1ml gold standard is 2-6 μg. the

The present invention detects the preparation method of the gold label immunochromatography test strip of equine encephalitis virus antibody, and the method comprises:

(1) Spray equine encephalitis virus protein on the nitrocellulose membrane at a certain spraying speed with a cross-flow spraying gold scribing machine as the detection strip and goat anti-rabbit IgG or rabbit anti-recombinant equine encephalitis virus protein IgG as the quality control strip bring;

(2) Prepare a gold-labeled antibody protective film containing gold-labeled SPA or recombinant equine encephalitis virus protein, apply gold-labeled SPA or recombinant equine encephalitis virus protein evenly on the glass fiber membrane, and bake After drying or freeze-drying, a gold-labeled antibody protective film is made.

In the preparation method of the present invention, in the step of spraying the coating film, the film spraying speed is 10-100 mm/s. the

In the preparation method of the present invention, the added amount of the recombinant equine encephalitis virus protein is 0.1-5 mg/ml, and the diluent of the protein can be PB solution. the

In the preparation method of the present invention, the concentration of goat anti-rabbit IgG or rabbit anti-recombinant equine encephalitis virus protein IgG in the quality control strip is 0.1-5 mg/ml. the

In the preparation method of the invention, the gold-labeled SPA or recombinant equine encephalitis virus protein is uniformly coated on the glass fiber membrane, and the pH is 6.0-6.4. the

The present invention relates to the application of the test paper in the detection of equine encephalitis virus antibody, which includes mixing the sample to be tested with the sample diluent, and then adding the sample mixture into the sample hole 7 of the test paper, and the liquid in the sample moves upward by siphon action, The results can be interpreted in 10-15 minutes. the

The invention relates to the immunochromatographic test paper for detecting equine encephalitis virus antibody prepared by the above method. the

Immunochromatography test strip consumables are used in the present invention, consumables such as glass fiber binding pads, nitrocellulose membranes (such as NC membranes, SHF 1350225), sample pads, absorbent pads, and filter paper, which can be purchased from Millipore. the

The above-mentioned gold-labeled immunochromatographic test paper for detecting equine encephalitis virus antibodies involved in the present invention also includes a gold-labeled antibody protective film. the

The above-mentioned gold standard immunochromatographic test paper for detecting equine encephalitis virus antibody involved in the present invention, wherein the reaction support is a PVC board. the

The above-mentioned gold standard immunochromatographic test paper for detecting equine encephalitis virus antibody involved in the present invention, wherein the water-absorbing pad is selected from filter paper. the

The above-mentioned gold-labeled immunochromatographic test paper for detecting equine encephalitis virus antibody involved in the present invention, wherein the gold-labeled antibody protective film is selected from polyester film, glass fiber or filter paper fiber. the

The method of the present invention can achieve the purpose of quantitative detection, and the experimental instrument used is, for example, a gold standard immunoassay analyzer, which can be obtained from the Chinese Academy of Inspection and Quarantine. Quantitative detection results and judgment: Use gold-labeled immunochromatographic test strips to detect the samples to be tested, and the reacted test strips are scanned by the gold-labeled immunoanalyzer in turn, and the corresponding reflected light intensity is recorded. The optical density detection value T and quality control Value C and T/C said. the

(1) Determine the judgment value (Cut-off)

The DJM-3 quantitative gold standard immunoassay analyzer was used to detect 20 negative values, and the cut-off value (Cut-off) was determined by calculating the ratio of T/C. Cut-off=average (Average)+3×standard deviation (Stdeva). Greater than or equal to Cut-off is positive.

(2) Fitting curve establishment

Take several different concentrations (including negative) of the standard substance of the target to be tested as the abscissa, and the T/C ratio measured by the gold-labeled strip analyzer after detecting each concentration with the gold-labeled test strip as the ordinate, and establish a fitting curve , and determine the quantitative detection sensitivity.

(3) Quantitative detection

The concentration of the sample can be calculated by substituting the T/C ratio measured by the gold standard analyzer of the sample to be tested into the fitting curve, so as to obtain the concentration of the sample and achieve quantitative detection.

1. Preparation of gold-labeled immunochromatographic test strips

Materials and methods

1. Antigen and antibody

Equine encephalitis virus protein antigen and purified rabbit anti-equine encephalitis virus antibody were prepared by our laboratory.

2. Immunochromatography test strip consumables

Bonding pad (glass fiber), nitrocellulose membrane (NC membrane, SHF 1350225), sample pad, absorbent pad, and filter paper were purchased from Millipore.

3. Preparation of gold-labeled immunochromatographic test strips

3.1 Gold Label Bonding Pad

SPA or recombinant equine encephalitis virus protein with a pH value of 6.0-6.4 and a concentration of 0.2mg/ml was labeled with 25nm gold-labeled particles prepared by the sodium citrate method, and dried at 37°C.

3.2 Nitrocellulose membrane

Detection band: recombinant equine encephalitis virus protein 1 mg/ml; quality control band: goat anti-rabbit IgG or rabbit anti-recombinant equine encephalitis virus protein IgG: 1 mg/ml, dry at 37°C.

3.3 Assembly

Paste the sample pad, bonding pad, and water-absorbing pad on the backing card with the release agent in sequence, cut into 0.4cm strips, dry, pack in a shell, and store at room temperature for later use.

2. Sample detection

1. Processing of simulated samples

Human serum samples are used for detection, and the samples are diluted 100 times with a sample treatment solution as samples to be tested; in addition, rabbit anti-equine encephalitis virus antibodies are added to the diluted human serum for detection at the same time.

2 sample detection

Add 100 μl of the treated sample and sample treatment solution (as a negative sample) to the end of the prepared chromatographic strip sample pad, let it stand for 15 minutes, and observe the results. If both the test strip and the quality control strip appear red, it is judged as positive, and if only the quality control strip appears red, it is negative.

3. Antibody Sensitivity Test of Eastern Equine Encephalitis Virus

1. Intuitive detection sensitivity evaluation

Gold-labeled immunochromatographic test strips were prepared according to the determined optimal reaction conditions to detect antibodies to EEEV at different concentrations. Dilute Eastern equine encephalitis virus antibody with sample treatment solution to concentrations of 15 ng/mL, 30 ng/mL, 60 ng/mL, 75 ng/mL, 150 ng/mL, 300 ng/mL, 600 ng/mL , 1200ng/mL, while testing. The results showed that the sensitivity of direct visual inspection was 60 ng/mL.

2. Sensitivity of quantitative detection

Detecting different concentrations of eastern equine encephalitis virus antibody, the gold standard analyzer reads the calculated cut-off value (Cut-off) is 0.012, and the average T/C ratio of eastern equine encephalitis virus antibody with a concentration lower than 60 ng/ml is 0, 0.006, lower than 0.012, it is negative; the average T/C ratio of Eastern equine encephalitis virus antibody of 60~1200ng/ml is 0.067, 0.079, 0.111, 0.221, 1.32, 1.331, all higher than 0.012, it is positive ; so the quantitative detection sensitivity is 60ng/ml (Table 1).

the

Table 1 EEV gold standard immunochromatographic test paper detection results of each concentration East Malaysia virus antibody concentration (ng/mL) Detection value T (V) Quality control value C (V) T/C value result 0 0 2.437 0 - 15 0.001 2.364 0 - 30 0.014 2.475 0.006 - 60 0.115 2.328 0.067 + 75 0.184 2.726 0.079 + 150 0.257 2.311 0.111 + 300 0.523 2.376 0.221 + 600 4.451 3.370 1.32 + 1200 4.392 3.298 1.331 +

3. Sample practicability test

The serum of healthy people was diluted 100 times with the sample treatment solution and used as the sample to be tested. The diluted serum of healthy people was tested by adding different concentrations of Eastern Equine Encephalitis virus antibody. The results showed that the serum of healthy people was negative, and Eastern Equine Encephalitis The sample sensitivity of inflammatory virus antibody was 60 ng/ml.

4. Western Equine Encephalitis Virus Antibody Detection Sensitivity Test

1. Intuitive detection sensitivity evaluation

Colloidal gold immunochromatographic test strips were prepared according to the determined optimal reaction conditions to detect antibodies to Western equine encephalitis virus at different concentrations. Dilute Western equine encephalitis virus antibody with sample treatment solution to concentrations of 90 ng/mL, 120 ng/mL, 180 ng/mL, 300 ng/mL, 450 ng/mL, 600 ng/mL, 900 ng/mL, 1200 ng/mL, while testing.

The results showed that the direct visual sensitivity of 120 ng/mL showed a clearer color. the

2. Sensitivity of quantitative detection

Detecting different concentrations of Western equine encephalitis virus antibody, the calculated cut-off value (Cut-off) is 0.011, and the average T/C ratio of Western equine encephalitis virus antibody with a concentration of 120ng/ml is higher than 0.011. The results are: Positive; when the Western equine encephalitis virus antibody concentration is greater than 120ng/ml, the average T/C ratio is greater than 0.011, so the sensitivity of quantitative detection is 120ng/ml.

the

Table 2 The detection results of Western equine encephalitis virus antibody gold-labeled immunochromatographic test paper at each concentration Western horse virus antibody concentration (ng/mL) T/C value result 0 0 - 90 0.002 - 120 0.014 + 180 0.025 + 300 0.042 + 450 0.054 + 600 0.074 + 900 0.114 + 1200 0.139 +

3. Standard curve

With the antibody concentration of Simma virus as the abscissa (X), and the gold standard analyzer reading value T/C ratio (Y) as the ordinate, a colloidal gold detection standard curve was established. When the concentration of the West Horse virus antibody is 120-1200 ng/ml, there is a good linear relationship between the concentration of the antibody and the T/C ratio read by the gold standard analyzer, and the linear regression equation is Y = 0.0001X+0.0025, R ² is 0.9947.

5. Sensitivity test of Venezuelan equine encephalitis virus antibody detection

1. Intuitive detection sensitivity evaluation

Colloidal gold immunochromatographic test strips were prepared according to the determined optimal reaction conditions to detect antibodies to Venezuelan equine encephalitis virus at different concentrations. Dilute the rabbit serum of Weimao virus E2 with the sample treatment solution to a concentration of 1/700, 1/1000, 1/3000, 1/7000, 1/10000, 1/20000, and 1/30000 times dilutions for simultaneous detection .

The results show that the sensitivity of direct visual inspection is 1/20000, and the color rendering is clearer. the

6. Specificity test

1. Oriental equine encephalitis virus antibody gold standard immunochromatographic test paper 

The prepared gold-labeled immunochromatographic test strips were used to detect Venezuelan equine encephalitis virus E2-19AA rabbit serum, Venezuelan equine encephalitis virus E2 rabbit serum, western equine encephalitis virus E1C antibody, and dengue virus Deng-E-3 rabbit serum. Serum, influenza A H1N1 virus antibody and West Nile virus NS1 rabbit serum showed no positive reaction, indicating that there was no cross-reaction with these antibodies and serum. 5 rabbit sera, 15 mouse sera, and 51 healthy Human serum, the results were all negative, indicating that the detection method has good specificity.

2. Venezuelan Equine Encephalitis Virus Antibody Gold Label Immunochromatographic Test Paper

Prepare gold-labeled immunochromatographic test strips according to the determined optimal reaction conditions, and use the sample diluent to treat the prepared colloidal gold immunochromatographic test strips to detect horse Qiubo GP2 virus rabbit serum and eastern equine encephalitis virus E1C rabbit serum , Western Equine Encephalitis Virus E1C Rabbit Serum, Dengue Virus E2 Rabbit Serum, Influenza A H1N1 Antibody, West Nile Virus NS1 Rabbit Serum. There was no positive reaction in the test results, which proved that there was no cross-reaction with these antibodies, and the specificity of the detection method was good.

3. Western equine encephalitis virus antibody gold standard immunochromatographic test paper

Prepare gold-labeled immunochromatographic test strips according to the determined optimal reaction conditions, and detect rabbit anti-Maqiubo GP2 protein, smallpox M1R protein, avian influenza NP protein, dengue virus Deng-E-D3 protein, common Rickettsia 120N protein antibody, 63 healthy mouse sera and 105 healthy human sera. The prepared gold-labeled immunochromatographic test strip was used for detection, and was compared with the Western equine encephalitis virus antibody sample of the same concentration, and the blank control was detected at the same time. The results showed that the specific detection effect on western equine encephalitis virus antibody was good. 63 samples of healthy mouse serum were diluted 1:10 with the sample treatment solution for detection, and the results showed that 6 samples were positive, and the false positive rate was 9.52%; for 105 Serum samples from healthy people were tested, and 7 samples were detected positive, with a false positive rate of 6.67%. The detection test strip has no non-specific phenomenon for these antibodies. the

7. Stability test

Store the newly prepared gold-labeled immunochromatographic test strips at 37°C for 2 weeks, and store them at room temperature for 4 weeks. Compared with the newly prepared test strips, the specificity and sensitivity did not decrease significantly, and the specificity was good. .

Claims (10)

1. gold-marking immunity tomographic system that detects equine encephalitis virus antibody; It is characterized in that; This gold-marking immunity tomographic system comprises gold-marking immunity analyser, the gold-marking immunity test strips A that is used to detect eastern equine encephalitis virus antibody, the gold-marking immunity test strips B that is used to detect western equine encephalitis virus antibody that is used for detection by quantitative, the gold-marking immunity test strips C that is used to detect Venezuelan equine encephalitis virus antibody; Three test strips include the reaction stilt, closely are connected in the nitrocellulose membrane of reaction stilt upper surface middle part; The upper surface of nitrocellulose membrane initiating terminal overlap with adsorptive pads be connected, upper surface that nitrocellulose membrane is terminal overlaps with golden labeling antibody diaphragm and is connected the overlapping sample pad that is connected with of the upper surface portion of golden labeling antibody diaphragm;

Wherein, Golden labeling antibody diaphragm among the gold-marking immunity test strips A comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization eastern equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-eastern equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of eastern equine encephalitis near its end near its initiating terminal place on the nitrocellulose membrane;

Golden labeling antibody diaphragm among the gold-marking immunity test strips B comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization western equine encephalitis virus E1C antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of western equine encephalitis virus near its end near its initiating terminal place on the nitrocellulose membrane;

Golden labeling antibody diaphragm among the gold-marking immunity test strips C comprises membrane body and is uniformly coated on the staphylococcal protein A or the reorganization Venezuelan equine encephalitis virus E antigen coating of the gold mark mark on it, is provided with Quality Control band that goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit encapsulate, is provided with the antigen coated test strip of Venezuelan equine encephalitis virus near its end near its initiating terminal place on the nitrocellulose membrane.

2. gold-marking immunity tomographic system as claimed in claim 1 is characterized in that, said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C are made into integration side by side.

3. gold-marking immunity tomographic system as claimed in claim 2 is characterized in that, said reaction holder is the PVC plate, and adsorptive pads is a filter paper for oil; Said golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.

4. gold-marking immunity tomographic system as claimed in claim 2 is characterized in that, said sample pad is glass fibre membrane, polyester film or filter paper fibre.

5. gold-marking immunity tomographic system as claimed in claim 2; It is characterized in that; Said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C wrap up separately whole outside and are provided with shell; Be provided with sample well corresponding to said sample pad place on this shell, be provided with view window corresponding to said Quality Control band, test strip place on the shell.

6. preparation method of gold-marking immunity test strips according to claim 1 is characterized in that this preparation method is specially:

1) preparation collaurum;

2) utilize the collaurum difference corresponding staphylococcal protein A of mark or reorganization eastern equine encephalitis virus E1C antigen, staphylococcal protein A or reorganization western equine encephalitis virus E1C antigen, staphylococcal protein A or the reorganization Venezuelan equine encephalitis virus E antigen that makes;

3) with at a distance from stream metal spraying pen machine with the initiating terminal setting spray film speed and respectively goat anti-rabbit igg or the anti-eastern equine encephalitis virus IgG of rabbit are sprayed to nitrocellulose membrane one with the initiating terminal that forms Quality Control band one, goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit are sprayed to nitrocellulose membrane two with the initiating terminal that forms Quality Control band two, goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit are sprayed to nitrocellulose membrane three forming Quality Control band three, the end that reorganization equine encephalitis albumen is sprayed to three nitrocellulose membranes is to form the relevant detection band;

4) with step 2) in the three kinds of albumen or the antigen of the colloid gold label that makes be uniformly coated on respectively on three glass fibre membranes, three corresponding golden labeling antibody diaphragms are processed in and oven dry or freeze drying;

5) in reaction stilt upper surface middle part three nitrocellulose membranes in the step 3) are set; The upper surface of three nitrocellulose membrane initiating terminals is overlapped with corresponding adsorptive pads respectively and is connected; Three terminal upper surfaces of nitrocellulose membrane are overlapped with corresponding golden labeling antibody diaphragm and are connected the corresponding sample pad of the overlapping connection of the upper surface portion of golden labeling antibody diaphragm;

6) test paper that comprises three test strips that forms in the step 5) is cut into the shape of sized, carries out drying, dress shell, to form test strips.

7. preparation method as claimed in claim 6 is characterized in that, the concrete steps of preparation collaurum are in the said step 1):

1) preparation HAuCl ₄The WS: add HAuCl under the magnetic agitation ₄, adding trisodium citrate aqueous solution simultaneously, colour stable continued heating is cooled to and adds pure water after the room temperature and complement to setting value, and 4 ℃ keep in Dark Place;

2) use K ₂CO ₃Adjust pH is 6.0-6.4;

3) collaurum is transferred to pH 6.0-6.4, with PB dilution SPA to 0.2 mg/ml;

4) keep collaurum stable.

8. preparation method as claimed in claim 6 is characterized in that, in the said step 3), spray film speed is 10-100mm/s; The addition of said reorganization equine encephalitis albumen is 0.1-5mg/ml, and the dilution of this recombinant antigen can be PBS; The concentration of the anti-eastern equine encephalitis virus IgG of goat anti-rabbit igg or rabbit, goat anti-rabbit igg or the anti-western equine encephalitis virus IgG of rabbit, goat anti-rabbit igg or the anti-Venezuelan equine encephalitis virus IgG of rabbit is 0.1-5 mg/ml.

9. preparation method as claimed in claim 6 is characterized in that, is 6.0-6.4 with the albumen of colloid gold label or the pH of antigen; Said sample pad adopts the phosphate buffer that contains NP 40 that the plain film of spun glass is carried out pre-service.

10. application of wanting 1 described gold-marking immunity test strips like right; Said gold-marking immunity test strips A, gold-marking immunity test strips B and gold-marking immunity test strips C are become gold-marking immunity chromatography detection by quantitative system with gold mark analyser organic combination; Reacted test strips scans through the gold-marking immunity analyser; Write down corresponding reflective light intensity, represent, with this test strips testing result of correct interpretation with optical density detected value T, Quality Control value C and T/C.

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