CN110927384A

CN110927384A - Fluorescence immunochromatography test strip and preparation method and application thereof

Info

Publication number: CN110927384A
Application number: CN201911205664.0A
Authority: CN
Inventors: 彭洁; 严俊; 张变强; 周泽奇; 粟艳
Original assignee: Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2020-03-27
Anticipated expiration: 2039-11-29
Also published as: CN110927384B

Abstract

The invention provides a fluorescence immunochromatographic test strip and a preparation method and application thereof. The fluorescence immunochromatographic test strip comprises a sample pad, a fluorescence pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is coated with a shielding line, a detection line and a quality control line, and a shielding liquid for preparing the shielding line comprises an anti-HAMA antibody, calcium ions, casein, a protein stabilizer and a buffer solution. When the fluorescence immunochromatographic test strip provided by the invention is used for detecting the aspergillus or candida antigens, the interference substances in the sample to be detected are adsorbed through the shielding line, so that the influence of the interference substances in the sample to be detected is reduced, the combination of the aspergillus or candida antigens and the fluorescent particles is more stable, the chromatographic background is clearer, the detection result has better sensitivity, specificity and stability, and the detection result is accurate and reliable.

Description

Fluorescence immunochromatography test strip and preparation method and application thereof

Technical Field

The invention belongs to the technical field of immunodetection and analysis, particularly relates to a fluorescence immunochromatographic test strip and a preparation method and application thereof, and particularly relates to a fluorescence immunochromatographic test strip for detecting antigens of aspergillus and candida as well as a preparation method and application thereof.

Background

Aspergillus (Aspergillus) is an important pathogenic bacterium for the condition, often infecting patients with low or defective immunity. Aspergillus fumigatus is the most common pathogenic bacterium causing serious deep Aspergillus infection of immunosuppressed patients, and the death rate of the disease is high. In recent years, the incidence and mortality of invasive deep fungal infections has become higher with the massive abuse of antibiotics and the widespread clinical use of immunosuppressants. Among them, Aspergillus, particularly Aspergillus fumigatus, has become an important pathogenic fungus in clinical practice.

Galactomannans have been shown to be present on the aspergillus cell wall as the major antigenic component of aspergillus and are also the major antigenic component of the cell wall. Aspergillus fumigatus galactomannan is found in high amounts in animal models of infection and in serum of patients with invasive Aspergillus, and thus is used as a marker for diagnosing Aspergillus infection. The detection of the aspergillus is realized by an antibody specifically aiming at the galactomannan antigen of the aspergillus fumigatus, and the blood sensitivity of the detection reaches more than 80 percent. At present, the number of clinical immunoassay products aiming at aspergillus fumigatus in the international market is small, and the most typical product is a double-antibody sandwich ELISA immunoassay kit of Bio-Rad company.

Candida, which is one of the most common pathogenic bacteria in fungi, mainly comprises candida albicans, candida tropicalis, candida glabrata, candida parapsilosis and candida krusei, and accounts for more than 80% of clinical medical samples. Mannan (Mannan, Mn) is the main antigenic component of candida cell walls. Candida is often parasitic on human skin, oral cavity, vagina, intestinal mucosa and other places, and candidiasis is easily caused when the immune function of an organism is reduced.

CN105866409A discloses a candida mannan antigen immunodetection kit, which comprises a Mn-coated enzyme label plate and an anti-Mn polyclonal enzyme labeled antibody. The kit comprises the steps of coating Mn on an enzyme label plate, competitively binding a sample to be detected or a standard antigen and the coated antigen to a limited antibody binding site, carrying out chromogenic reaction on the sample to be detected or the standard antigen and a substrate through horseradish peroxidase, drawing a standard curve according to a Mn standard substance, and calculating the concentration value of the antigen to be detected according to the standard curve. The immunoassay kit provided by the invention adopts Enzyme-Linked immunosorbent Assay (ELISA) to detect antigen, and the steps of processing and detecting are complex and the processing time is long.

Immunochromatography (Immuno chromatographic Assay, ICA) is a novel membrane detection technique based on antigen-antibody specific immune reactions. The technology takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) as a stationary phase, a test solution as a mobile phase, a fluorescence labeled antibody or antigen fixed on a connecting pad, and an analyte to be analyzed moves on the chromatography strip through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with a plurality of antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is combined with a coating antibody to form a double-antibody sandwich-type macromolecular substance when reaching a detection line.

The immunofluorescence technology (fluoroscience immuno chromatography Assay) is a novel immunolabeling technology which applies Fluorescence as a tracer marker to an antigen antibody, and the Fluorescence microsphere coated with Eu chelate and subjected to surface carboxylation modification is covalently bonded with amino on the surface of the antibody to form an immunolabeling compound. The fluorescence chromatography test strip has the following advantages: the kit is convenient and quick to use, low in cost, convenient for basic level use and field use, wide in application range and suitable for various detection conditions; meanwhile, a color development reagent is not required to be added, the steps of an enzyme-labeled carcinogenic substrate and a stop solution are omitted, and the kit is harmless to a human body.

However, when the fluorescence chromatography test strip is used to detect the antigens of aspergillus and candida, the sample to be detected needs to be pretreated by EDTA before loading, so that residual EDTA exists in the sample to be detected, and the processed sample still has interfering substances, such as polysaccharides and glycoproteins, which affect the immunoreaction on the detection line during the chromatography process, so that the accuracy of the detection result is low.

Therefore, in the field of clinical detection of aspergillus and candida antigens, an immunochromatographic product which is simple in operation steps and high in accuracy is urgently needed for detecting the aspergillus and candida antigens.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a fluorescence immunochromatographic test strip and a preparation method and application thereof. According to the fluorescence immunochromatographic test strip provided by the invention, a shielding wire is added, the shielding wire can adsorb interfering substances in a sample to be detected, the influence of the interfering substances in the sample to be detected in the detection process is reduced, the chromatographic background of the test strip is clearer, and the detection result is more sensitive and accurate. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a fluorescence immunochromatographic test strip, which comprises a sample pad, a fluorescence pad, a nitrocellulose membrane (NC membrane) and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is coated with a shielding line, a detection line (T line) and a quality control line (C line), and a shielding solution for preparing the shielding line comprises an anti-HAMA antibody, casein, calcium ions, a protein stabilizer and a buffer solution.

According to the fluorescent immunochromatographic test strip provided by the invention, a shielding line is added before a traditional detection line and a quality control line, and components of the shielding line are reasonably screened, so that the fluorescent immunochromatographic test strip can adsorb interfering substances which may influence a detection result in a sample to be detected, such as Ethylene Diamine Tetraacetic Acid (EDTA), human anti-mouse antibody (HAMA) and the like, and the sensitivity and accuracy of an immunochromatographic result are improved.

In a preferred embodiment of the present invention, the anti-HAMA antibody in the masking liquid may be present at a concentration of 0.5 to 2% by mass, for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2%, preferably 1%.

Preferably, the mass concentration of calcium ions in the shielding liquid is 0.5 to 2%, and may be, for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, or the like, and preferably 1%.

Preferably, the mass concentration of casein in the masking liquid is 0.5-2%, for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, or 2%, and preferably 1%.

In a preferred embodiment of the present invention, the protein stabilizer in the masking liquid is one or a combination of two or more of sucrose, trehalose, BSA (bovine serum albumin), mannitol, and glycerol, preferably trehalose.

Preferably, the protein stabilizer is present in a concentration of 1-3% by mass, e.g., 1%, 1.2%, 1.5%, 1.6%, 1.7%, 1.8%, 2%, etc., preferably 2%.

Preferably, the buffer in the shielding solution has a pH of 8.0 to 9.0, for example, 8.0, 8.2, 8.4, 8.5, 8.7, 8.9, or 9.0, and preferably 8.5.

Preferably, the buffer is at a molarity of 0.01-0.05M, such as 0.01M, 0.015M, 0.02M, 0.025M, 0.03M, 0.035M, 0.04M, or 0.05M, and preferably 0.02M.

Preferably, the buffer is any one of Tris-HCl buffer, PBS buffer, PB buffer or CBS buffer, and is preferably Tris-HCl buffer.

As a preferable technical scheme of the invention, the fluorescent pad is embedded with a fluorescently-labeled candida antibody or aspergillus antibody.

Preferably, the detection line is a detection line containing candida antibodies or aspergillus antibodies.

Preferably, the candida or aspergillus antibody is a murine antibody.

The murine antibody is the most common antibody form in the current commercial antibodies, and has the advantages of wide source, low cost, good specificity and stability.

Preferably, the quality control line is a quality control line containing an anti-mouse antibody, preferably a goat anti-mouse IgG antibody.

As a preferred technical scheme, the candida antibodies are monoclonal antibodies and/or polyclonal antibodies of the candida mannan antigen, and preferably the monoclonal antibodies of the candida mannan antigen.

Preferably, the aspergillus antibody is a monoclonal antibody against aspergillus galactomannan antigen and/or a polyclonal antibody against aspergillus galactomannan antigen, preferably a monoclonal antibody against aspergillus galactomannan antigen.

Antibodies are classified into polyclonal antibodies and monoclonal antibodies, and methods for preparing the polyclonal antibodies or the monoclonal antibodies are well known to those skilled in the art. Polyclonal antibodies may have the problem of being less specific than monoclonal antibodies, and may have false positive results. In particular, although polyclonal antibodies can be used as the antibodies of the invention, monoclonal antibodies have significant advantages.

As a preferable technical solution of the present invention, the shielding line on the nitrocellulose membrane is located at one end close to the fluorescent pad, the quality control line is located at one end close to the absorbent pad, and the detection line is located between the shielding line and the quality control line.

Preferably, the distance between the shield wire and the detection wire is 3-5mm, and may be, for example, 3mm, 3.2mm, 3.5mm, 4mm, 4.2mm, 4.5mm, 4.8mm, or 5 mm.

Preferably, the distance between the quality control line and the detection line is 3-5mm, and may be, for example, 3mm, 3.2mm, 3.5mm, 4mm, 4.2mm, 4.5mm, 4.8mm, or 5 mm.

In a preferred embodiment of the present invention, the sample pad is a glass fiber film or a polyvinyl acetate film, preferably a glass fiber film.

Preferably, the fluorescent pad is a glass fiber film.

Preferably, the fluorescence immunochromatographic test strip further comprises a bottom plate.

Preferably, the bottom plate is a polyvinyl chloride (PVC) rubber plate.

In a second aspect, the present invention further provides a method for preparing the fluorescent immunochromatographic test strip of the first aspect, wherein the method for preparing comprises: preparing a fluorescent pad, preparing a shielding solution, preparing a nitrocellulose membrane, and assembling a fluorescent immunochromatographic test strip.

The preparation method of the fluorescent pad comprises the following steps:

adding candida antibodies or aspergillus antibodies into the activated fluorescent microsphere solution to the final concentration of 10 mug/mL, and standing for 120min for marking; after completion, a solution containing 10% BSA was added to the above solution as a blocking solution and allowed to stand for 60 min. And centrifuging the obtained fluorescent solution for 20min at 10000rpm, removing the supernatant after centrifugation, adding 100mL of complex solution into the precipitate, fully mixing the complex solution uniformly, and performing ultrasonic treatment on the mixture for 60s to obtain the candida or aspergillus antibody labeling solution. Spraying candida or aspergillus antibody marking solution onto a glass fiber membrane by using a gold spraying and membrane scratching instrument according to the volume of 15 mu L/cm; and (3) drying the obtained glass fiber membrane in an oven at 37 ℃ for 180min, adding a drying agent after drying, and sealing and storing to obtain the fluorescent pad.

The preparation method of the shielding liquid comprises the following steps:

weighing certain mass of Tris (hydroxymethyl) aminomethane (Tris), trehalose, casein and CaCl₂Putting into a beaker, adding deionized water, adding anti-HAMA antibody, and mixing to obtain a shielding solution containing 0.5-2% anti-HAMA antibody, 1-3% trehalose, 0.01-0.05M TRIS (pH 8.5), 0.1M calcium ion and 0.5-2% casein. For example, 0.24g Tris, 1.5g trehalose, 1g casein, 1.1g CaCl are weighed₂Placing into a beaker, adding 100mL of deionized water, and adding 1g of anti-HAMA antibody to obtain a shielding solution containing 1% of anti-HAMA antibody, 1.5% of trehalose, 0.015M TRIS (pH 8.5), 0.1M of calcium ions and 1% of casein.

The nitrocellulose membrane comprises the following steps:

scribing a shielding solution (as a shielding line), a solution (as a detection line) containing a candida antibody or an aspergillus antibody and a solution (as a quality control line) containing an anti-candida antibody or an aspergillus antibody on an NC membrane by using a fluorescence-spraying membrane scribing instrument at the condition of 1 mu L/cm; and (3) drying the NC membrane in an oven at 37 ℃ for 4h, adding a drying agent after drying, and sealing and storing.

The assembly of the fluorescence immunochromatographic test strip comprises the following steps:

assembling the coated NC film, the fluorescent pad, the water absorption pad and the sample pad on a PVC plate; cutting the obtained assembled plate into fluorescent test strips with the thickness of 4 +/-0.2 mm by using a strip cutting device; and placing the obtained test strip into a sealed bag, and adding a drying agent for preservation.

In a third aspect, a test kit comprises the fluorescent immunochromatographic strip of the first aspect.

In a fourth aspect, the use of the fluorescence immunochromatographic test strip of the first aspect and/or the detection kit of the third aspect for detecting candida or aspergillus is provided.

Preferably, the candida includes any one or a combination of two or more of candida albicans, candida tropicalis, candida parapsilosis, candida krusei or candida glabrata.

Preferably, the aspergillus includes any one or a combination of more than two of aspergillus fumigatus, aspergillus flavus, aspergillus terreus or aspergillus niger.

In the invention, the use method of the test strip comprises the following steps:

1. pretreatment of aspergillus antigen and candida antigen detection samples: adding 300 mu L of serum to be detected into a centrifuge tube, adding 100 mu L of 0.08M EDTA solution sample treatment solution into the centrifuge tube, carrying out vortex oscillation for 10s to thoroughly mix the serum and the EDTA solution, putting the centrifuge tube into a water bath kettle, heating the centrifuge tube at 100 ℃ for 180 +/-10 s, taking the centrifuge tube out of the water bath kettle, putting the centrifuge tube into a centrifuge, and centrifuging the centrifuge tube at 10000g for 10min at 4 ℃;

2. detecting a sample to be detected: sucking 90 mu L of a sample to be detected, slowly dripping the sample into a sample adding hole of a test strip, scanning a detection area by using a fluorescence immunochromatography analyzer to obtain a fluorescence signal, wherein an I value appears after the detection is finished, the result is read within 20-25min, and the read result after 25min is unreliable;

3. and (4) analyzing results: if the I value is less than 0.5, determining that the sample is negative, and indicating that the sample to be detected does not contain aspergillus antigens or candida antigens; if the I value is greater than 0.5, the test sample is determined to be positive, and the test sample contains the aspergillus antigen or the candida antigen.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between any of the above-recited numerical ranges not recited, and for the sake of brevity and clarity, the present invention is not intended to be exhaustive of the specific numerical values encompassed within the range.

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

the fluorescence immunochromatographic test strip provided by the invention adopts a sandwich method to detect the aspergillus or candida antigens, has better sensitivity, specificity and stability, the components in the added shielding lines can adsorb interfering substances in a sample to be detected, and the influence of the interfering substances in the sample to be detected during detection is reduced, so that the combination of the aspergillus or candida antigens and fluorescent particles is more stable, the chromatographic background is clearer, and the detection result is accurate and reliable.

Drawings

Fig. 1 is a schematic structural diagram of the fluorescence immunochromatographic test strip provided by the present invention.

FIG. 2 is a graph showing fluorescence signals obtained after detection of Aspergillus antigen positive samples in example 1.

FIG. 3 is a graph showing fluorescence signals of comparative example 1 after detection of an Aspergillus antigen positive sample.

FIG. 4 is a graph showing fluorescence signals of Candida positive samples in example 5.

FIG. 5 is a graph showing fluorescence signals of a positive Candida antigen sample in comparative example 6.

The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.

Sources of reagents and instrumentation used in the following examples: the antibody used in the experiment is prepared by dana (Tianjin) Biotechnology limited company by a conventional method; the gold spraying and film scratching instrument is purchased from Shanghai gold-labeled Biotech limited; the slitter is purchased from Hai jin Biao Biotechnology GmbH; the fluorescence immunochromatography analyzer is purchased from Guangzhou blue Bob Biotechnology GmbH; PVC plywood, absorbent paper, fiberglass membrane and cardboard were purchased from Biotech, Bikenlebo, Hangzhou.

Example 1

The embodiment provides an aspergillus antigen fluorescence immunochromatographic test strip and a preparation method thereof.

In the embodiment, the fluorescence pad of the test strip is embedded with fluorescence-labeled aspergillus galactomannan antibody with the concentration of 10 mug/mL;

the nitrocellulose membrane is embedded with a shielding line, a detection line (T line) and a quality control line (C line), and the detection line is prepared from an aspergillus galactomannan antibody solution with the concentration of 2 mg/mL; the quality control line is prepared by a goat anti-mouse IgG antibody solution with the concentration of 2 mg/mL; the formulation of the shielding solution used for preparing the shielded wire is shown in table 1 (wherein% represents mass concentration, and M represents molar concentration mol/L).

TABLE 1

Components	Concentration of
		anti-HAMA antibodies	1％
Trehalose
		2％
TRIS			0.02M(pH 8.5)
	Calcium ion	0.1M
Casein protein
		1％

The fluorescence immunochromatographic test strip can be prepared by the following method:

one end of the PVC rubber plate is sequentially and mutually lapped and stuck with a sample pad, a fluorescent pad, a nitrocellulose membrane and water absorption filter paper, then the PVC rubber plate and the stuck material are cut into test strips with the width of 4 +/-0.2 mm, the test strips are loaded into a card shell, and a sample adding area and a detection area (an observation area) are arranged on the card shell.

Wherein the sample pad is a glass fiber membrane; and (3) scratching a shielding solution (serving as a shielding line), a solution (serving as a detection line) containing an aspergillus antibody and a solution (serving as a quality control line) containing an anti-aspergillus antibody on an NC membrane by using a fluorescence spraying and membrane scratching instrument under the condition of 1 mu L/cm, drying the NC membrane in an oven at 37 ℃ for 4h, adding a drying agent, and sealing and storing.

Assembling the fluorescent test strip: assembling the coated NC film, the fluorescent pad, the water absorption pad and the sample pad on a PVC plate; cutting the obtained assembled plate into fluorescent test strips with the thickness of 4 +/-0.2 mm by using a strip cutting device; and placing the obtained test strip into a sealed bag, and adding a drying agent for preservation.

As shown in fig. 1, the detection principle of the fluorescence immunochromatographic test strip of the present invention is as follows:

(1) after the sample to be detected is added into the sample adding area (sample pad), the sample to be detected moves into the fluorescence pad through capillary action, if the sample to be detected contains the antigen of aspergillus, the antigen in the sample is combined with the antibody of aspergillus in the fluorescence to form an antigen-antibody-fluorescence combination, and the combination continuously moves to one end of the water absorption pad;

(2) when the detection sample moves to the shielding wire, interference factors such as HAMA, polysaccharide, EDTA and the like in the detection sample react with corresponding antibodies, calcium ions and casein in the shielding wire, stay at the shielding wire and do not move continuously along with the detection sample, and the antigen-antibody-fluorescence conjugate in the detection sample does not influence and continuously moves;

(3) when the test sample moves to the detection line, the antigen-antibody-fluorescent conjugate is captured, an antibody-antigen-antibody-fluorescent conjugate is generated at the detection line, and upon detection using a fluorescence immunochromatographic analyzer, a fluorescent band can be observed: if the antigen of the aspergillus does not exist in the detected actual sample, an immune complex cannot be formed, and a fluorescent strip cannot appear;

(4) when the detection sample continues flowing and flows to a quality control line, namely the goat anti-mouse IgG antibody, a fluorescence strip appears no matter whether the sample to be detected contains the aspergillus antigen or not, and the effectiveness of the test strip is proved. As long as the quality control line does not develop color, the test strip is invalid and the actual sample needs to be retested.

Example 2

The difference from the example 1 is that the formulation of the shielding liquid is shown in table 2, and the rest components and the preparation method are the same as the example 1.

TABLE 2

Components	Concentration of
		anti-HAMA antibodies	0.5％
Trehalose
		2％
TRIS			0.01M(pH 8.0)
	Calcium ion	0.05M
Casein protein
		2％

Example 3

The difference from the example 1 is that the formulation of the shielding liquid is shown in table 3, and the rest components and the preparation method are the same as the example 1.

TABLE 3

Components	Concentration of
		anti-HAMA antibodies	2％
Trehalose	0.5％
		TRIS	0.05M(pH 9.0)
Calcium ion	0.2M
		Casein protein	0.5％

Example 4

The difference from the example 1 is that the formulation of the shielding liquid is shown in table 4, and the rest components and the preparation method are the same as the example 1.

TABLE 4

Components	Concentration of
		anti-HAMA antibodies	1％
Trehalose	1.5％
		TRIS	0.02M(pH 8.5)
Calcium ion	0.12M
		Casein protein	1.8％

Comparative example 1

This example provides an aspergillus antigen fluorescence immunochromatographic test strip, which is different from example 1 only in that the test strip does not contain a shielding line.

Comparative example 2

This example provides an aspergillus antigen fluorescence immunochromatographic test strip, which is different from example 1 only in that the shielding solution does not contain an anti-HAMA antibody.

Comparative example 3

This example provides an aspergillus antigen fluorescence immunochromatographic test strip, which is different from example 1 only in that the shielding solution does not contain casein.

Comparative example 4

This example provides an aspergillus antigen fluorescence immunochromatographic test strip, which is different from example 1 only in that the shielding solution does not contain calcium ions.

Comparative example 5

This example provides an aspergillus antigen fluorescence immunochromatographic test strip, which is different from example 1 only in that the mass concentration of the anti-HAMA antibody in the shielding solution is 0.2%.

Test example 1

Repeatability detection experiment of immunochromatographic test paper

The immunochromatographic test strips provided in examples 1 to 4 and comparative examples 1 to 5 were used to detect the Aspergillus galactomannan antigen positive reference substance and the Aspergillus galactomannan antigen negative reference substance 10 times each, and differences in the detection results were observed. The detection result of each test strip on the positive reference substance is shown in table 5: (M represents the mean value; SD represents the standard deviation; CV represents the coefficient of variation). The smaller the SD and CV, the smaller the degree of dispersion representing the measurement, and the more accurate the measurement.

TABLE 5

The test results of each test strip for the negative reference are shown in table 6:

TABLE 6

As can be seen from the detection results in tables 5 and 6, the test strips for fluorescence immunochromatography provided in examples 1 to 4 all have an I value greater than 1 when detecting a positive reference substance, and have an I value less than 0.5 when detecting a negative reference substance, indicating that there is no error in the detection results. Meanwhile, the detection results of the standard deviation SD and the dispersion degree CV of the detection results of the embodiments 1 to 4 are obviously smaller than those of the test strips provided by the comparative examples 1 to 5, which shows that the repeatability of the detection results of the fluorescence immunochromatographic test strip provided by the invention is obviously improved, and the influence of non-specific adsorption of the sample is eliminated.

Test example 2

Sensitivity and specificity experiments

The immunochromatography test strip provided in the embodiments 1 to 4 and the comparative examples 1 to 5 is used for simultaneously detecting 60 aspergillus antigen positive samples and 72 aspergillus antigen negative samples, and the sensitivity and the specificity of the test strip are calculated according to the negative and positive results of the test strip, wherein the sensitivity is the proportion of correctly judging a true positive sample as a true positive sample; the specificity is a ratio of correctly judging a true negative sample as a true negative.

The results of the sensitivity and specificity measurements are shown in Table 7.

TABLE 7

The results in table 7 show that the sensitivity of the immunochromatographic test strip provided by the invention is higher than 86% when detecting positive and negative samples, and the specificity of the obtained test strip is higher than 86%, which indicates that the specificity is also better.

Test example 3

Background chromatography

The immunochromatography test strips provided in examples 1-4 and comparative examples 1-5 were used to simultaneously detect the Aspergillus antigen samples, count the chromatography time, and observe the chromatography background.

According to the observation results, the chromatography time of the fluorescence immunochromatographic test strip prepared in the examples 1 to 4 is 15 to 20min (wherein the moving time of the sample on the NC membrane is 60 to 90s), the background of fluorescence chromatography is clean, and no layering phenomenon exists; and the chromatographic time of the test strip prepared in the comparative examples 1-5 is 18-25min (wherein the moving time of the sample on the NC membrane is 90-110s), the fluorescence residue is obvious, and the fluorescent particles are shunted with the buffer system.

In the fluorescence signal spectra obtained by the fluorescence quantitative analyzer after the test strips of example 1 and comparative example 1 detect the aspergillus antigen positive sample are shown in fig. 2 and 3, wherein the abscissa represents the displacement (scanning position) and the ordinate represents the fluorescence signal intensity. In FIG. 2, a relatively distinct peak appears at a position around the displacement 350, which represents the fluorescence intensity of the detection line on the test strip provided in example 1, and a small peak appears at a position around 450, which represents the fluorescence intensity of the quality control line of the test strip; correspondingly, the position of about 350 of displacement in fig. 3 represents the fluorescence intensity of the detection line on the test strip provided in the comparative example 1, and the position of about 450 represents the fluorescence intensity of the quality control line, and as can be seen from fig. 2 and fig. 3, the peak of the test strip detection line provided in the embodiment 1 is more obvious, that is, the fluorescence intensity is higher, the fluorescence intensities of other positions of the test strip are lower, and the chromatographic background is clear.

Example 5

The embodiment provides a candida antigen fluorescence immunochromatographic test strip and a preparation method thereof.

In the embodiment, the fluorescence pad of the test strip is embedded with a candida mannan antibody with a fluorescence mark and a concentration of 15 mug/mL;

the nitrocellulose membrane is embedded with a shielding wire, a detection line and a quality control line, wherein the detection line is prepared from a candida mannan antibody solution with the concentration of 0.8 mg/mL; the quality control line is prepared by a goat anti-mouse IgG antibody solution with the concentration of 1.5 mg/mL; the formulation of the shielding solution used to prepare the shielded wire is shown in table 8.

TABLE 8

The preparation method of the test strip is the same as that of the example 1.

Example 6

The difference from example 5 is that the formulation of the shielding liquid is shown in Table 9, and the rest components and preparation method are the same as example 5.

TABLE 9

Example 7

The difference from example 5 is that the formulation of the shielding liquid is shown in Table 10, and the rest components and preparation method are the same as example 5.

Watch 10

Example 8

The difference from example 5 is that the formulation of the shielding liquid is shown in Table 11, and the rest components and preparation method are the same as example 5.

TABLE 11

Comparative example 6

The present embodiment provides a candida antigen fluorescence immunochromatographic test strip, which is different from that in embodiment 5 in that the test strip does not contain a shielding line.

Comparative example 7

This example provides a candida antigen fluorescence immunochromatographic test strip, which is different from example 5 only in that the shielding solution does not contain anti-HAMA antibody.

Comparative example 8

This example provides a candida antigen fluorescence immunochromatographic test strip, which is different from example 5 only in that the shielding solution does not contain casein.

Comparative example 9

This example provides a candida antigen fluorescence immunochromatographic test strip, which is different from example 5 only in that the shielding solution does not contain calcium ions.

Comparative example 10

This example provides a candida antigen fluorescence immunochromatographic test strip, which is different from example 5 only in that the mass concentration of the anti-HAMA antibody in the shielding solution is 0.2%.

Test example 4

Repeatability detection experiment of immunochromatographic test paper

The immunochromatography test strips provided in examples 5-8 and comparative examples 6-10 were used to detect candida mannan antigen positive and negative reference samples 10 times each, and differences in the detection results were observed. Wherein, the detection result of each test strip for the negative reference substance is shown in table 12:

TABLE 12

The test results of the test strips for the positive reference substances are shown in table 13:

watch 13

As can be seen from the test results in tables 12 and 13, the test strips for fluorescence immunochromatography provided in examples 5 to 8 all have an I value greater than 2 when detecting a positive reference, and have an I value less than 0.5 when detecting a negative reference, indicating that there is no error in the test results. Meanwhile, the detection results of the standard deviation SD and the dispersion degree CV of the detection results of the embodiments 5 to 8 are obviously smaller than those of the test strips provided by the comparative examples 6 to 10, which shows that the repeatability of the detection results of the fluorescence immunochromatographic test strip provided by the invention is obviously improved, and the influence of non-specific adsorption of the sample is eliminated.

Test example 5

Sensitivity and specificity experiments

82 candida antigen negative samples and 65 candida antigen positive samples were simultaneously detected by using the immunochromatographic test strips provided in examples 5 to 8 and comparative examples 6 to 10, and the sensitivity and specificity were calculated according to the negative and positive detection results, and the final calculation results are shown in table 14.

TABLE 14

As can be seen from the results in Table 14, the immunochromatographic test strip provided by the invention has a sensitivity of more than 80% when detecting positive and negative samples, and is higher in sensitivity, and the specificity of the obtained test strip is more than 82%, which indicates that the specificity is also better.

Test example 6

Background chromatography

The immunochromatography test strips provided in examples 5-8 and comparative examples 6-10 were used to simultaneously detect candida antigen samples, count the chromatography time, and observe the chromatography background.

The experimental results are as follows: the chromatography time of the fluorescence immunochromatographic test strip prepared in examples 5-8 is 15-20min (wherein the moving time of the sample on the NC membrane is 60-90 s); the chromatographic time of the test strips prepared in comparative examples 6-10 was 18-25min (wherein the moving time of the sample on the NC membrane was 90-110s), the fluorescence residue was significant, and the fluorescent particles were shunted away from the buffer system.

The fluorescence signal spectra obtained by the fluorescence quantitative analyzer after the test strips of example 5 and comparative example 6 detect positive samples are shown in fig. 4 and 5, wherein the abscissa in the figures represents the displacement (scanning position) and the ordinate represents the fluorescence signal intensity. In FIG. 4, a relatively distinct peak appears at a position around the displacement 350, which represents the fluorescence intensity of the detection line on the test strip provided in example 5, and a small peak appears at a position around 450, which represents the fluorescence intensity of the quality control line of the test strip; correspondingly, the position around 350 in fig. 5 represents the fluorescence intensity of the detection line on the test strip provided in the comparative example 6, and the position around 450 represents the fluorescence intensity of the quality control line, and it can be seen from fig. 4 and fig. 5 that the fluorescence intensity of the detection line of the test strip provided in the embodiment 5 is higher, the fluorescence intensity of other positions of the test strip is lower, and the chromatographic background is clear.

It can be known from the above examples and test examples that, after a shielding line containing an anti-HAMA antibody, calcium ions, casein, a protein stabilizer and a buffer is added to the immunochromatography test strip, the detection accuracy and sensitivity of the test strip are improved, and the detection result is more accurate and reliable.

The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A fluorescence immunochromatographic test strip is characterized by comprising a sample pad, a fluorescence pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped,

the nitrocellulose membrane is coated with a shielding wire, a detection wire and a quality control wire,

the shielding solution for preparing the shielding wire comprises an anti-HAMA antibody, casein, calcium ions, a protein stabilizing agent and a buffer solution.

2. The fluorescence immunochromatographic test strip according to claim 1, wherein the mass concentration of the anti-HAMA antibody in the shielding solution is 0.5-2%, preferably 1%;

preferably, the mass concentration of calcium ions in the shielding liquid is 0.5-2%, preferably 1%;

preferably, the mass concentration of casein in the shielding liquid is 0.5-2%, preferably 1%.

3. The fluorescence immunochromatographic test strip according to claim 1 or 2, wherein the protein stabilizer in the shielding solution is one or a combination of two or more of sucrose, trehalose, BSA, mannitol or glycerol, preferably trehalose;

preferably, the mass concentration of the protein stabilizer is 1-3%, preferably 2%;

preferably, the pH value of the buffer solution in the shielding solution is 8.0-9.0, and 8.5 is preferable;

preferably, the buffer has a molarity of 0.01-0.05M, preferably 0.02M;

4. The fluorescence immunochromatographic strip according to any one of claims 1 to 3, wherein a fluorescently-labeled Candida antibody or Aspergillus antibody is embedded on the fluorescent pad;

preferably, the detection line is a detection line containing candida antibodies or aspergillus antibodies;

preferably, the candida or aspergillus antibody is a murine antibody;

5. The fluorescence immunochromatographic test strip according to any one of claims 1 to 4, wherein the Candida antibodies are monoclonal antibodies against Candida mannan antigens and/or polyclonal antibodies against Candida mannan antigens, preferably monoclonal antibodies against Candida mannan antigens;

6. The fluorescence immunochromatographic test strip according to any one of claims 1 to 5, wherein the shielding line on the nitrocellulose membrane is located at one end near the fluorescent pad, the quality control line is located at one end near the absorbent pad, and the detection line is located between the shielding line and the quality control line;

preferably, the distance between the shielding wire and the detection wire is 3-5 mm;

preferably, the distance between the quality control line and the detection line is 3-5 mm.

7. The fluorescence immunochromatographic test strip according to any one of claims 1 to 6, wherein the sample pad is a glass fiber membrane or a polyester fiber membrane, preferably a glass fiber membrane;

preferably, the fluorescent pad is a glass fiber film;

preferably, the fluorescence immunochromatographic test strip further comprises a bottom plate;

preferably, the bottom plate is a polyvinyl chloride rubber plate.

8. The preparation method of the fluorescent immunochromatographic test strip according to any one of claims 1 to 7, which comprises the steps of:

preparing a fluorescent pad, preparing a shielding liquid, preparing a nitrocellulose membrane, and assembling a fluorescence test immunochromatography paper strip.

9. A test kit comprising the fluorescent immunochromatographic test strip of any one of claims 1 to 7.

10. Use of the immunofluorometric chromatographic test strip according to any one of claims 1 to 7 and/or of the test kit according to claim 9 for the detection of candida or aspergillus;

preferably, the candida includes any one or a combination of more than two of candida albicans, candida tropicalis, candida parapsilosis, candida krusei or candida glabrata;