CN110927384B

CN110927384B - Fluorescent immunochromatography test strip and preparation method and application thereof

Info

Publication number: CN110927384B
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: 2023-08-08
Anticipated expiration: 2039-11-29
Also published as: CN110927384A

Abstract

The invention provides a fluorescent immunochromatography test strip and a preparation method and application thereof. The fluorescent immunochromatography test strip comprises a sample pad, a fluorescent pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein a shielding wire, a detection wire and a quality control wire are coated on the nitrocellulose membrane, and shielding liquid for preparing the shielding wire comprises an anti-HAMA antibody, calcium ions, casein, a protein stabilizer and a buffer solution. When the fluorescent immunochromatographic test strip provided by the invention is used for detecting aspergillus or candida antigens, the interference substances in the sample to be detected are adsorbed by the shielding wire, so that the influence of the interference substances in the sample to be detected during detection is reduced, the combination of aspergillus or candida antigens and 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

Fluorescent immunochromatography test strip and preparation method and application thereof

Technical Field

The invention belongs to the technical field of immunodetection analysis, and particularly relates to a fluorescent immunochromatography test strip and a preparation method and application thereof, in particular to a fluorescent immunochromatography test strip for detecting aspergillus and candida antigens and a preparation method and application thereof.

Background

Aspergillus (Aspergillus) is an important conditional pathogen, often infecting patients with hypoimmunity or immunocompetence. Aspergillus fumigatus is the most common pathogenic bacteria causing severe deep Aspergillus infection of immunosuppressed patients, and the death rate is high. In recent years, with the extensive abuse of antibiotics and the widespread clinical use of immunosuppressants, the incidence and mortality of invasive deep fungal infections has increased. Among them, aspergillus, in particular aspergillus fumigatus (Aspergillus fumigatus), has become an important pathogenic fungus in clinic.

Galactomannans have been shown to be present on the cell wall of aspergillus as the main antigenic component of aspergillus, as well as the cell wall. Aspergillus fumigatus galactomannans are found in high levels in serum of animal models of infection and of patients with invasive Aspergillus and therefore serve as markers for diagnosing Aspergillus infections. The detection of aspergillus is realized by the antibody specific to aspergillus fumigatus galactomannan antigen, and the detection blood sensitivity reaches more than 80%. The number of clinical immunodetection products aiming at aspergillus fumigatus in the international market is small at present, and the most typical product is a double-antibody sandwich ELISA immunodetection kit of Bio-Rad company.

Candida is one of the most common conditional pathogenic bacteria in fungi, mainly including candida albicans, candida tropicalis, candida glabrata, candida parapsilosis and candida krusei, which account for more than 80% of clinical medical samples. Mannans (Mannan, mn) are the major antigenic component of candida cell walls. Candida is often deposited on the skin, mouth, vagina, intestinal mucosa and other sites of a person, and when the immune function of the body is reduced, candidiasis is easily caused.

CN105866409a discloses a candida mannan antigen immunodetection kit, which comprises an Mn coated elisa plate and an anti-Mn polyclonal elisa antibody. According to the kit, mn is coated on an ELISA plate, a sample to be detected or a standard antigen and the coated antigen compete for binding with a limited antibody binding site, then a chromogenic reaction is carried out between horseradish peroxidase and a substrate, a standard curve is drawn according to the Mn standard substance, and the concentration value of the antigen to be detected is calculated according to the standard curve. The immune detection kit provided by the invention adopts an Enzyme-linked immunosorbent assay (Enzyme-Linked Immuno Sorbent Assay, ELISA) to detect the antigen, and has complex treatment and detection steps and long treatment time.

Immunochromatography (Immuno chromatographic Assay, ICA) is a novel membrane detection technique based on an antigen-antibody specific immune reaction. The technology uses strip fiber chromatographic materials fixed with detection lines (coated antibodies or coated antigens) and quality control lines (anti-antibodies) as stationary phases, test liquid as mobile phases, fluorescent labeled antibodies or antigens are fixed on a connecting pad, and analytes are enabled to move on chromatographic strips 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 fluorescent labeled antibody under the action of a mobile phase, and then is combined with a coated antibody to form a sandwich type macromolecular substance with double-antibody sandwich when reaching a detection line.

The immunofluorescence technology (Fluorescence immuno chromatography Assay) is a novel immunolabeling technology which uses fluorescence as a tracer marker to be applied to antigen and antibody, and the fluorescent microsphere with the Eu chelate coated on the surface modified by carboxylation is covalently combined with amino groups on the surface of the antibody to form an immunolabeling compound. The fluorescence chromatography test strip has the following advantages: the use is convenient and quick, the cost is low, the use of a base layer and the use on site are convenient, the application range is wide, and the method can adapt to various detection conditions; meanwhile, the step of adding a chromogenic reagent, namely an enzyme-labeled carcinogenic substrate and a stop solution is omitted, and the method is harmless to human bodies.

However, when using fluorescent chromatographic test strips for detecting aspergillus and candida antigens, the sample to be detected needs to be pretreated with EDTA before being sampled, so that residual EDTA is present in the sample to be detected, and interference substances such as polysaccharide, glycoprotein and the like are still present in the treated sample, and the interference substances and the residual EDTA influence immune reaction on the detection line in the chromatographic process, so that the accuracy of the detection result is low.

Therefore, in the field of clinical detection of aspergillus and candida antigens, there is an urgent need for an immunochromatographic product for detecting aspergillus and candida antigens, which has the advantages of simple operation steps and high accuracy.

Disclosure of Invention

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

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

According to the fluorescent immunochromatography test strip provided by the invention, one shielding wire is added before the traditional detection wire and quality control wire, and the components of the shielding wire are reasonably screened, so that the shielding wire can adsorb interference substances, such as ethylenediamine tetraacetic acid (EDTA), human anti-mouse antibody (HAMA) and the like, which possibly influence the detection result in a sample to be detected, thereby improving the sensitivity and accuracy of the immunochromatography result.

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

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

The mass concentration of casein in the shielding liquid is preferably 0.5 to 2%, for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2%, etc., preferably 1%.

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

Preferably, the mass concentration of the protein stabilizer is 1 to 3%, for example, 1%, 1.2%, 1.5%, 1.6%, 1.7%, 1.8% or 2%, etc., preferably 2%.

Preferably, the pH of the buffer in the shielding solution is 8.0-9.0, for example, 8.0, 8.2, 8.4, 8.5, 8.7, 8.9 or 9.0, etc., preferably 8.5.

Preferably, the molar concentration of the buffer is 0.01-0.05M, for example, 0.01M, 0.015M, 0.02M, 0.025M, 0.03M, 0.035M, 0.04M or 0.05M, etc., preferably 0.02M.

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

As a preferable technical scheme of the invention, the fluorescent pad is embedded with fluorescent-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.

Murine antibodies are the most common antibody form in current commercial antibodies, and are widely available, low in cost, and good in 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 embodiment of the present invention, the candida antibody is a monoclonal antibody against candida mannan antigen and/or a polyclonal antibody against candida mannan antigen, preferably a monoclonal antibody against candida mannan antigen.

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

Antibodies are classified into polyclonal antibodies and monoclonal antibodies, and methods for preparing the polyclonal antibodies or monoclonal antibodies are well known to those skilled in the art. Polyclonal antibodies may have a problem of weak specificity compared to monoclonal antibodies, and may have false positive results. In particular, although polyclonal antibodies may be employed, monoclonal antibodies have more significant advantages.

As a preferable technical scheme of the invention, the shielding wire on the nitrocellulose membrane is positioned at one end close to the fluorescent pad, the quality control wire is positioned at one end close to the water absorption pad, and the detection wire is positioned between the shielding wire and the quality control wire.

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

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

As a preferred embodiment of the present invention, the sample pad is a glass fiber film or a polyethylene fiber film, preferably a glass fiber film.

Preferably, the fluorescent pad is a glass fiber membrane.

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

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

In a second aspect, the present invention also provides a method for preparing the fluorescent immunochromatographic test strip according to the first aspect, the method comprising: preparing a fluorescent pad, preparing shielding liquid, preparing a nitrocellulose membrane and assembling a fluorescent immunochromatography test strip.

Wherein, the preparation of the fluorescent pad comprises the following steps:

adding candida antibody or aspergillus antibody into the activated fluorescent microsphere solution to a final concentration of 10 mug/mL, and standing for 120min for marking; after completion, a 10% BSA-containing solution was added to the above solution and allowed to stand for 60 minutes as a blocking solution. Centrifuging the obtained fluorescent solution at 10000rpm for 20min, removing supernatant, adding 100mL of complex solution into the precipitate, fully mixing, and ultrasonic treating for 60s to obtain candida or aspergillus antibody marker solution. Spraying candida or aspergillus antibody marking liquid on a glass fiber membrane according to 15 mu L/cm by using a metal spraying film drawing instrument; and (3) drying the obtained glass fiber membrane in an oven at 37 ℃ for 180min, and adding a drying agent for sealing and preserving after drying to obtain the fluorescent pad.

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

weighing a certain mass of Tris (hydroxymethyl) aminomethane (Tris), trehalose, casein and CaCl ₂ Placing into a beaker, adding deionized water, adding an anti-HAMA antibody into the beaker, and uniformly mixing to obtain the shielding liquid containing 0.5-2% of the anti-HAMA antibody, 1-3% of trehalose, 0.01-0.05M TRIS (pH 8.5), 0.1M calcium ions and 0.5-2% of casein. For example, 0.24g Tris, 1.5g trehalose, 1g casein, 1.1g CaCl were weighed out ₂ Put into a beaker, 100mL of deionized water is added, and then 1g of anti-HAMA antibody is added to the beaker, so that a shielding liquid containing 1% of anti-HAMA antibody, 1.5% of trehalose, 0.015M TRIS (pH 8.5), 0.1M calcium ions and 1% of casein in mass concentration is obtained.

The nitrocellulose membrane comprises the following steps:

masking liquid (as masking line), solution containing candida antibody or aspergillus antibody (as detecting line) and solution containing anti candida antibody or aspergillus antibody (as quality control line) are marked on NC film according to the condition of 1 muL/cm by using a fluorescence film marking instrument; and (3) drying the NC film for 4 hours at 37 ℃ in an oven, and adding a drying agent for sealing and preserving after drying.

The assembly of the fluorescent immunochromatography 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 assembly plate into fluorescent test strips with the length of 4+/-0.2 mm by using a strip cutting device; and (5) placing the test strip in a sealed bag, adding a drying agent, and preserving.

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

In a fourth aspect, the use of a fluorescent immunochromatographic strip according to the first aspect and/or a detection kit according to the third aspect for detecting candida or aspergillus.

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

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

In the invention, the using 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 liquid into the centrifuge tube, vortex vibrating for 10s to thoroughly mix uniformly, putting the centrifuge tube into a water bath kettle, heating for 180+/-10 s at 100 ℃, taking the centrifuge tube out of the water bath kettle, putting the centrifuge tube into the centrifuge, and centrifuging for 10min at 10000g at 4 ℃;

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

3. analysis of results: if the I value is less than 0.5, the sample is negative, which indicates that the sample to be tested does not contain aspergillus antigen or candida antigen; if the I value is more than 0.5, the sample is positive, which indicates that the sample to be tested contains aspergillus antigen or candida antigen.

The numerical ranges recited herein include not only the above-listed point values, but also any point values between the above-listed numerical ranges that are not listed, and are limited in space and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that the stated ranges include.

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

the fluorescence immunochromatography test strip provided by the invention adopts a sandwich method to detect aspergillus or candida antigens, has better sensitivity, specificity and stability, and the components in the added shielding line can adsorb interfering substances in a sample to be detected, so that the influence of the interfering substances in the sample to be detected during detection is reduced, 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 a fluorescent immunochromatographic test strip provided by the invention.

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

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

FIG. 4 is a graph of fluorescent signals after detection of candida antigen positive samples in example 5.

FIG. 5 is a graph of fluorescent signals after detection of candida antigen positive samples in comparative example 6.

The present invention will be described in further detail below. The following examples are merely illustrative of the present invention and are not intended to represent or limit the scope of the invention as defined in the claims.

Detailed Description

The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings.

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

Sources of reagents and instrumentation used in the following examples: the antibody used in the experiment is prepared by a conventional method from Dana (Tianjin) biotechnology limited company; the metal spraying and film drawing instrument is purchased from Shanghai gold mark biotechnology limited company; slitter was purchased from Hai jin sign Biotechnology Co., ltd; fluorescent immunochromatography analyzers were purchased from blue-boy biotechnology limited, guangzhou; PVC plywood, absorbent paper, fiberglass membrane and cardboard were purchased from Hangzhou Bi Kenlai Bo Biotechnology Co.

Example 1

The embodiment provides an aspergillus antigen fluorescent immunochromatography test strip and a preparation method thereof.

The fluorescent pad of the test strip in this example is embedded with a fluorescent-labeled Aspergillus galactomannan antibody at a concentration of 10. Mu.g/mL;

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

TABLE 1

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

The fluorescent immunochromatography test strip can be prepared according to the following method:

and (3) attaching a sample pad, a fluorescent pad, a nitrocellulose membrane and water-absorbing filter paper to one end of the PVC rubber plate in a sequential and overlapping manner, cutting the PVC rubber plate and attached materials into test strips with the width of 4+/-0.2 mm, and placing the test strips into a clamping shell, wherein a sample adding area and a detection area (observation area) are formed in the clamping shell.

Wherein the sample pad is a glass fiber membrane; the shielding liquid (as shielding line), the solution containing the aspergillus antibody (as detection line) and the solution containing the anti-aspergillus antibody (as quality control line) are marked on the NC film by using a fluorescence-spraying film marking instrument according to the condition of 1 mu L/cm, the NC film is placed in an oven at 37 ℃ for drying for 4 hours, and a drying agent is added for sealing and storing after drying.

Assembling a 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 assembly plate into fluorescent test strips with the length of 4+/-0.2 mm by using a strip cutting device; and (5) placing the test strip in a sealed bag, adding a drying agent, and preserving.

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

(1) After a detected sample is added into a sample adding area (sample pad), the detected sample moves into a fluorescent pad through capillary action, if the detected sample contains aspergillus antigen, the antigen in the sample is combined with aspergillus antibody in fluorescence to form an antigen-antibody-fluorescence conjugate, and the antigen-antibody-fluorescence conjugate continues to move towards one end of a water absorbing pad;

(2) When the detection sample moves to the shielding line, interference factors in the detection sample, such as HAMA, polysaccharide, EDTA and the like, react with corresponding antibodies, calcium ions and casein in the shielding line, stay at the shielding line and do not move continuously with the detection sample, and antigen-antibody-fluorescence conjugates in the detection sample are not affected to move continuously;

(3) When the detection sample moves to the detection line, the antigen-antibody-fluorescent conjugate is captured, the antibody-antigen-antibody-fluorescent conjugate is generated at the detection line, and when the detection is performed by using a fluorescence immunochromatographic analyzer, a fluorescent band can be observed: if the antigen of aspergillus is not present in the detected actual sample, immune complex is not formed, and fluorescent strip is not generated;

(4) When the detection sample continues to flow to the quality control line, namely the goat anti-mouse IgG antibody, no matter whether the sample to be detected contains the aspergillus antigen or not, a fluorescent strip appears, and the effectiveness of the test strip is proved. As long as the quality control line does not develop, the test strip is not effective and the actual sample needs to be re-detected.

Example 2

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

TABLE 2

Component (A)	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 example 1 is that the formulation of the shielding liquid is shown in table 3, and the rest of the components and the preparation method are the same as example 1.

TABLE 3 Table 3

Component (A)	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 example 1 is that the formulation of the shielding liquid is shown in table 4, and the rest of the components and the preparation method are the same as example 1.

TABLE 4 Table 4

Component (A)	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

The present example provides an aspergillus antigen fluorescent immunochromatographic test strip, which differs from example 1 only in that the test strip does not contain a shielding wire.

Comparative example 2

The present example provides an aspergillus antigen fluorescent immunochromatographic test strip, which differs from example 1 only in that the shielding liquid does not contain an anti-HAMA antibody.

Comparative example 3

This example provides an aspergillus antigen fluorescent immunochromatographic test strip, which differs from example 1 only in that the shielding liquid does not contain casein.

Comparative example 4

The present embodiment provides an aspergillus antigen fluorescent immunochromatographic test strip, which is different from embodiment 1 only in that the shielding liquid does not contain calcium ions.

Comparative example 5

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

Test example 1

Repetitive detection experiment of immunochromatography test paper

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

TABLE 5

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

TABLE 6

As can be seen from the test results in tables 5 and 6, the fluorescent immunochromatographic test strips provided in examples 1 to 4 have I values greater than 1 when detecting positive reference substances and I values less than 0.5 when detecting negative reference substances, indicating that the test results are error-free. Meanwhile, the standard deviation SD and the discrete degree CV of the detection results of the embodiments 1-4 are obviously smaller than those of the test strips provided by the comparative examples 1-5, which shows that the repeatability of the detection results of the fluorescent immunochromatography test strip provided by the invention is obviously improved, and the influence of non-specific adsorption of a sample is eliminated.

Test example 2

Sensitivity and specificity experiments

60 cases of aspergillus antigen positive samples and 72 cases of aspergillus antigen negative samples are detected simultaneously by using the immunochromatographic test strips provided in examples 1-4 and comparative examples 1-5, and the sensitivity and the specificity of the test strips are calculated according to the yin-yang of the detection results, wherein the sensitivity is the proportion of correctly judging a true positive sample as true positive; the specificity is the ratio at which a true negative sample is correctly judged to be true negative.

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

TABLE 7

As shown in the results of Table 7, the sensitivity of the immunochromatography test strip provided by the invention is more than 86% when detecting negative and positive samples, the sensitivity is higher, and the specificity of the obtained test strip is more than 86%, which indicates that the specificity is also better.

Test example 3

Chromatographic background experiment

The immunochromatographic test strips provided in examples 1 to 4 and comparative examples 1 to 5 were used to simultaneously detect an Aspergillus antigen sample, and the chromatographic time was counted and the chromatographic background was observed.

As shown by the observation results, the chromatographic time of the fluorescent immunochromatographic test strip prepared in the examples 1-4 is 15-20min (the moving time of the sample on the NC film is 60-90 s), the fluorescent chromatographic background is clean, and the layering phenomenon is avoided; the test strips prepared in comparative examples 1-5 had a chromatography time of 18-25min (wherein the sample was moved over the NC membrane for 90-110 s), a distinct fluorescent residue, and fluorescent particles were shunted to the buffer system.

Among them, the fluorescent signal patterns obtained by the fluorescent quantitative analyzer after detecting the positive sample of the aspergillus antigen by the test strip of example 1 and comparative example 1 are shown in fig. 2 and 3, in which the abscissa represents the displacement (scanning position) and the ordinate represents the fluorescent signal intensity. In fig. 2, a relatively obvious peak appears at a position around the displacement 350, the peak 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, the peak represents the fluorescence intensity of the quality control line of the test strip; the position about the displacement 350 in fig. 3 shows the fluorescence intensity of the detection line on the test strip provided in comparative example 1, and the position about 450 shows the fluorescence intensity of the quality control line, and fig. 2 and fig. 3 show that the peak of the detection line of the test strip provided in embodiment 1 is more obvious, that is, the fluorescence intensity is higher, the fluorescence intensity of other positions of the test strip is lower, and the chromatographic background is clear.

Example 5

The embodiment provides a candida antigen fluorescent immunochromatography test strip and a preparation method thereof.

The fluorescent pad of the test strip in the embodiment is embedded with fluorescent-labeled candida mannan antibody with the concentration of 15 mug/mL;

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

TABLE 8

The preparation method of the test strip is the same as that of 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 of the components and the 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 of the components and the preparation method are the same as example 5.

Table 10

Example 8

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

TABLE 11

Comparative example 6

The present example provides a candida antigen fluorescent immunochromatographic test strip, which differs from example 5 in that the test strip does not contain a shielding wire.

Comparative example 7

This example provides a candida antigen fluorescent immunochromatographic test strip which differs from example 5 only in that the shielding liquid does not contain an anti-HAMA antibody.

Comparative example 8

This example provides a candida antigen fluorescent immunochromatographic test strip, which differs from example 5 only in that the shielding liquid does not contain casein.

Comparative example 9

The present embodiment provides a candida antigen fluorescent immunochromatographic test strip, which is different from embodiment 5 only in that the shielding liquid contains no calcium ions.

Comparative example 10

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

Test example 4

Repetitive detection experiment of immunochromatography test paper

The immunochromatographic test strips provided in examples 5 to 8 and comparative examples 6 to 10 were used to detect the candida mannan antigen positive reference and the candida mannan antigen negative reference 10 times each, and differences in the detection results were observed. The test results of each test strip on the negative reference are shown in table 12:

table 12

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The test results of each test strip on the positive reference are shown in table 13:

TABLE 13

As can be seen from the test results in tables 12 and 13, the fluorescent immunochromatographic test strips of examples 5 to 8 have I values of more than 2 when detecting positive reference substances and I values of less than 0.5 when detecting negative reference substances, indicating that the test results are error-free. Meanwhile, the standard deviation SD and the discrete degree CV of the detection results of the embodiments 5-8 are obviously smaller than those of the test strips provided by the comparative examples 6-10, which shows that the repeatability of the detection results of the fluorescent immunochromatography test strip provided by the invention is obviously improved, and the influence of non-specific adsorption of a sample is eliminated.

Test example 5

Sensitivity and specificity experiments

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

TABLE 14

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

Test example 6

Chromatographic background experiment

The immunochromatographic test strips provided in examples 5-8 and comparative examples 6-10 were used to simultaneously detect candida antigen samples, the chromatographic time was counted, and the chromatographic background was observed.

The experimental results are: the chromatographic time of the fluorescent immunochromatographic test strip prepared in examples 5 to 8 is 15 to 20 minutes (wherein the movement time of the sample on the NC membrane is 60 to 90 seconds); the test strips prepared in comparative examples 6-10 had a chromatography time of 18-25min (wherein the sample was moved over the NC membrane for 90-110 s), a distinct fluorescent residue, and fluorescent particles were shunted to the buffer system.

The fluorescent signal patterns obtained by the fluorescent quantitative analyzer after the positive samples were detected by the test strips of example 5 and comparative example 6 are shown in fig. 4 and 5, in which the abscissa represents displacement (scanning position) and the ordinate represents fluorescent signal intensity. In fig. 4, a relatively obvious peak appears at a position around the displacement 350, the peak 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, the peak represents the fluorescence intensity of the quality control line of the test strip; correspondingly, the position about 350 in fig. 5 shows the fluorescence intensity of the detection line on the test strip provided in comparative example 6, the position about 450 shows the fluorescence intensity of the quality control line, and fig. 4 and fig. 5 show that the fluorescence intensity of the detection line on the test strip provided in example 5 is higher, the fluorescence intensity of the other positions of the test strip is lower, and the chromatographic background is clear.

By combining the above examples and test examples, it is known that when a shielding line containing an anti-HAMA antibody, calcium ions, casein, a protein stabilizer and a buffer solution is added to an immunochromatographic test strip, the detection accuracy and sensitivity of the test strip are improved, and the detection result is more accurate and reliable.

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

Claims

1. A fluorescent immunochromatographic test strip is characterized by comprising a sample pad, a fluorescent 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,

preparing shielding liquid of the shielding wire, wherein the shielding liquid comprises an anti-HAMA antibody, casein, calcium ions, a protein stabilizer and a buffer solution;

the mass concentration of the anti-HAMA antibody in the shielding liquid is 0.5-2%;

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

the mass concentration of casein in the shielding liquid is 0.5-2%;

the protein stabilizer in the shielding liquid is any one or the combination of more than two of sucrose, trehalose, BSA, mannitol or glycerol;

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

the pH value of the buffer solution in the shielding solution is 8.0-9.0;

the molar concentration of the buffer solution is 0.01-0.05M;

the buffer solution is any one of Tris-HCl buffer solution, PBS buffer solution, PB buffer solution or CBS buffer solution.

2. The fluorescent immunochromatographic test strip according to claim 1, in which the mass concentration of the anti-HAMA antibody in the shielding solution is 1%.

3. The fluorescent immunochromatographic test strip according to claim 1, in which the mass concentration of calcium ions in the shielding liquid is 1%.

4. The fluorescent immunochromatographic test strip according to claim 1, in which the mass concentration of casein in the shielding solution is 1%.

5. The fluorescent immunochromatographic strip according to claim 1, in which the protein stabilizer in the shielding liquid is trehalose.

6. The fluorescent immunochromatographic test strip according to claim 1, in which the mass concentration of the protein stabilizing agent is 2%.

7. The fluorescent immunochromatographic test strip according to claim 1, in which the pH value of the buffer solution in the shielding solution is 8.5.

8. The fluorescent immunochromatographic strip according to claim 1, in which the molar concentration of the buffer is 0.02M.

9. The fluorescent immunochromatographic strip according to claim 1, in which the buffer is Tris-HCl buffer.

10. The fluorescent immunochromatographic test strip according to claim 1, in which the fluorescent pad is embedded with a fluorescent-labeled candida antibody or aspergillus antibody.

11. The fluorescent immunochromatographic strip of claim 10, in which the detection line is a detection line containing a candida antibody or an aspergillus antibody, which is a murine antibody.

12. The fluorescent immunochromatographic strip according to claim 11, in which the quality control line is a quality control line containing an anti-mouse antibody, and the anti-mouse antibody is a goat anti-mouse IgG antibody.

13. The fluorescent immunochromatographic test strip according to claim 12, in which the candida antibody is a monoclonal antibody against candida mannan antigen and/or a polyclonal antibody against candida mannan antigen.

14. The fluorescent immunochromatographic test strip according to claim 13, in which the candida antibody is a monoclonal antibody against candida mannan antigen.

15. The fluorescent immunochromatographic strip according to claim 12, in which the aspergillus antibody is a monoclonal antibody against aspergillus galactomannan antigen and/or a polyclonal antibody against aspergillus galactomannan antigen.

16. The fluorescent immunochromatographic strip according to claim 15, in which the aspergillus antibody is a monoclonal antibody against aspergillus galactomannan antigen.

17. The fluorescent immunochromatographic test strip according to claim 1, in which the shielding line on the nitrocellulose membrane is positioned at one end close to the fluorescent pad, the quality control line is positioned at one end close to the water absorbing pad, and the detection line is positioned between the shielding line and the quality control line.

18. The fluorescent immunochromatographic test strip according to claim 17, in which the distance between the shielding line and the detection line is 3 to 5mm.

19. The fluorescent immunochromatographic test strip according to claim 18, in which the distance between the quality control line and the detection line is 3 to 5mm.

20. The fluorescent immunochromatographic test strip according to claim 1, in which the sample pad is a glass fiber membrane or a polyester fiber membrane.

21. The fluorescent immunochromatographic test strip according to claim 20, in which the sample pad is a glass fiber membrane.

22. The fluorescent immunochromatographic test strip according to claim 1, in which the fluorescent pad is a glass fiber membrane.

23. The fluorescent immunochromatographic test strip according to claim 1, further comprising a bottom plate.

24. The fluorescent immunochromatographic test strip according to claim 23, in which the bottom plate is a polyvinyl chloride gel plate.

25. A method of preparing a fluorescent immunochromatographic test strip according to any one of claims 1 to 24, comprising:

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

26. A test kit comprising the fluorescent immunochromatographic strip according to any one of claims 1 to 24.

27. Use of a fluorescent immunochromatographic strip according to any one of claims 1 to 24 and/or a detection kit according to claim 26 for the preparation of a product for detecting candida or aspergillus.

28. The use of claim 27, wherein the candida comprises any one or a combination of two or more of candida albicans, candida tropicalis, candida parapsilosis, candida krusei, or candida glabrata.

29. The use according to claim 27, wherein the aspergillus comprises any one or a combination of two or more of aspergillus fumigatus, aspergillus flavus, aspergillus terreus or aspergillus niger.