AU2021104253A4

AU2021104253A4 - Fluorescence immunochromatography test strip for detecting tryptase and use thereof, and tryptase detection method

Info

Publication number: AU2021104253A4
Application number: AU2021104253A
Authority: AU
Inventors: Chuhan Chen; Shanshan Chen; Lei Cheng; Yi Liu; Huahao SHEN; Yifei Wang; Shandong Wu; Zhoujie Wu; Mingzhi Zhu
Original assignee: Hangzhou Zheda Dixun Biological Gene Engineering Co Ltd
Current assignee: Hangzhou Zheda Dixun Biological Gene Engineering Co Ltd
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-09-09
Anticipated expiration: 2029-07-16

Abstract

OF THE DISCLOSURE Disclosed is a fluorescence immunochromatography test strip for detecting tryptase, use thereof, and a tryptase detection method, relates to the technical field of FICA. The fluorescence immunochromatography test strip of the present disclosure includes a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper which are sequentially lapped and adhered on the bottom plate, wherein the conjugate pad is coated with a first anti-tryptase antibody labeled with fluorescent microspheres; the NC membrane is coated sequentially with a second anti-tryptase antibody (T line) and an anti-rabbit IgG antibody (C line); wherein the T line is the test line and the C line is the control line. In the present disclosure, the tryptase content in samples may be accurately and quantitatively detected, thereby being used for auxiliary diagnosis of bronchial asthma, chronic urticaria, anaphylactic shock, MC storage disease, etc., and the diagnosis has the advantages of simple operation, short detection time, and low cost. 1/2 co CL) Ut cz) -n. QF 2*

Description

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FLUORESCENCE IMMUNOCHROMATOGRAPHY TEST STRIP FOR DETECTING TRYPTASE AND USE THEREOF, AND TRYPTASE DETECTION METHOD TECHNICAL FIELD

[01] The present disclosure belongs to the technical field of fluorescence immunochromatography assay (FICA), and specifically relates to a fluorescence immunochromatography test strip for detecting tryptase and use thereof, and a tryptase detection method.

BACKGROUNDART

[02] Tryptase is a neutral protease released by mast cells (MCs). During MC degranulation, tryptase is released along with other mediators to participate in an allergic inflammatory process. Tryptase has the functions of promoting airway repair, regulating the tension and responsiveness of airway smooth muscle cells (SMCs), stimulating the activation of MCs, and so on. The detection of tryptase can be used as an indicator for reflecting the activity of MCs. In many MC-related diseases, the tryptase content in serum is increased with the degranulation process of MCs, and the increase of tryptase content in serum is related to the severity of clinical symptoms to some extent.

[03] It can be seen that the number of MCs increases when allergic inflammation occurs, and MCs release a variety of inflammatory mediators after activating, causing symptoms and signs of anaphylaxis. After an acute allergic reaction, the tryptase content in the peripheral blood increases. In the serum of patients with systemic anaphylaxis, the tryptase content is significantly increased. Therefore, the tryptase content level in serum may be used as one of the laboratory diagnostic indicators for drug anaphylactic shock and insect venom anaphylactic shock, and may provide a basis for clinical diagnosis and treatment. MC storage disease is difficult to diagnose, and the patients are not suitable for allergen desensitization treatment. The detection of tryptase level in blood may assist in the diagnosis of MC storage disease.

[04] However, there are few products for detecting tryptase in blood, and the detection method is basically enzyme-linked immunosorbent assay (ELISA), which has the disadvantages of cumbersome operations, long time consumption, and high cost. For example, Fluoroenzyme-immunome-trieassay, which is widely used abroad, uses ImmunoCAP T M Tryptase reagent and requires four reaction steps and takes more than two hours to complete one test, and requires special automation equipment to complete the test, resulting in an automation equipment investment of not less than 600,000 yuan and a reagent cost of not less than 80 yuan/person.

SUMMARY OF THE INVENTION

[05] Preferred embodiments of the present invention seek to provide a fluorescence immunochromatography test strip for detecting tryptase and use thereof, which can quantitatively detect the tryptase content in a blood sample. The technical scheme of the present disclosure may help doctors confirm the applicability of a treatment plan to patients based on the changes of tryptase content in patients before and after treatment. The fluorescence immunochromatography test strip has the advantages of simple operation, short detection time, and low cost.

[06] The present disclosure provides the following technical schemes.

[07] The present disclosure provides a fluorescence immunochromatography test strip for detecting tryptase, including a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper which are sequentially lapped and adhered on the bottom plate; wherein the conjugate pad is coated with a first anti-tryptase antibody labeled with fluorescent microspheres;

[08] the NC membrane includes a test line and a control line; the test line is coated with a second anti-tryptase antibody; the control line is coated with an anti-rabbit IgG antibody; and

[09] the first anti-tryptase antibody labeled with fluorescent microspheres is paired with the second anti-tryptase antibody.

[10] In some embodiments, the particle size of the fluorescent microspheres is in a range of 50 nm to 500 nm.

[11] In some embodiments, the concentration of the first anti-tryptase antibody labeled with fluorescent microspheres coated on the conjugate pad is in a range of 2 mg/mL to 10 mg/mL; the concentration of the anti-rabbit IgG antibody coated on the control line is in a range of 1 mg/mL to 5 mg/mL; and the concentration of the second anti-tryptase antibody coated on the test line is in a range of 1 mg/mL to 5 mg/mL.

[12] The present disclosure also provides use of the fluorescence immunochromatography test strip described in the above technical scheme in the preparation of a kit for detecting tryptase content in samples.

[13] The present disclosure also provides a method for detecting tryptase content in samples, including the following steps: adding 70 L to 120 L of a blood sample solution dropwise on the sample pad of the fluorescence immunochromatography test strip described in the above scheme, reading the fluorescence signal ratio on the fluorescence immunochromatography test strip 10 min to min later, and determining the tryptase content according to a standard curve.

[14] The standard curve is:

84 13 2

[15] y = 5.41925 + (0.18367-5.41925)/(1 + (x/118.84607)o ), R2 = 0.99542,

[16] wherein x represents the concentration of tryptase in the blood sample, with a unit of ng/mL, and y represents the fluorescence signal ratio.

[17] The present disclosure provides a fluorescence immunochromatography test strip for detecting tryptase, including a bottom plate, and a sample pad, a conjugate pad, an NC membrane, and an absorbent paper which are sequentially lapped and adhered on the bottom plate. The conjugate pad is coated with a first anti-tryptase antibody labeled with fluorescent microspheres; the NC membrane is coated sequentially with a second anti-tryptase antibody (T line) and an anti-rabbit IgG antibody (C line); wherein the T line is the test line and the C line is the control line. In the present disclosure, the first anti-tryptase antibody labeled with fluorescent microspheres is used as the secondary antibody, and the second anti-tryptase antibody coated on the NC membrane is used as the primary antibody; and the first anti-tryptase antibody labeled with fluorescent microspheres is paired with the second anti-tryptase antibody. In the present disclosure, the tryptase content in samples may be accurately and quantitatively detected, and the technical scheme of the present disclosure has the advantages of simple operation (only one-step sample addition is required), rapid tryptase quantification (the result can be read 10 min to 15 min after sample addition), low cost (only a dry fluorescence reader is required for supporting instrument, with the price of not higher than 3,000 yuan; the reagent cost of not higher than 5 yuan/person), high detection accuracy, and high sensitivity (detection limit is less than 0.2 ng/mL).

BRIEF DESCRIPTION OF DRAWINGS

[18] FIG. 1 is the structural diagram of the fluorescence immunochromatography test strip in the present disclosure;

[19] FIG. 2 shows the standard curve for FICA.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[20] The present disclosure provides a fluorescence immunochromatography test strip for detecting tryptase, including a sample pad, a conjugate pad, an NC membrane, and an absorbent paper which are connected end-to-end and sequentially fixed on a PVC bottom plate; the conjugate pad is coated with a first anti-tryptase antibody labeled with fluorescent microspheres;

[21] the NC membrane includes a test line and a control line; the test line is coated with a second anti-tryptase antibody; the control line is coated with an anti-rabbit IgG antibody; and

[22] the first anti-tryptase antibody labeled with fluorescent microspheres is paired with the second anti-tryptase antibody.

[23] The structure of the fluorescence immunochromatography test strip of the present disclosure is preferably as shown in FIG. 1, wherein a sample pad, a conjugate pad, an NC membrane, and an absorbent paper are sequentially lapped on a bottom plate.

[24] In the present disclosure, the bottom plate preferably includes a PVC bottom plate.

[25] In the present disclosure, the particle size of the fluorescent microspheres is preferably in a range of 50 nm to 500 nm, more preferably in a range of 100 nm to 300 nm, and most preferably 200 nm.

[26] In the present disclosure, the fluorescent microspheres preferably include inorganic/organic fluorescent microspheres, organic/inorganic fluorescent microspheres, inorganic/inorganic fluorescent microspheres, and organic/organic fluorescent microspheres. In the present disclosure, for the inorganic/organic fluorescent microspheres, an inorganic material is used as a carrier and an organic compound is used as a fluorescent substance; for the organic/inorganic fluorescent microspheres, an organic material is used as a carrier and an inorganic compound is used as a fluorescent substance; for the inorganic/inorganic fluorescent microspheres, an inorganic material is used as a carrier and bound to an inorganic fluorescent nanocrystal with active groups; and the organic/organic fluorescent microspheres are polymer fluorescent microspheres formed by polymerization of organic fluorescent substances and organic material carriers.

[27] In the present disclosure, the fluorescent microsphere is preferably time-resolved fluorescent microspheres. In the specific implementation of the present disclosure, the fluorescent microsphere is MD033 purchased from Nanjing Microdetection Bio-Tech Co., Ltd.

[28] In the present disclosure, the first anti-tryptase antibody is preferably a biotinylated anti tryptase antibody, which is purchased from Shanghai Xuanya Biotechnology Co., Ltd., with Item No.: XY-KT-2780.

[29] In the present disclosure, a method for coating the first anti-tryptase antibody labeled with fluorescent microspheres on the conjugate pad preferably includes the following steps:

[30] (a) mixing the fluorescent microspheres and the first anti-tryptase antibody to obtain the first anti-tryptase antibody labeled with fluorescent microspheres;

[31] (b) spray-coating the first anti-tryptase antibody labeled with fluorescent microspheres on the conjugate pad.

[32] In the present disclosure, the fluorescent microspheres and the first anti-tryptase antibody are firstly mixed to obtain the first anti-tryptase antibody labeled with fluorescent microspheres. In the present disclosure, the volume ratio of the fluorescent microspheres and the first anti-tryptase antibody is preferably 10:1. There is no special limitation on the sources of the fluorescent microspheres and the first anti-tryptase antibody, and conventional commercially-available products will do.

[33] In the present disclosure, after the first anti-tryptase antibody labeled with fluorescent microspheres is obtained, the first anti-tryptase antibody labeled with fluorescent microspheres is spray-coated on the conjugate pad. In the present disclosure, the spray amount of the first anti-tryptase antibody labeled with fluorescent microspheres on the conjugate pad is preferably in a range of 2 mg/mL to 10 mg/mL and more preferably in a range of 5 mg/mL to 8 mg/mL. In the present disclosure, the first anti-tryptase antibody labeled with fluorescent microspheres is used as a secondary antibody.

[34] In the present disclosure, the control line and test line on the NC membrane are preferably arranged in parallel; the number of the test line is preferably one; the number of the control line is preferably one; the test line (T line) is coated with a second anti-tryptase antibody, and the coating concentration of the second anti-tryptase antibody is preferably in a range of 1 mg/mL to 5 mg/mL and more preferably in a range of 2 mg/mL to 3 mg/mL. The control line (C line) is coated with an anti-rabbit IgG antibody, and the coating concentration of the anti-rabbit IgG antibody is preferably in a range of 1 mg/mL to 5 mg/mL, more preferably in a range of 2 mg/mL to 3 mg/mL. In the present disclosure, the second anti-tryptase antibody coated on the test line on the NC membrane is used as a primary antibody, which is paired with the first anti-tryptase antibody labeled with fluorescent microspheres (secondary antibody). In the specific implementation of the present disclosure, the second anti-tryptase antibody is preferably ZY-1303Ab (Item No.) purchased from Shanghai Zeye Biotechnology Co., Ltd.

[35] In the present disclosure, there is no special limitations on the preparation method of the NC membrane, and the preparation method preferably includes: diluting the second anti-tryptase antibody and anti-rabbit IgG antibody with 10 mM phosphate buffered saline (PBS, pH 7.4), separately; and streaking the obtained two diluted antibody solutions on an NC membrane in parallel, separately, a testing area coated with the second anti-tryptase antibody and a control area coated with the anti rabbit IgG antibody are formed when the antibodies penetrate into the NC membrane. In the present disclosure, the preferred conditions for the step of streaking on testing area are as follows: the concentration of the diluted antibody is 3 mg/mL, the liquid output of peristaltic pump is 0.5 mL/cm, and streaking speed is 50 m/20 min, and the NC membrane is forced air drying at 37°C for 12 h; and the preferred conditions for the step of streaking on control area are as follows: the concentration of the diluted antibody is 5 mg/mL the liquid output of peristaltic pump is 0.5 mL/cm, and streaking speed is 50 m/20 min, the control line, parallel to the line in the testing area, is streaked on the NC membrane which is forced air drying at 37°C for 12 h. After the streaking, the preparation method preferably further includes: confining the NC membrane at 37°C for 60 min with a confining liquid, drying the membrane at 37°C for 2 h, and packaging for use.

[36] In the present disclosure, the sample pad, the conjugate pad, the NC membrane, and the absorbent filter paper are preferably assembled and adhered on the PVC bottom plate sequentially, and a resulting product is cut into test strips shown in FIG. 1 as required (4 mm) with a strip cutter.

[37] The present disclosure also provides use of the fluorescence immunochromatography test strip described above in the preparation of a kit for detecting tryptase content in samples.

[38] The present disclosure also provides a method for detecting tryptase content in samples, including the following steps: adding 70 L to 120 L of a sample solution dropwise on the sample pad of the fluorescence immunochromatography test strip described above, reading the fluorescence signal ratio (T line/C line) on the fluorescence immunochromatography test strip 10 min to 15 min later, and determining the tryptase content according to a standard curve.

[39] The standard curve is:

[40] y =5.41925+(0.18367-5.41925)/(1 + (x/118.84607) 0.84132) , R 2 =0.99542,

[41] wherein x represents the concentration of tryptase in the sample solution (ng/mL), and y is a fluorescence signal ratio (T/C).

[42] In the present disclosure, 70 L to 120 L of the sample to be tested is added dropwise on the sample pad, and the test strip is placed in a special fluorescence signal reader to read the fluorescence signal ratio after reaction in the control area is completed 10 min to 15 minutes later,, thus achieving quantitative determination. In the present disclosure, after being added dropwise on the sample pad, the sample to be tested is reacted and binds to the first anti-tryptase antibody labeled with fluorescent microspheres on the conjugate pad, then reacts with the second anti-tryptase antibody on the testing area, and finally reaches the control area where the reaction is completed.

[43] The fluorescence immunochromatography test strip for detecting tryptase, use thereof, and a tryptase detection method provided in the present disclosure will be described in further detail with reference to examples, but these examples cannot be understood as limiting the scope of protection of the present disclosure.

[44] Example 1

[45] 1. Preparation of the conjugate pad

[46] 1) Preparation of the anti-tryptase antibody labeled with fluorescent microspheres

[47] Fluorescent microspheres (Nanjing Microdetection Bio-Tech Co., Ltd., MD033) were mixed with the biotinylated anti-tryptase antibody (a first anti-tryptase antibody, Shanghai Xuanya Biotechnology Co., Ltd., XY-KT-2780) in a volume ratio of 10:1; the mixture was Centrifuged after reaction for 30 min; and the obtained precipitate was dissolved in a 20 mM boric acid buffer and diluted to the original volume.

[48] 2) The first anti-tryptase antibody labeled with fluorescent microspheres was spray-coated on the conjugate pad at an amount of 2 L/cm to 10 L/cm.

[49] 2. Preparation of an NC membrane

[50] 1) Membrane treatment: a piece of NC membrane was taken and marked, and the marked membrane was soaked in a membrane treatment solution (Tris-HCl buffer salt solution, pH 7.4) for 5 min to 10 min.

[51] 2) Assembly of the spotting device: the soaked NC membrane was placed on a flat mat, and an antibody spotting template was placed, leaving a place for labelingattaching marker paper.

[52] 3) Preparation of the second anti-tryptase antibody testing area: the second anti-tryptase antibody (Shanghai Zeye Biotechnology Co., Ltd., Item No.: ZY-1303Ab) was streaked at a concentration of 2 mg/mL, liquid output of peristaltic pump of 0.5 mL/cm, and streaking speed of 50 m/20 min, and the NC membrane after streaking was forced air drying at 37°C for 12 h.

[53] 4) Preparation of the control area: the anti-rabbit IgG antibody (sigma, Item No.: AP132) was streaked on the NC membrane at a concentration of 2 mg/mL, liquid output of peristaltic pump of 0.5 mL/cm, and streaking speed of 50 m/20 min, and the control line is parallel to the line in testing area, the NC membrane after streaking was forced air drying at 37°C for 12 h.

[54] 5) The NC membrane was confined at 37°C for 60 min with a confining liquid (prepared withl00 mL of PBS and 0.5 g of BSA), after taking it out, the NC membrane was dried at 37°C for 2 h and packaged for use.

[55] 3. Assembly of the test strip

[56] The sample pad, the conjugate pad, the NC membrane, and the absorbent filter paper were sequentially assembled and adhered on a PVC bottom plate, and a resulting product was cut into test strips shown in FIG. 1 as required (4 mm) with a strip cutter.

[57] Example 2

[58] 1. The assembled test strips in Example 1 were put into a plastic case with top and bottom parts for detecting the antigen to be tested:

[59] 100 L of the sample to be tested was added dropwise on the sample pad, the sample to be tested was reacted and bound to the anti-tryptase antibody labeled with fluorescent microspheres on the conjugate pad, then reacted with the anti-tryptase antibody on the testing area, and finally reached the control area where the reaction was completed. Followed, the test strip was put in a special fluorescence signal reader to read a fluorescence signal ratio (T/C), thus achieving quantitative determination.

[60] 2. Linearity of the dosage-response curve: A series of calibration solution prepared from tryptase calibrators were detected at the concentrations of 200.00 ng/mL, 100.00 ng/mL, 50 ng/mL, ng/mL, 10 ng/mL, and 1 ng/mL; and the S curve was used for fitting. The results showed that, within a concentration range of 1 ng/mL to 200 ng/mL, the relationship between concentrations and test values showed a smooth growth curve, the curve equation was shown in FIG. 2:

84 13 2

[61] y = 5.41925 + (0.18367-5.41925)/(1 + (x/118.84607)o ), R2 = 0.99542.

[62] 3. Detection of positive samples and negative control samples

[63] 100 pL of the sample to be tested (from the Allergy Department of a First-Class Hospital at Grade 3) was added dropwise on the sample pad, the result was read using a dry fluorescence immunoassay analyzer (Guangzhou Lanbo Biological Technology Co., Ltd., AFS-1000) after counting the timer for 10 min to 15 min with a timer.

[64] ImmunoCAPTMTryptase (under the brand of phadia, Thermofisher) is a regent used worldwide in the art, and the methodology involved is of fluorescence ELISA, which needs four reaction steps and takes more than two hours to complete one test, and the method requireds special automation equipment to complete the test, resulting in an automation equipment investment of not less than 600,000 yuan and a reagent cost of not less than 80 yuan/person.

[65] The comparison between the detection result of the present disclosure and the detection result of ImmunoCAPTM Tryptase is shown in Table 1.

[66] Table 1 Detection results of positive samples and negative control samples

Product of the present disclosure ImmunoCAPTM Tryptase No. Sample type result (ng/mL) positive/negative result (ng/mL) positive/negative

1 Positive sample 113.6 + 122.1

+ 1

2 Positive sample 87.3 + 96.9

+ 2

3 Positive sample 21.8 + 21.2

+ 3

4 Positive sample 42.4 + 45.1

+ 4

Positive sample 36.8 + 32.6

+ 5

6 Healthy control 3.8 - 3.5 1

7 Healthy control 7.2 - 7.7 2

8 Healthy control 2.3 - 2.1 3

9 Healthy control 8.8 - 7.5 4

10 Healthy control 4.9 - 4.6 5

[67] The results in Table 1 show that detection results of the present disclosure are 100% consistent with those using ImmunoCAPTM Tryptase, indicating that the present disclosure can achieve the same clinical performance as the imported fluorescent ELISA reagent under the condition of short detection time and low cost.

[68] The above descriptions are only preferred embodiments of the present disclosure. It should be noted by those skilled in the art that, several improvements and modifications can be made without departing from the principle of the present disclosure, and these improvements and modifications also should be regarded as falling within the protection scope of the present disclosure.

[69] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[70] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

WHAT IS CLAIMED IS:

1. A fluorescence immunochromatography test strip for detecting tryptase, comprising a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper which are sequentially lapped and adhered on the bottom plate; wherein the conjugate pad is coated with a first anti-tryptase antibody labeled with fluorescent microspheres;

the NC membrane comprises a test line and a control line; the test line is coated with a second anti-tryptase antibody; the control line is coated with an anti-rabbit IgG antibody; and

the first anti-tryptase antibody labeled with fluorescent microspheres is paired with the second anti-tryptase antibody.

2. The fluorescence immunochromatography test strip according to claim 1, wherein a particle size of the fluorescent microspheres is in a range of 50 nm to 500 nm.

3. The fluorescence immunochromatography test strip according to claim 1, wherein the concentration of the first anti-tryptase antibody labeled with fluorescent microspheres coated on the conjugate pad is in a range of 2 mg/mL to 10 mg/mL; the concentration of the anti-rabbit IgG antibody coated on the control line is in a range of1 mg/mL to 5 mg/mL; and the concentration of the second anti-tryptase antibody coated on the test line is in a range of 1 mg/mL to 5 mg/mL.

4. Use of the fluorescence immunochromatography test strip according to any one of claims I to 3 in the preparation of a kit for detecting tryptase content in samples.

5. A method for detecting tryptase content in samples, comprising the following steps: adding 70 pL to 120 L of a blood sample solution dropwise on the sample pad of the fluorescence immunochromatography test strip according to any one of claims 1 to 3, reading the fluorescence signal ratio on the fluorescence immunochromatography test strip 10 min to 15 min later, and determining the tryptase content according to a standard curve; wherein

the standard curve is: y = 5.41925 + (0.18367-5.41925)/(1 + (x/118.84607) 8 4 13 2 ), R2 = 0.99542, wherein x represents the concentration of the tryptase in the blood sample, with a unit of ng/mL, and y represents the fluorescence signal ratio.