CN114252596A

CN114252596A - Fecal lactoferrin detection kit and preparation method thereof

Info

Publication number: CN114252596A
Application number: CN202111460507.1A
Authority: CN
Inventors: 郭亚; 黄春英; 林燕清
Original assignee: ZHUHAI KEYU BIOLOGICAL ENGINEERING CO LTD
Current assignee: ZHUHAI KEYU BIOLOGICAL ENGINEERING CO LTD
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-03-29

Abstract

The invention provides a fecal lactoferrin detection kit and a preparation method thereof, and the fecal lactoferrin detection kit comprises a PVC base plate, wherein a sample pad, a colloidal gold-labeled pad, a nitrocellulose membrane and absorbent paper are sequentially overlapped from left to right on the PVC base plate; the nitrocellulose membrane is internally provided with a detection line T, a contrast line R and a quality control line C which are sequentially distributed from left to right, and a detection area T, a contrast area T and a quality control area C are respectively formed on the nitrocellulose membrane at the corresponding detection line T, contrast line R and quality control line C; the right side end part of the sample pad is lapped on the upper side of the left side end part of the colloidal gold label pad, the right side end part of the colloidal gold label pad is lapped on the upper side of the left side end part of the nitrocellulose membrane, and the left side end part of the absorbent paper is lapped on the upper side of the right side end part of the nitrocellulose membrane; the method has the advantages of simple overall operation and reliable performance, realizes quick and accurate qualitative detection of fecal lactoferrin, intuitively judges whether a sample to be detected contains lactoferrin, is low in cost and is suitable for clinical quick detection.

Description

Fecal lactoferrin detection kit and preparation method thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to a medical detection technology, in particular to a fecal lactoferrin detection kit and a preparation method thereof.

[ background of the invention ]

Diarrhea is a common symptom in clinical practice, however, there are many difficulties and problems in diagnosing and treating diarrhea. The causes of diarrhea are diverse and many invasive diagnostic methods are not necessary if it can be determined whether the diarrhea is inflammatory by a simple assay.

The leucocyte in the excrement represents inflammation in the intestinal tract, so the detection of the leucocyte in the excrement is particularly important, but the detection method has strict requirements and is not easy to operate clinically. Lactoferrin (1actoferrin, LF) is a glycoprotein found in recent years that binds to iron, is secreted from most mucous membranes, is a major component of polymorphonuclear neutrophil secondary granules, is mainly distributed and expressed in neutrophils, epithelial cells, and tissue fluid, and has various functions such as promoting iron absorption, resisting bacteria, regulating immunity, resisting infection, resisting oxidation, and resisting viruses. The existing research shows that the lactoferrin participates in inflammatory reaction and tumor immunity and has wide biological activity, and during intestinal inflammation, leucocytes infiltrate mucosa to increase the concentration of the lactoferrin in feces. Lactoferrin has a strong stability, and feces can be kept for a long time, and lactoferrin can be detected by a simple and inexpensive method, and is highly clinically useful.

The lactoferrin is extremely stable in feces, so that the lactoferrin is far superior to the conventional feces marker, particularly compared with the feces DNA detection of tumors, the specimen does not need to be stored in a freezing way, the lactoferrin is extremely convenient in clinical application, the lactoferrin is detected by adopting a colloidal gold method, the method is simpler and faster than enzyme-linked immunosorbent assay, the result can be interpreted in 10-15 minutes, meanwhile, the detection repeatability is good, the finished product is stable in structure and good in use performance, the existing invasive detection means can be partially replaced, and the clinical practicability is high.

In recent years, colloidal gold method in vitro diagnostic reagent is an important direction for IVD development, the technology is rapidly developed, is widely applied in the biomedical field, particularly medical examination, receives high and wide attention, and is accepted by the world medical examination world. Therefore, in the aspect of result judgment, how to judge whether a sample contains a lactoferrin antigen by using a colloidal gold method in-vitro diagnostic reagent is a technical problem which needs to be solved urgently for detecting fecal lactoferrin at present.

[ summary of the invention ]

The fecal lactoferrin detection kit provided by the invention is simple to operate, reliable in performance, low in cost and suitable for clinical rapid detection, and can be used for intuitively and rapidly judging whether a sample contains a lactoferrin antigen without the aid of any instrument.

In order to achieve the purpose of the invention, the technical scheme is as follows:

a fecal lactoferrin detection kit comprises a PVC base plate, wherein a sample pad, a colloidal gold-labeled pad, a nitrocellulose membrane and absorbent paper are sequentially overlapped from left to right on the PVC base plate;

the nitrocellulose membrane is internally provided with detection lines T and quality control lines C which are sequentially distributed from left to right, and a detection area T and a quality control area C are respectively formed on the nitrocellulose membrane at the corresponding detection lines T and quality control lines C;

the right side end part of the sample pad is lapped on the upper side of the left side end part of the colloidal gold label pad, the right side end part of the colloidal gold label pad is lapped on the upper side of the left side end part of the nitrocellulose membrane, and the left side end part of the absorbent paper is lapped on the upper side of the right side end part of the nitrocellulose membrane;

the left side tip of colloidal gold mark pad is located the inboard of sample pad, the left side tip of nitrocellulose membrane is located the inboard of colloidal gold mark pad, the right side end of nitrocellulose membrane is located the inboard of absorbent paper.

Preferably, the colloidal gold labeled pad is made of glass fiber.

Preferably, the material of the sample pad is polyester film or glass fiber.

Preferably, the strip width of the sample pad, the colloidal gold-labeled pad, the nitrocellulose membrane and the absorbent paper is not less than 2.5 mm.

A preparation method of a fecal lactoferrin detection kit comprises the following steps:

A. preparing a sample pad:

horizontally placing glass fiber on glass or a preservative film, pouring a proper volume of prepared sample pad treatment solution on the glass fiber, uniformly coating the solution on the glass fiber, placing the coated sample pad on a nail plate blister for drying, drying for 12-24 hours in an environment that the humidity of a drying room is less than or equal to 25%, and placing the dried sample pad on a self-sealing bag for sealing for later use;

B. preparing a colloidal gold-labeled pad:

preparing a colloidal gold solution, a labeled lactoferrin antibody and a labeled antibody, spraying the colloidal gold solution on glass fibers, and drying to obtain a colloidal gold labeled pad;

C. preparing a nitrocellulose membrane:

diluting a detection line coating antibody and a quality control line coating antibody by using a coating buffer solution to prepare coating working solution, namely detection line coating solution and quality control line coating solution, respectively coating the prepared detection line coating solution and quality control line coating solution on a nitrocellulose membrane by using a membrane spraying instrument to form a detection line T and a quality control line C, and obtaining the nitrocellulose membrane containing the detection line T and the quality control line C;

D. pasting a plate and cutting:

and sequentially overlapping the sample pad, the colloidal gold label pad, the nitrocellulose membrane and the absorbent paper on the PVC base plate, and cutting the sample pad, the colloidal gold label pad, the nitrocellulose membrane and the absorbent paper into strips with the width not less than 2.5mm according to the specification requirement.

Further, the sample pad treatment solution in step A consists of 0.3-0.7% Tween 20, 0.3-0.7% Triton X-100, 3.5-4.5% BSA, 0.25% preservative and 0.05mol/L Tris-HCl buffer solution with pH of 8.0.

Further, the material of nail dish blister in step A is polyvinyl chloride, and the specification of nail dish blister is: 550 x 310mm, consisting of 2mm nail tips, with a 2cm spacing between nails. During the use, the sample pad that the coating is good is put on nail dish blister and is dried, and whole sample pad is unsettled and is favorable to gaseous circulation, makes the sample pad dry more homogeneous, and drying time is shorter.

Further, the step B specifically includes the following steps:

a. firing the colloidal gold solution;

b. preparing a fecal lactoferrin colloidal gold solution and a marker colloidal gold solution;

c. coating: uniformly coating a proper amount of liquid on the glass fiber, and drying the coated glass fiber;

d. and (5) after drying the colloidal gold label pad, sealing and storing the colloidal gold label pad for later use by using an aluminum foil bag.

Further, in the step B, after the colloidal gold labeled pad is prepared, the labeled pad contains a fecal lactoferrin labeled antibody and a marker antibody.

Further, the step C specifically includes the following steps:

e. preparing solutions of a lactoferrin detection line coating antibody and a quality control line coating object according to the formula requirements, correspondingly and respectively forming a detection line coating solution and a quality control line coating solution, wherein the spraying amount is 0.8-1.2 muL/cm, and the concentration of the detection line coating solution and the concentration of the quality control line coating solution are 1.0 mg/mL;

f. taking a PVC bottom plate adhered with the nitrocellulose membrane, and marking out a detection line T and a quality control line C at intervals; wherein the specification width of the nitrocellulose membrane is 20 mm;

g. the distance between the quality control line C and the detection line T is 5mm, and the error is less than or equal to +/-0.5 mm; the distance from the detection line T to the bottom end of the nitrocellulose membrane is 7mm, and the error is less than or equal to +/-0.5 mm;

h. setting a drying temperature and humidity, drying at the humidity of less than or equal to 25% and the temperature of less than or equal to 30 ℃, wherein the drying time is 12-24 hours;

i. and (5) placing the dried nitrocellulose membrane in a self-sealing bag for sealing for later use.

Further, the step D specifically includes the following steps:

j. checking the humidity of the production workshop, and ensuring that the humidity is less than or equal to 25 percent to start working;

k. tearing off release paper on the upper surface of the PVC base plate, adhering the colloidal gold-labeled pad on the PVC base plate, overlapping the upper edge of the colloidal gold-labeled pad with the lower edge of the nitrocellulose membrane by 1-2 mm, adhering the sample pad below the colloidal gold-labeled pad, and overlapping the upper edge of the sample pad with the lower edge of the colloidal gold-labeled pad by 1-2 mm;

1. tearing off release paper on the upper surface of the PVC base plate, and linearly sticking absorbent paper on the PVC base plate, so that the absorbent paper is overlapped with the upper edge of the nitrocellulose membrane by 2 +/-0.5 mm;

m, pasting an invisible adhesive tape, and overlapping the lower edge of the invisible adhesive tape and the lower edge of the nitrocellulose membrane pad by 1-1.5 mm;

and n, operating the automatic cutting machine to cut according to the required width.

The invention has the advantages that:

compared with the prior art, the method adopts the principle of a double-antibody sandwich method to determine whether the fecal sample contains lactoferrin or not, does not need any instrument and equipment, is simple, convenient and quick to operate, can judge whether the sample contains lactoferrin antigen or not through manual operation and naked eye reading, and does not cause harm to the body of a detected person; in the detection process, the sample pad is processed before detection, so that the effect of reducing the interference of other pathogens can be effectively achieved, false positive is eliminated, missing detection is prevented, the detection result has good specificity and high sensitivity, and the detection method can be used for screening in hospitals at all levels; meanwhile, the lactoferrin detection kit can be used for manual operation, and can also be matched with a fecal analyzer to realize automatic detection.

During testing, the sample is dripped into the sample adding part, if the sample contains lactoferrin antigen, lactoferrin can be combined with the lactoferrin antibody marked by colloidal gold to form immune complex, and the complex and the sample move from bottom to top under the action of chromatography. When the complex passes through the detection zone T, the complex is captured by the lactoferrin detection line-coated antibody immobilized on the membrane, and a mauve strip appears at the position of the detection zone T. And the color intensity of the detection zone T is related to the content of lactoferrin in the sample. No matter whether the sample contains lactoferrin or not, a mauve strip appears in the quality control area C, and the strip of the quality control area is used for judging whether the chromatography process is normal or not and is also used as an internal control standard for reagent detection.

[ description of the drawings ]

FIG. 1 is a schematic sectional view of the kit of the present invention;

FIG. 2 is a schematic top view of the reagent cartridge of the present invention;

FIG. 3 is a schematic top view of the nail plate blister of the present invention;

FIG. 4 is a graph showing the results of a test of a first formulation of a sample pad treatment fluid according to the present invention;

FIG. 5 is a graph showing the results of a second formulation of the sample pad treatment solution of the present invention;

FIG. 6 is a graph showing the results of a third formulation of the sample pad treatment solution of the present invention;

FIG. 7 is a graph of test results for a first drying means of a sample pad in accordance with the present invention;

FIG. 8 is a graph of test results for a second drying tool of a sample pad of the present invention;

[ detailed description ] embodiments

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

A fecal lactoferrin detection kit is shown in figures 1 and 2 and comprises a PVC base plate 1, wherein a sample pad 2, a colloidal gold label pad 3, a nitrocellulose membrane 4 and absorbent paper 5 are sequentially overlapped on the PVC base plate 1 from left to right; the nitrocellulose membrane 4 is internally provided with a detection line T6 and a quality control line C7 which are sequentially distributed from left to right, and a detection area T and a quality control area C are respectively formed on the nitrocellulose membrane 4 at the corresponding detection line T6 and quality control line C7; the right side end part of the sample pad 2 is lapped on the upper side of the left side end part of the colloidal gold label pad 3, the right side end part of the colloidal gold label pad 3 is lapped on the upper side of the left side end part of the nitrocellulose membrane 4, and the left side end part of the absorbent paper 5 is lapped on the upper side of the right side end part of the nitrocellulose membrane 4; the left end of the colloidal gold pad 3 is positioned at the inner side of the sample pad 2, the left end of the nitrocellulose membrane 4 is positioned at the inner side of the colloidal gold pad 3, and the right end of the nitrocellulose membrane 4 is positioned at the inner side of the absorbent paper 5.

Wherein, the colloidal gold label pad 3 is made of glass fiber, and the sample pad 2 is made of polyester film or glass fiber; and the strip widths of the sample pad 2, the colloidal gold-labeled pad 3, the nitrocellulose membrane 4 and the absorbent paper 5 are not less than 2.5 mm.

The preparation method of the fecal lactoferrin detection kit is shown in figures 1 to 3 and comprises the following steps:

A. preparation of sample pad 2:

the glass fiber is flatly placed on the glass or the preservative film, the prepared sample pad treatment solution with proper volume is poured on the glass fiber, the liquid is evenly coated on the glass fiber, and the coated sample pad is dried.

Optimally selecting the proportion of the sample pad treatment solution:

the first formulation consisted of < 0.3% Tween 20, < 0.3% Triton X-100, < 3.5% BSA, < 0.25% preservative in 0.05mol/L Tris-HCl buffer pH 8.0, as shown in FIG. 4, with the test results shown by the bar lines in the figure.

The second formula comprises 0.3-0.7% of Tween 20, 0.3-0.7% of Triton X-100, 3.5-4.5% of BSA, 0.25% of preservative and 0.05mol/L Tris-HCl buffer solution with the pH value of 8.0. Preferably, the sample pad treatment solution consists of 0.6% Tween 20, 0.5% Triton X-100, 4% BSA, 0.25% preservative in 0.05mol/L Tris-HCl buffer pH 8.0, as shown in FIG. 5, and the test results are shown by the bar lines in the figure.

The third formulation consisted of > 0.7% Tween 20, > 0.7% Triton X-100, > 4.5% BSA, > 0.25% preservative in 0.05mol/L Tris-HCl buffer pH 8.0, as shown in FIG. 6, with the test results shown by the bar lines.

In summary, the formula proportion of the sample pad treatment solution in fig. 5 is the optimal proportion, and positive omission and false positive cannot be caused.

Sample pad drying tool optimal selection:

first dry instrument is for leveling the rack: the material of level and smooth rack is polyvinyl chloride, and the specification of level and smooth rack is: 550 × 310mm, as shown in fig. 7, and dried for 12 hours, 16 hours, 20 hours, and 24 hours to test whether the color development of 10 sheets of reagents is consistent, and the results are shown by the bar lines in the figure.

The second drying tool is a nail plate blister: the material of nail dish blister is polyvinyl chloride, and the specification of nail dish blister is: 550 mm by 310mm, composed of 2mm nail tips, with a spacing of 2cm between the nails, as shown in fig. 8, and dried for 12 hours, 16 hours, 20 hours, 24 hours to test whether 10 reagents developed color consistently, the results are shown by the bar lines in the figure.

In summary, the drying tool shown in fig. 8 is the optimal tool, the coated sample mat is placed on the nail plate blister for drying, the whole sample mat is suspended in the air to facilitate the circulation of air, so that the sample mat is dried more uniformly, the drying time is shorter, the sample mat is dried for 12-24 hours in the environment that the humidity of a drying room is less than or equal to 25%, and the dried sample mat can be placed in the self-sealing bag for sealing and standby.

B. Preparing a colloidal gold label pad 3:

preparing a colloidal gold solution, a labeled lactoferrin antibody and a labeled antibody, spraying the colloidal gold solution on glass fibers, and drying to obtain a colloidal gold labeled pad; the method comprises the following specific steps:

a. firing the colloidal gold solution;

And after the colloidal gold-labeled pad is prepared, the fecal lactoferrin-labeled antibody and the marker antibody are contained.

C. Preparation of nitrocellulose membrane 4:

diluting the detection line coated antibody and the quality control line coated antibody with a coating buffer solution to prepare coating working solution, namely detection line coated solution and quality control line coated solution, respectively coating the prepared detection line coated solution and quality control line coated solution on a nitrocellulose membrane by adopting a membrane spraying instrument to form a detection line T6 and a quality control line C7, and obtaining a nitrocellulose membrane 4 containing the detection line T6 and the quality control line C7;

the method specifically comprises the following steps:

D. Pasting a plate and cutting:

and sequentially overlapping the sample pad 2, the colloidal gold label pad 3, the nitrocellulose membrane 4 and the absorbent paper 5 on the PVC base plate 1, and cutting the sample pad into strips with the width not less than 2.5mm according to the specification requirement.

The method specifically comprises the following steps:

k. tearing off release paper on the upper surface of the PVC base plate 1, adhering the colloidal gold-labeled pad to the PVC base plate 1, overlapping the upper edge of the colloidal gold-labeled pad 3 with the lower edge of the nitrocellulose membrane 4 by 1-2 mm, adhering the sample pad 2 below the colloidal gold-labeled pad 3, and overlapping the upper edge of the sample pad 2 with the lower edge of the colloidal gold-labeled pad 3 by 1-2 mm;

l, tearing off release paper on the upper surface of the PVC base plate 1, and linearly sticking the absorbent paper 5 on the PVC base plate 1 to enable the absorbent paper 5 to be overlapped with the upper edge of the nitrocellulose membrane 4 by about 2 mm;

The above-mentioned embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, except for the cases listed in the specific embodiments; all equivalent variations of the methods and principles of the present invention are intended to be within the scope of the present invention.

Claims

1. The utility model provides a excrement and urine lactoferrin detect reagent box, includes PVC bottom plate (1), its characterized in that:

a sample pad (2), a colloidal gold label pad (3), a nitrocellulose membrane (4) and absorbent paper (5) are sequentially overlapped from left to right on the PVC base plate (1);

the nitrocellulose membrane (4) is internally provided with detection lines T (6) and quality control lines C (7) which are sequentially distributed from left to right, and the detection lines T (6) and the quality control lines C (7) which correspond to each other form a detection area T and a quality control area C on the nitrocellulose membrane (4) respectively;

the right side end of the sample pad (2) is lapped on the upper side of the left side end of the colloidal gold label pad (3), the right side end of the colloidal gold label pad (3) is lapped on the upper side of the left side end of the nitrocellulose membrane (4), and the left side end of the absorbent paper (5) is lapped on the upper side of the right side end of the nitrocellulose membrane (4);

the left side tip of colloidal gold mark pad (3) is located the inboard of sample pad (2), the left side tip of nitrocellulose membrane (4) is located the inboard of colloidal gold mark pad (3), the right side tip of nitrocellulose membrane (4) is located the inboard of absorbent paper (5).

2. The fecal lactoferrin detection kit according to claim 1, wherein the colloidal gold labeled pad (3) is made of glass fiber.

3. The fecal lactoferrin detection kit according to claim 1, wherein the sample pad (2) is made of polyester film or glass fiber.

4. The fecal lactoferrin detection kit according to claim 1, wherein the widths of the sample pad (2), the colloidal gold labeled pad (3), the nitrocellulose membrane (4) and the absorbent paper (5) are not less than 2.5 mm.

5. The preparation method of the fecal lactoferrin detection kit according to any one of claims 1 to 4, comprising the steps of:

A. preparation of sample pad (2):

flatly placing glass fibers on glass or a preservative film, pouring a proper volume of prepared sample pad treatment liquid on the glass fibers, uniformly coating the liquid on the glass fibers, placing the coated sample pad on a nail plate blister (8) for drying, drying for 12-24 hours in an environment that the humidity of a drying room is less than or equal to 25%, and placing the dried sample pad on a self-sealing bag for sealing for later use;

B. preparing a colloidal gold label pad (3):

C. preparation of nitrocellulose membrane (4):

diluting a detection line coating antibody and a quality control line coating antibody by using a coating buffer solution to prepare coating working solution, namely detection line coating solution and quality control line coating solution, respectively coating the prepared detection line coating solution and quality control line coating solution on a nitrocellulose membrane by using a membrane spraying instrument to form a detection line T (6) and a quality control line C (7), and obtaining the nitrocellulose membrane (4) containing the detection line T (6) and the quality control line C (7);

D. pasting a plate and cutting:

and sequentially overlapping the sample pad (2), the colloidal gold label pad (3), the nitrocellulose membrane (4) and the absorbent paper (5) on the PVC base plate (1), and cutting into strips with the width not less than 2.5mm according to the specification requirement.

6. The method for preparing a fecal lactoferrin detection kit of claim 5, wherein the sample pad treatment solution in step A is composed of 0.3% -0.7% Tween 20, 0.3% -0.7% Triton X-100, 3.5% -4.5% BSA, 0.25% preservative and 0.05mol/L Tris-HCl buffer solution with pH 8.0.

7. The preparation method of the fecal lactoferrin detection kit according to claim 5, wherein in the step A, the material of the nail plate blister (8) is polyvinyl chloride, and the specification of the nail plate blister is as follows: 550 x 310mm, consisting of 2mm nail tips, with a 2cm spacing between nails.

8. The method for preparing a fecal lactoferrin detection kit according to claim 5, wherein the step B specifically comprises the following steps:

a. firing the colloidal gold solution;

9. The method for preparing a fecal lactoferrin detection kit according to claim 7, wherein in step B, the colloidal gold labeled pad is prepared and then contains the fecal lactoferrin marker antibody and the marker antibody.

10. The method for preparing a fecal lactoferrin detection kit according to claim 5, wherein the step C specifically comprises the following steps:

f. taking a PVC bottom plate adhered with the nitrocellulose membrane, and marking out a detection line T and a quality control line C at intervals;

11. The method for preparing a fecal lactoferrin detection kit according to claim 5, wherein the step D specifically comprises the following steps:

k. tearing off release paper on the upper surface of the PVC base plate (1), adhering the colloidal gold label pad on the PVC base plate (1), overlapping the upper edge of the colloidal gold label pad (3) with the lower edge of the nitrocellulose membrane (4) for 1-2 mm, adhering the sample pad (2) below the colloidal gold label pad (3), and overlapping the upper edge of the sample pad (2) with the lower edge of the colloidal gold label pad (3) for 1-2 mm;

l, tearing off release paper on the upper surface of the PVC base plate (1), and linearly sticking the absorbent paper (5) to the PVC base plate (1) to enable the absorbent paper (5) and the upper edge of the nitrocellulose membrane (4) to be overlapped by 2 +/-0.5 mm;