CN115407060A - Immunoassay reagent for testing procalcitonin level in oral sample and application - Google Patents

Abstract

The invention discloses an immunodetection reagent for testing the level of procalcitonin in an oral sample and application thereof. The sampling head is immersed in an oral cavity sample, the sampling handle is pushed, the sample is concentrated in the sample extraction structure, the sample flows into the immunoassay reaction structure through the extraction structure, and the level of procalcitonin in the sample is detected. The invention provides an oral cavity sampling mode and a detection reagent of procalcitonin, improves the detection efficiency and accuracy and has important clinical significance.

Description

Immunoassay reagent for testing procalcitonin level in oral sample and application

Technical Field

The invention relates to the technical field of medical instruments, in particular to an immunoassay reagent for testing the level of procalcitonin in an oral cavity sample and application thereof.

Background

Procalcitonin (PCT) is a hormone-inactive glycoprotein of 116 amino acids, which was first detected in the serum of septic patients in the 90 s of the 20 th century. Produced by thyroid-C cells under physiological conditions, and very little can be detected in the serum of healthy people; when the body is seriously infected, including bacterial infection, fungal infection and the like, and the function failure of multiple organs, the level of procalcitonin is increased, so that the procalcitonin is generally used as the strongest index of bacterial infection in children and old patients clinically for differential diagnosis of bacterial infection, and can be used for effectively distinguishing bacterial infection from viral infection.

Clinical studies show that the level of procalcitonin in an oral sample is correlated with the level of procalcitonin in a blood sample, and the sampling of the oral sample is a non-invasive way without worrying about injury or infection possibly caused by invasive sampling.

Procalcitonin is widely applied to diagnosis of children infected diseases, and the existing procalcitonin detection products are blood detection and need to undergo venous blood sampling, which is a painful process that children are difficult to adapt to and parents are unwilling to accept. And even cause fear for many children and apprehension for parents. Compared with blood sampling, oral cavity sampling has the advantages of easy collection, no harm to the body, easier acceptance by children and the like. The detection reagent has the advantages of convenience in carrying, high detection efficiency, low operation difficulty and the like, and has wide clinical application value.

Disclosure of Invention

In view of the above problems, the present invention aims to provide an immunoassay reagent for measuring procalcitonin level in an oral sample, which has high efficiency and convenient use. In order to achieve the purpose, the invention provides an immunodetection reagent for testing the level of procalcitonin in an oral cavity sample, which consists of an oral cavity sample sampling structure, a sample to be detected extracting structure, an immunodetection reaction structure and an immunodetection detection structure.

Furthermore, the oral cavity sample sampling structure consists of a sampling head and a sampling handle, and the sampling head and the sampling handle are detachably connected;

furthermore, the sample extraction structure to be detected is a tubular structure and an elastic structure and consists of a liquid phase containing casein, which is arranged in the elastic structure;

furthermore, the immunoassay reaction structure is an immunochromatography detection structure and comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a supporting bottom sheet, wherein an anti-human procalcitonin antibody marked by an indicator is arranged on the sample pad, the anti-human procalcitonin antibody marked by the indicator is combined with procalcitonin antigen in a sample to be detected to form an indicator-anti-human procalcitonin antibody-procalcitonin antigen complex, and the sample pad is connected with the nitrocellulose membrane; the proximal end of the nitrocellulose membrane is connected with the combination pad, the distal end of the nitrocellulose membrane is connected with the water absorption pad, a detection T line is arranged on the nitrocellulose membrane, and the T line is prepared by coating an anti-human procalcitonin antibody which has pairing immune binding activity and is marked by an indicator and arranged on the sample pad;

further, the immunodetection structure is a detection structure for immunodetection reaction results, and comprises at least one of a colloidal gold detector, a fluorescence detector and visual detection.

Further, the sampling head is at least one of an oral cavity sterile sampling swab, a biological sampling cotton swab and a flocking swab.

The invention also provides an application scheme of a sampling device for collecting a sample from virus detection of a human body, which is characterized by comprising the following steps:

1) Holding the sampling head in the mouth and collecting saliva sufficiently;

2) Then rubbing the gum part to carry out gum sampling;

3) Pushing the sampling handle to concentrate the sample into the sample extraction structure;

4) Flowing the sample into the immunoassay reaction structure via the extraction structure;

5) Reacting and determining the level of procalcitonin in the sample.

Due to the adoption of the technical scheme, the invention has the following advantages:

(1) Compared with blood sample sampling, the sampling mode of the invention is more acceptable, and has the advantages of easy acquisition, no harm to the body, easier acceptance by children and the like;

(2) The detection reagent has the advantages of convenience in carrying, high detection efficiency, low operation difficulty and the like.

Drawings

FIG. 1 is a schematic view of a sampling head of the present invention;

FIG. 2 is a schematic view of the sampling handle of the present invention;

FIG. 3 is a schematic diagram of the structure of the sample to be examined;

FIG. 4 is a schematic diagram of the immunoassay reaction of the present invention;

FIG. 5 is a schematic view of the immunoassay clip of the present invention;

fig. 6 is a cross-sectional schematic view of a sampling handle of the present invention.

Detailed Description

To further illustrate the technical means and effects of the present invention for achieving the predetermined purpose, the following embodiments are further described with reference to the accompanying drawings, but the present invention is not limited to the following description.

First embodiment, swab sampling and sample Collection

(1) Sampling: pinching the sterile external package of the swab head, tearing the packaging plastic package bag from the end of the swab handle, removing the swab handle, connecting the connecting rod 1 (shown in figure 1) to the end of the sampling handle 3 (shown in figure 2), holding the sampling handle, putting the swab head into the oral cavity of a detected person, fully absorbing saliva, and then scraping the gum parts on the left and right sides to fully sample.

(2) Sample collection and processing: after sampling, the sampling handle is held by hand to connect with the end of the sample extraction structure 4 (shown in fig. 3) to be detected, so that the swab head filled with the sample is mixed with the liquid phase containing casein in the elastic structure 5 (shown in fig. 3), and the elastic structure 5 is squeezed, so that the liquid phase flows into the immunoassay reaction structure 6 (shown in fig. 4).

Example two Procalcitonin assay

(1) Preparation of sample pad

The sample pad is made of a glass cellulose membrane, salt ions such as NaCl and BSA and proteins are dissolved by using buffer solution Tris and the like, then a small amount of surfactant Tween20 is added, the pH value is adjusted to 7-8, the sample pad is uniformly paved on glass fibers according to the water absorption capacity of the glass fibers, and the glass fibers are placed at 37 ℃ for drying for 8 hours to obtain the sample pad.

(2) Preparation of the conjugate pad

Taking out the glass cellulose membrane, transferring the colloidal gold labeled mouse anti-procalcitonin monoclonal antibody on a membrane printing instrument, starting the membrane printing instrument, setting the moving speed of the spray pen to be 30mm/s and the liquid propelling amount to be 1ul/mm. And then loading a goat anti-procalcitonin polyclonal antibody, adjusting the position of a film printing nozzle on the glass cellulose film, starting film printing, taking out the sprayed glass cellulose film, putting the glass cellulose film into an electric heating vacuum drying oven, and drying for 8 hours at 37 ℃ to obtain the bonding pad.

(3) Coating and drying of procalcitonin nitrocellulose membrane

Diluting goat anti-mouse IgG antibody to the concentration of 1.0mg/mL by using 50mM PB buffer solution with pH7.2, and drawing a line at the marked position of the C line at the upper end of the NC membrane at the drawing rate of 0.8ul/cm to obtain a quality control line; diluting the goat anti-human calcitonin original polyclonal antibody to the concentration of 1.0mg/mL by using a dilution coating solution, scribing the T line mark position on an NC membrane at the scribing speed of 0.8ul/cm to obtain a quantitative detection line, wherein the interval between the detection line and the quality control line is 5mm, and drying at 37 ℃ for 8 hours after scribing is finished to obtain the goat anti-human calcitonin original polyclonal antibody.

(4) Preparing absorbent paper: the absorbent paper was cut into pieces of 30cm by 2.7 cm.

(5) And (3) sequentially sticking the treated sample pad, the microsphere mark combination pad, the nitrocellulose membrane and the water absorption pad on a PVC (polyvinyl chloride) base plate, cutting the stuck sample pad, the microsphere mark combination pad, the nitrocellulose membrane and the water absorption pad into test strips with the width of 4mm by using a slitter, and placing the test strips in a detection tubular structure.

(6) The elastic structure 6 in which the sample processing liquid prepared in example 1 was stored was pressed to flow the sample processing liquid into the detection site, and detection was performed, and the detection result was read.

EXAMPLE III clinical application study

The immunoassay reagent (the lowest detection limit is 50 ng/ml) for testing the level of procalcitonin in the oral sample is used for sampling and detecting the oral sample of 30 cases of 8-12 years old children, and the serum sample detection result is adopted for verification, so that the coincidence rate of the oral sample and the blood sample detection result reaches 100 percent.

Claims (3)

1. An immunodetection reagent for testing the level of procalcitonin in an oral sample, which is characterized by comprising an oral sample sampling structure, a sample to be detected extracting structure, an immunodetection reaction structure and an immunodetection reaction structure:

the oral cavity sample sampling structure consists of a sampling head and a sampling handle, and the sampling head is detachably connected with the sampling handle;

the sample extraction structure to be detected is a tubular and elastic structure and consists of a liquid phase containing casein, which is arranged in the elastic structure;

the immunoassay reaction structure is an immunochromatography detection structure and comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a supporting bottom sheet, wherein an anti-human procalcitonin antibody marked by an indicator is arranged on the sample pad, the anti-human procalcitonin antibody marked by the indicator is combined with procalcitonin antigen in a sample to be detected to form an indicator-anti-human procalcitonin antibody-procalcitonin antigen complex, and the sample pad is connected with the nitrocellulose membrane; the proximal end of the nitrocellulose membrane is connected with the combination pad, the distal end of the nitrocellulose membrane is connected with the water absorption pad, a detection T line is arranged on the nitrocellulose membrane, and the T line is prepared by coating an anti-human procalcitonin antibody which has pairing immune binding activity and is marked by an indicator and arranged on the sample pad;

the immunodetection structure is a detection structure for immunodetection reaction results and comprises at least one of a colloidal gold detector, a fluorescence detector and visual detection.

2. The immunoassay reagent for the testing of the level of procalcitonin in an oral sample of claim 1, wherein the sampling head is at least one of an oral sterile sampling swab, a biological sampling swab, or a flocked swab.

3. Use of an immunoassay reagent for the detection of procalcitonin levels in an oral sample comprising the steps of:

1) Holding the sampling head in the mouth and collecting saliva sufficiently;

2) Then rubbing the gum part to carry out gum sampling;

3) Pushing the sampling handle to collect the sample into the sample extraction structure;

4) Flowing the sample into the immunoassay reaction structure via the extraction structure;

5) Reacting and determining the level of procalcitonin in the sample.

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