CN105842452A - Preparation method of duplicate diagnosis gold-colloidal strip for hand-foot-mouth disease EV71 and CA16 - Google Patents

Abstract

The invention discloses a preparation method of a duplicate diagnosis gold-colloidal strip for hand-foot-mouth disease EV71 and CA16. The preparation method includes the following steps: diluting chloroauric acid to 0.01% with deionized water, heating to 95 DEG C, stabilizing for 23min, quickly adding 1mL of 1% trisodium citrate, reacting till the color becomes wine red, stabilizing for 3min prior to taking out, and continuing stirring to room temperature; under the condition that pH is 8.0, slowly adding colloidal gold solution according to the antibody amount proportionally, reacting at room temperature for 30min, adding 1% BSA for closing, centrifuging, discarding supernate, uniformly mixing precipitate with 1mL of TBS, centrifuging, discarding supernate, and resuspending; spraying detection line colloidal gold labelled antibodies and quality control line antibodies on a nitrocellulose membrane to complete assembling of the duplicate sandwich strip. The convenient and efficient detection means is provided, the defect that only single pathogen of hand-foot-mouth disease can be detected on the market is overcome, and cost of clinical examination is lowered.

Description

Hand-foot-mouth disease EV71 diagnoses the preparation method of gold test strip with CA16 bigeminy

Technical field

The present invention relates to medical field, be specifically related to a kind of hand-foot-mouth disease EV71 and CA16 bigeminy diagnosis gold mark The preparation method of reagent paper.

Background technology

HFMD (Hand-foot-mouth disease, HFMD) is global infectious disease, widely distributed,

Without obvious Regional Distribution.The pathogen of the hand-foot-mouth disease of early discovery is mainly CA16 type, 20th century At the beginning of the seventies, it was recently reported that hand-foot-mouth disease is relevant with EV71 infection, hereafter EV71 infects and infects with CA16 It is alternately present, becomes the main pathogens of hand-foot-mouth disease.In recent years, EV71 with CA16 carries in Southeast Asia one Popular, cause more serious symptom and death.Since two thousand eight, hand-foot-mouth disease stream in Chinese children Row trend is increasingly severe.Only 2010, health ministry statistical data showed: China's Mainland occurs altogether 1,774,669 example hand-foot-mouth disease case, causes 905 examples death and more than 20,000 example serious symptoms.But there is no at present For preventative vaccine or the curative drug of hand-foot-mouth disease, bring great prestige to the life and health of child The side of body.

Hand-foot-mouth disease is popular betides annual summer, Qiu Liangji, the 6-9 month is peak period.This disease premorbid number My god, just can find virus at throat position with feces, the most i.e. have infectivity, infectivity in morbidity one week after The strongest；And patient is sustainable discharges virus via intestinal, as long as 8-12 week.Incubation period is 2-10 days, the most about 3-5 days.Their early stage, does not has obvious advanced symptoms, most patients to rise suddenly Disease, about half patient are generated heat while premorbid 1～2d or morbidity, how at about 38 DEG C, have upper sense Symptom, such as cough, watery nasal discharge, Nausea and vomiting etc..Morbidity later stage hands, foot, mouth, four positions of buttocks go out Existing herpes.The most much like with herpangina, herpetic gingivostomatitis and foot and mouth disease, child once occurs The mortality complication such as myocarditis, pulmonary edema, AME are difficult to cure.Therefore, in clinic Upper early diagnosis is just particularly important.There is presently no the reagent of data display Clinical detection hand-foot-mouth disease Box.Clinical Laboratory is mainly carried out with cell inoculation, neonatal rat inoculation, serum neutralization test and RT-PCR method Diagnosis, these methods are the longest, and testing cost is high, RT-PCR often has false positive results, give clinical morning Phase diagnosis brings certain difficulty.

Colloidal gold method, is a kind of novel immunity being applied to antigen-antibody using gold colloidal as tracer label thing Labelling, this technology is easy to use quickly, is responded and can complete in 15 minutes；Low cost, it is not necessary to Special instrument and equipment；Applied range, is suitable for multiple testing conditions；Label is stable, labelling sample More than 4 DEG C of 2 year years of storage, no signal relaxation phenomenon；Gold colloidal is originally as redness, it is not necessary to adds and sends out Color reagent, eliminates enzyme target carcinogenecity substrate and the step of stop buffer, to human non-toxic's evil.Detection specimen Kind is many: can be used for having a blood test, urine or feces, thus is suitable for various inspections.

Summary of the invention

It is an object of the invention to provide the system of a kind of hand-foot-mouth disease EV71 and CA16 bigeminy diagnosis gold test strip Preparation Method, gained reagent paper is prepared bigeminy gold label test strip with colloid gold label EV71 with CA16 monoclonal antibody, can be answered For early diagnosis and the examination of clinical hand-foot-mouth disease, for the therapic opportunity that the treatment offer of this disease is optimal, make More infant is away from the threat of this disease.

For achieving the above object, the technical scheme that the present invention takes is:

Hand-foot-mouth disease EV71 and the preparation method of CA16 bigeminy diagnosis gold test strip, comprise the steps:

S1, with deionized water, 1ml 1% gold chloride is diluted to 0.01%100ml, is heated to 95 DEG C, Stablizing 23min, rapidly join the trisodium citrate 1mL of 1%, reaction is claret to color, stablizes 3min Rear taking-up, continues stirring to room temperature；

S2, under conditions of pH is 8.0, by 0.2mg/ml antibody and colloidal gold solution with 1: 1 ratio Example mixes, room temperature reaction 30min, by the dosage of every milliliter of addition 10%BSA 600ul, closes 30min, 4 DEG C of 12000r/min are centrifuged 30min, abandon supernatant, and precipitate 1mL TBS mixes, 4 DEG C 12000 R/min, centrifugal 30min, abandon supernatant, and 0.1mL TBS is resuspended, keeps in Dark Place standby；

S3, carry out on nitrocellulose filter detect line colloidal gold labeled monoclonal antibody, the spraying of nature controlling line antibody, Detection line is respectively EV71 monoclonal antibody 1mg/mL, CA16 monoclonal antibody 1mg/mL；Nature controlling line is sheep anti-mouse antibody 1mg/mL；

S4, sample pad, pad, gained nitrocellulose filter, adsorptive pads are attached to successively with adhesive PVC backboard on, be cut into the wide reagent paper of 4mm, drying at room temperature.

The method have the advantages that

Convenient, efficient detection means can not only be provided, and compensate for can only detecting on market hand-foot-mouth disease The defect of single pathogen.Greatly reduce Clinical detection expense, it is simple to popularize at basic hospital, for brothers The prevention and control of stomatosis and treatment provide favourable foundation.

Accompanying drawing explanation

Fig. 1 is the structural representation of embodiment of the present invention hand-foot-mouth disease EV71 and CA16 bigeminy diagnosis gold test strip Figure.

Detailed description of the invention

In order to make objects and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is carried out Further describe.Should be appreciated that specific embodiment described herein only in order to explain the present invention, It is not intended to limit the present invention.

Embodiment

(1) prepared by colloidal gold solution

With deionized water, 1ml 1% gold chloride is diluted to 0.01%100ml, is heated to 95 DEG C, stable 23min, rapidly joins the trisodium citrate 1mL of 1%, and reaction to color is claret, after stablizing 3min Take out, continue the colloid gold particle diameter that stirring is prepared at electricity Microscopic observation to room temperature.

(2) optimization of colloidal gold labeled monoclonal antibody optimum condition

A: the mensuration of gold labeling antibody optimum pH

Take same amount of colloidal gold solution to put in 1.5mL centrifuge tube, use 0.1mol/LK₂CO₃Adjust pH To 8.0, taking ELISA Plate 8 hole, add the gold colloidal 200ul shop fixtures of different pH respectively, every hole is distinguished again Adding monoclonal antibody 10ul, after concussion reaction 30min, every hole adds 10%NaCl solution 20ul, and concussion is anti- After answering 15min, it is the suitableeest for detecting at that pH that 520nm wavelength OD value is the highest by microplate reader pH。

B: the optium concentration of gold labeling antibody determines

Antibody is diluted to 0.2mg/mL, takes ELISA Plate, every hole 200ul gold colloidal shop fixtures, take not consubstantiality Long-pending 4H8,10B2 monoclonal antibody is separately added into, and deionized water supplies volume, after concussion reaction 30min, often Hole adds 10%NaCl solution 20ul, and concussion reaction 15min, it is quantitative that gold colloidal and antibody reach minimum steady Each hole still keep red constant, detect under 520nm wavelength condition by microplate reader simultaneously, OD value is surely The fixed minimum hole containing antibody is the amount of antibody needed for 200ul gold colloidal.

C: the preparation of gold labeling antibody

Under conditions of pH is 8.0,0.2mg/ml antibody is mixed with 1: 1 ratio with colloidal gold solution, Room temperature reaction 30min, by every milliliter add 10%BSA 600ul dosage, close 30min, 4 DEG C 12000r/min is centrifuged 30min, abandons supernatant, and precipitate 1mL TBS mixes, 4 DEG C 12000 R/min, centrifugal 30min, abandon supernatant, and 0.1mL TBS is resuspended, keeps in Dark Place standby；

Nitrocellulose filter carries out detecting line colloidal gold labeled monoclonal antibody, the spraying of nature controlling line antibody, inspection Survey line is respectively EV71 monoclonal antibody 1mg/mL, CA16 monoclonal antibody 1mg/mL；Nature controlling line is sheep anti-mouse antibody 1mg/mL；

The assembling of the sandwich reagent paper of bigeminy

Sample pad, pad, gained nitrocellulose filter, adsorptive pads are attached to successively with adhesive On PVC backboard, it is cut into the wide reagent paper of 4mm, drying at room temperature.

The detection of gold label test strip judges with result

By the sample of ELISA test positive, take 60ul sample drop and be added to the sample pad of colloidal gold strip On, observed result after 15min, detection line and nature controlling line all occur that red line is that the positive, only nature controlling line occur Red line is negative, as quality control band does not occurs then for losing efficacy.The relatively accuracy rate of gold test strip detection.

Gained hand-foot-mouth disease EV71 and CA16 bigeminy diagnosis gold test strip as it is shown in figure 1, include PVC base plate, On shown PVC base plate, end face is sequentially provided with sample pad, NC film (nitrocellulose filter) from left to right and inhales Water filter paper, described NC film (nitrocellulose filter) upper end is sequentially provided with containing EV71+CA16 mono-from left to right Anti-colloidal gold pad, T1 line (EV71 antibody), T2 line (CA16 antibody) and control line (C line), and Colloidal gold pad side containing EV71+CA16 monoclonal antibody is positioned at the lower section of sample pad side.

We have collected 326 parts of doubtful serum samples in clinic, simultaneously with indirect ELISA and bigeminy gold mark ELISA test strip EV71 and CA16, result shows that the recall rate of two kinds of detection methods is consistent.Use indirect ELISA Method has detected sample for about 8 hours, and the detection that test strips has only used 45 minutes to complete all samples.

The above is only the preferred embodiment of the present invention, it is noted that common for the art For technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, These improvements and modifications also should be regarded as protection scope of the present invention.

Claims (1)

1. hand-foot-mouth disease EV71 and CA16 bigeminy diagnose the preparation method of gold test strip, it is characterised in that Comprise the steps:

S1, with deionized water, 1ml 1% gold chloride is diluted to 0.01%100ml, is heated to 95 DEG C, Stablizing 23min, rapidly join the trisodium citrate 1mL of 1%, reaction is claret to color, stablizes 3min Rear taking-up, continues stirring to room temperature；

S2, under conditions of pH is 8.0, by 0.2mg/ml antibody and colloidal gold solution with 1: 1 ratio Example mixes, room temperature reaction 30min, by the dosage of every milliliter of addition 10%BSA 600ul, closes 30min, 4 DEG C of 12000r/min are centrifuged 30min, abandon supernatant, and precipitate 1mL TBS mixes, 4 DEG C 12000 R/min, centrifugal 30min, abandon supernatant, and 0.1mL TBS is resuspended, keeps in Dark Place standby；

S3, carry out on nitrocellulose filter detect line colloidal gold labeled monoclonal antibody, the spraying of nature controlling line antibody, Detection line is respectively EV71 monoclonal antibody 1mg/mL, CA16 monoclonal antibody 1mg/mL；Nature controlling line is sheep anti-mouse antibody 1mg/mL；

S4, sample pad, pad, gained nitrocellulose filter, adsorptive pads are attached to successively with adhesive PVC backboard on, be cut into the wide reagent paper of 4mm, drying at room temperature.