CN102253210A - Test strip for detecting enterovirus 71 (EV71) IgM antibody - Google Patents

Abstract

The invention provides a rapid test strip for detecting an enterovirus 71 (EV71) IgM antibody. In the invention, EV71 gene antigenic vascular adhesion protein-1 (VAP1) and anti-mouse IgG are coated on a nitrocellulose (NC) membrane in combination with a colloidal gold labeled anti-human IgM monoclonal antibody, and the specific EV71 IgM antibody in an infected human specimen is detected by utilizing a membrane chromatography capture method. The rapid test strip provided by the invention has the advantages of being simple, convenient and rapid in operation, having no need of special instrument and equipment and professional training, ensuring clear and easily-distinguished results and being easy for popularization; and the rapid test strip is applicable to a basic level as well as field test and early diagnosis, and plays a role in assisting the EV71 infection diagnosis.

Description

A kind of enterovirns type 71 (EV71) IgM antibody test test strips

Technical field

The invention belongs to field of biological detection, be specifically related to a kind of enterovirns type 71 (EV71) IgM antibody test test strips and application thereof.

Background technology

Enterovirns type 71 (EV71) belongs to enterovirus and belongs to the A type, be the close relative of Ke Saqi virus A 16 (CA16), the infection of EV71 can cause hand-foot-and-mouth disease (hand, foot and mouth disease, HFMD), brain (encephalitis), aseptic meningitis (asepicmeningitis) and the polio sample paralysis multiple diseases relevant such as (poliomyelitis-like paralysis) with nervous system.At present, the mankind are the unique known natural reservoir (of bird flu viruses) of EV71 virus, and virus is main by the close contact transmission between the crowd.According to the difference of nucleotide sequence, EV71 can be divided into 3 genotype such as A, B, C, Type B and C type can be further divided into B1, B2, B3, B4 and C1 and C2 hypotype again.The 7 strain EV71 viruses (SHZH03, SHZH98, SH2F1, SH2F2, SH2H25, SH2H26 and CHN287) that the China's Mainland has been found compare with EV71 virus C genotype representative strains, homology is between 89.3%～94.6%, comparatively approaching, and compare with A, B genotype representative strains, then homology is between 81.3%～84.0%.The 10 strain EV71 viruses that the China's Mainland is described all belong to EV71 virus C genotype.

The virion of EV71 is a spherical structure, no coating and projection, and the capsid of virion is formed very complicated, at first by 4 coat protein of EV71 viral gene group coding, VP1, VP2, VP3 are exposed to the surface of virus, by being assembled into protomer and subunit, finally form virus coat.Albumen VP4 then is embedded in the virus coat inboard and plays the connection effect.

Three polypeptide of VP1, VP2 and VP3 are exposed to the surface of virus coat, and its epitope mainly concentrates on VP1, VP2 and the VP3.VP1 not only has significant gene pleiomorphism, and in having concentrated and antigenic determinant, the antigenicity of directly influence virus.The VP1 gene order not only can be used as different serotypes The classification basis in the enterovirus genus, and can be used as the classification reference that does not belong to together in the Picornaviridae, so the VP1 gene is the main research contents of EV71 molecular epidemiology.

Coat protein VP1 is made up of 891 nucleotide, exist EV71 main in and epitope.Therefore obtain the expressing protein of this antigen by genetic engineering, and with its specific IgM antibodies as Detection of antigen EV71.

At present, the diagnosis of hand-foot-and-mouth disease, except the foundation clinical symptoms, laboratory diagnosis be indispensable as link.Breadboard diagnostic criteria is as follows:

1) total cellular score of blood test is general normal or higher, divides the time-like lymphocyte higher, and neutrophil leucocyte is lower.When 2) central nervous system complication being arranged, the cerebrospinal fluid cell number can increase, and albumen raises.3) morbidity back is separated or is detected correlated virus from ight soil, throat gargle.4) separate or detect correlated virus from cerebrospinal fluid or bleb liquid.5) from early stage blood serum, detect correlated virus IgM antibody.6) the convalescent serum neutralizing antibody had 〉=4 times growth than acute stage.

Yet up to the present, also do not have a kind of can be convenient, fast the report of test strips of detection EV71 specific IgM antibodies.

Summary of the invention

(1), the technical matters that will solve

The purpose of invention provides a kind of easy to use, quick, is used to detect the test strips of EV71 specific IgM antibodies, detects EV71 specific IgM antibodies in the infected patient sample, is used for the auxiliary diagnosis that people EV71 infects.

(2), technical scheme

The invention provides a kind of test strip, it comprises following composition:

(1) bag is by the reaction film of EV71 viral antigen VP1 and two bands of two anti-IgG;

(2) the bond release that contains the anti-people IgM monoclonal antibody of colloid gold label is filled up.

Above-mentioned two anti-can be that anti-mouse IgG two is anti-, described reaction film can be a nitrocellulose filter, it can be glass fibre membrane that described bond discharges pad.

The present invention also provides the method for the above-mentioned test strips of preparation, and it comprises the steps:

1) EV71 viral antigen VP1 and two anti-IgG are formed detection band and quality control band respectively on reaction film, standby;

2) with the anti-people IgM of colloid gold label monoclonal antibody, bag is discharged in the pad to bond;

3) with the reaction film for preparing and bond discharges pad and sample pad, adsorptive pads and reaction holder are assembled into test strips.

The invention also discloses described test strips in the application that detects the EV71 specific IgM antibodies.

The present invention program is: the EV71 gene engineering antigen VP1 and the anti-mouse IgG difference solid phase (NC film) on nitrocellulose membrane that adopt highly purified purifying, the anti-people IgM monoclonal antibody of association colloid gold mark is used rete and is analysed the EV71 specific IgM antibodies of catching in the ratio juris detection people sample.

(3), beneficial effect

Test strips of the present invention utilizes colloidal gold-labeled method and rete to analyse technology, is used for fast qualitative and detects the EV71 specific IgM antibodies that sample may exist, and reaches quick screening patient, the purpose of diagnoses and treatment in time.Can save a large amount of manpower and materials, easily and fast, simple and direct, not need special instruments and equipment, not need professional training, the result is clear easily to be distinguished, simple to operate, is easy to promote, be fit to basic unit, be suitable for on-the-spot the detection and early diagnosis, the Infect And Diagnose of EV71 is played booster action.

Description of drawings

Fig. 1: the front schematic view of A test strips of the present invention; The side schematic view of B test strips of the present invention.Wherein, 1: adsorptive pads; 2: nitrocellulose membrane (T:EV71 gene engineering antigen VP1; C: bag is by the Quality Control band of anti-mouse IgG); 3: the glass fibre membrane that contains the anti-people IgM monoclonal antibody of colloid gold label; 4: golden labeling antibody diaphragm; 5: the reaction holder.

Fig. 2: testing result synoptic diagram.Wherein, be followed successively by from left to right: two line positives of T, C; Line feminine gender of C; Two lines of T, C are negative or the T line positive is only arranged, and are invalid.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.

Embodiment 1:EV71 specific IgM antibodies test strip (colloidal gold method) (referring to Fig. 1)

EV71 gene engineering antigen VP1 purchases in Beijing peace baud gene technology company limited.

The reaction holder is 6.5cm * 0.4cm PCV plate; Adsorptive pads is the filter paper for oil of 2cm * 0.4cm; 2.0cm the nitrocellulose membrane of * 0.4cm wraps successively by anti-mouse IgG (2mg/ml), EV71 antigen VP1 (1.5mg/ml) contains the monoclonal antibody polyester film of the anti-people IgM (labelled amount 25 μ g/ml) of 0.4cm * 0.4cm colloid gold label; The glass fibre of sample pad 2.7cm * 0.4cm; Promptly formed enterovirns type 71 (EV71) IgM antibody test test strips (colloidal gold method).

Embodiment 2 detection methods (referring to Fig. 2)

Patient's whole blood, blood plasma or serum specimen 100-150 μ l directly are added dropwise to embodiment 1 test strips " 4 " locate, sample liquid is up along film, 10-15 minute sentence read result.

The result:

As detect the IgM antibody that contains EV71 in the sample, then with test strips on the anti-people IgM monoclonal antibody of colloid gold label form corresponding compound, up be coated on nitrocellulose membrane on EV71 specific antigen VP1 combine, form red lines, promptly form red stripes at the T place.

No matter whether contain corresponding antibody in the sample, the anti-people IgM monoclonal antibody of colloid gold label continues upwards to creep and the anti-mouse IgG that is coated on the film forms the red precipitate line, promptly locates to form red stripes at " C ".This line is a nature controlling line, loses efficacy as collaurum, and this line just can not occur, and illustrates that test strips lost efficacy.

Claims (7)

1. test strip is characterized in that it comprises:

(1) bag is by the reaction film of EV71 viral antigen VP1 and two bands of anti-mouse IgG;

(2) the bond release that contains the anti-people IgM monoclonal antibody of colloid gold label is filled up.

2. test strip as claimed in claim 1 is characterized in that, described reaction film is a nitrocellulose filter.

3. test strip as claimed in claim 1 is characterized in that, described bond discharges pad and is glass fibre membrane.

4. as claim 1 or 2 test strips as described in each, it is characterized in that described two anti-IgG are anti-mouse IgG.

5. method for preparing each described test strips of claim 1～4, it comprises the steps:

1) EV71 viral antigen VP1 and two anti-IgG are formed detection band and quality control band respectively on reaction film, standby;

2) with the anti-people IgM of colloid gold label monoclonal antibody, bag is discharged in the pad to bond;

3) with the reaction film for preparing and bond discharges pad and sample pad, adsorptive pads and reaction holder are assembled into test strips.

6.EV71 the application of viral antigen VP1 in gold-immunochromatographyreagent reagent for assay.

7. the application of the arbitrary described test strips of claim 1-4 in detecting the EV71 specific IgM antibodies.

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