CN102645538A - HA epitope colloidal gold rapid detection test strip of H5 subtype avian influenza virus antibody - Google Patents
HA epitope colloidal gold rapid detection test strip of H5 subtype avian influenza virus antibody Download PDFInfo
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Abstract
本发明公开了一种H5亚型禽流感病毒抗体的HA抗原表位胶体金快速检测试纸条。该试纸条可用于检测待测动物的H5亚型禽流感病毒的抗血清，由依次连接并固定于底板上的样品垫、胶体金结合垫、设有检测线和质控线的硝酸纤维素膜和吸水垫组成，所述检测线处的检测抗原为如下a)或b)的蛋白：a)由序列表序列2所示氨基酸序列组成的蛋白质；b)将序列表序列2的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可与H5亚型禽流感病毒的抗血清特异结合的蛋白。本发明的试纸条具有以下优点：抗原制备方法简单、安全、产量高；操作操作简单，特异性好、敏感性高、诊断结果可靠；适用于H5亚型禽流感病毒感染的现场检测，尤其适用基层单位或个人的现场快速检测。The invention discloses a colloidal gold rapid detection test strip for the HA antigen epitope of H5 subtype avian influenza virus antibody. The test strip can be used to detect the antiserum of the H5 subtype avian influenza virus of the animal to be tested. Composed of a membrane and a water-absorbent pad, the detection antigen at the detection line is the protein of a) or b) as follows: a) a protein composed of the amino acid sequence shown in Sequence Listing 2; b) the amino acid sequence of Sequence Listing 2 is processed One or several amino acid residues are substituted and/or deleted and/or added and can specifically bind to the antiserum of H5 subtype avian influenza virus. The test strip of the present invention has the following advantages: the antigen preparation method is simple, safe, and high in yield; the operation is simple, the specificity is good, the sensitivity is high, and the diagnosis result is reliable; it is suitable for on-site detection of H5 subtype avian influenza virus infection, especially It is suitable for on-site rapid detection of grassroots units or individuals.
Description
Technical field
The present invention relates to a kind of HA epitope colloidal gold fast detecting test paper strip of H5 subtype avian influenza virus antibody.
Background technology
(Avian influenza virus is a kind of A type influenza virus that can cause bird generation serious acute infectious disease AIV) to avian influenza virus, and its bird flu that causes is by type of classifying as the A of International Office of Epizootics (OIE) zoonosis.This virus is easy to morph, and has found 16 hemagglutinin (HA) and 9 neuraminidases (NA) hypotype so far.Wherein, H5 is a kind of highly pathogenic hypotype AIV, can cause the extensive death of bird and cause enormous economic loss to aviculture.Evidence suggests that H5 hypotype AIV can also propagate to human and other multiple mammal through number of ways, as: cat, dog, pig and tiger etc. cause its morbidity even death.This shows that H5 also has serious threat except having the serious harm supporting fowl production to people and animals' life security.Therefore, quick and precisely detecting the H5 subtype avian influenza virus infects significant to the investigation and the prevention and control of this disease.
The AIV genome by 8 independently RNA form the 10 kinds of albumen of encoding altogether.Wherein, HA albumen is the important surface antigen property albumen of of AIV; In the absorption of virus with wear aspects such as film, the viral pathogenicity of decision and play an important role, also be that virus stimulates body to produce the important antigen of neutralizing antibody, significant in the immunology diagnosis of AIV.
It is main agents with enzyme-labelled antigen or enzyme labelled antibody that EUSA (ELISA) is one type, through the substrate for enzymatic activity colour generation in the compound checking matter is carried out qualitative or quantitative immuno-labelling technique.That this technology has is highly sensitive, high specificity, accuracy good, characteristics such as simple to operation, is at present to the common technology of viral antigen and antibody test thereof.This technology was applied to the detection of bird flu early than 1974 by European countries, and the bird flu ELISA diagnostic method of China's research and development at present comprises avian flu virus detection ELISA and two kinds of avian influenza antibody detection ELISA.These two kinds of employed antigens of technology are totivirus concentrated antigens, need through concentrate, technologies such as purifying and deactivation prepare.Avian influenza virus is a kind of virus that is easy to make a variation, and has the harm of potentiality bio-safety.Especially the H5 subtype avian influenza virus is a kind of highly pathogenic virus, and preparation requires during antigen in the bio-safety facility of P3 level at least, to produce.Production cost is big, instrument and equipment requires height, can not satisfy needs of production, is unfavorable for applying of these technology.To these problems, it is that antigen is set up bird flu ELISA detection technique with hemagglutinin (HA) gene expression albumen that the scholar is arranged, and has proved that the HA hypotype that can be used for avian influenza virus carries out somatotype.But owing to contain more Escherichia coli rare codon in the HA gene, when the HA full-length gene was expressed in prokaryotic, the rare codon of dense distribution can have very big influence to Recombinant Protein Expression output in the sequence, can't satisfy the production needs; And these rare codons distributions extensively, it transformed need expend very big man power and material, increases considerably R&D costs.
Colloidal gold immunochromatographimethod is analyzed (gold-immunochromatography assay; GICA) be that a kind of that developed recently gets up is label with the collaurum, the biomacromolecule in the sample carried out the immune analysis technology of qualitative analysis through the antigen-antibody immunological response.Collaurum is applied to immune analysis as immune marker and starts from 1971; Along with 1989 being that the immune colloid gold Fast Detection Technique had obtained widespread use since the aids antibody diafiltration detectable of label exploitation was born in life medical diagnosis every field first with the collaurum.
The immune colloid gold quick detection test paper bar that the preparation of H5 subtype avian influenza virus epitope protokaryon recombinant expression protein is not arranged at present as yet.
Summary of the invention
The HA epitope colloidal gold fast detecting test paper strip that the purpose of this invention is to provide a kind of H5 subtype avian influenza virus antibody.Said test strips can be used for detecting the antiserum of the H5 subtype avian influenza virus of tested animal; Form by the sample pad, collaurum pad, the nitrocellulose filter that is provided with detection line and nature controlling line and the adsorptive pads that connect successively and be fixed on the base plate, the detection antigen at said detection line place be following a) or b) albumen:
A) protein of forming by amino acid sequence shown in the sequence table sequence 2;
B) with the amino acid sequence of sequence table sequence 2 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and can with the albumen of the antiserum specific bond of H5 subtype avian influenza virus;
Amino acid sequence shown in the sequence table sequence 2 is made up of 244 amino acid residues, contains the HA epitope of H5 subtype avian influenza virus.
Albumen in above-mentioned in order to make (a) is convenient to purifying, can connect label as shown in table 1 at the amino terminal or the carboxyl terminal of the protein of being made up of the amino acid sequence shown in the sequence table sequence 2;
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLA6 | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKL?ISEEDL |
Said detection antigen specifically can be the albumen that adds 6 histidines at the amino terminal of amino acid sequence shown in the sequence table sequence 2.
Be coated with the antibody of the anti-said tested animal IgG of colloid gold label on the said collaurum pad;
The detection antibody at said nature controlling line place is the antibody with the antibody specific bond of said anti-tested animal IgG;
When said tested animal was chicken, the coated antibody on the said collaurum pad was the antibody of the anti-chicken IgG of colloid gold label, and like the anti-chicken IgG of rabbit, the detection antibody at said nature controlling line place is anti-rabbit igg, like goat anti-rabbit igg;
When said tested animal was duck, the coated antibody on the said collaurum pad was the antibody of the anti-duck IgG of colloid gold label, and like the anti-duck IgG of rabbit, the detection antibody at said nature controlling line place is anti-rabbit igg, like goat anti-rabbit igg.
Said detection line is positioned at the end of said nitrocellulose filter near said collaurum pad;
Shown in nature controlling line be positioned at the end of said nitrocellulose filter near said adsorptive pads.
In above-mentioned test strips, said detection line is 0.5mm-1mm with the vertical range of an end margin that is connected said nitrocellulose filter of said collaurum pad.
In above-mentioned test strips, the vertical range of said nature controlling line and said detection line is 4mm-7mm.
In above-mentioned test strips, said sample pad was soaked with confining liquid second; The solvent of said confining liquid second is that PBS encapsulates damping fluid, and solute is bovine serum albumin(BSA), NaN
3, polyvinylpyrrolidone and sucrose, solute bovine serum albumin(BSA), NaN
3, polyvinylpyrrolidone and the concentration of sucrose in said confining liquid second is respectively 20g/L, 0.5g/L, 3g/L and 25g/L; The solvent that said PBS encapsulates damping fluid is a water, and solute is NaCl, KCl, KH
2PO
4And Na
2HPO
4, solute NaCl, KCl, KH
2PO
4And Na
2HPO
4The concentration that encapsulates in the damping fluid at said PBS is respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L.
In above-mentioned test strips, said detection antigen encapsulates on the said nitrocellulose filter according to the method that comprises the steps: encapsulate damping fluid with said PBS said detection antigen diluent is encapsulated on said nitrocellulose filter to 1.0-1.5mg/mL.
In above-mentioned test strips, the pH value that said PBS encapsulates damping fluid is 7.2.
In above-mentioned test strips, said tested animal can be chicken, duck or goose, but is not limited to above-mentioned animal.
Test strips of the present invention be with the HA epitope protokaryon recombinant expression protein of H5N1 subtype avian influenza virus as detecting antigen, combine with the colloidal gold immunochromatographimethod technology, have the following advantages: the antigen preparation method is simple, safety, output are high; Operating operation is simple, and specificity is good, susceptibility is high, diagnostic result is reliable; Be applicable to the scene detection that the H5 subtype avian influenza virus infects, especially suitable substrate unit or individual's field quick detection.
Description of drawings
Fig. 1 is the codon operating position table (critical value be 1%) of amino acid sequence in Escherichia coli of H5N1 subtype avian influenza virus HA gene code.Wherein, the full length sequence shown in the figure is a H5N1 subtype avian influenza virus HA gene ORFs amino acid sequence coded, and dash area is a HA antigen epitope genes amino acid sequence coded of the present invention; The Escherichia coli rare codon of frequency of utilization in the sequence≤1% shows with capitalization; The Escherichia coli rare codon of frequency of utilization≤0.28% shows with the capitalization of band frame.
Fig. 2 is the pcr amplification product electrophoretogram of ORFs (ORF) gene of H5 subtype avian influenza virus HA antigen epitope genes fragment and HA gene.Wherein, Swimming lane 1 contains the HA antigen epitope genes fragment of EcoR I restriction endonuclease recognition sequence for two ends; Swimming lane 2 is a molecular weight standard; Be followed successively by 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom, swimming lane 3 contains the HA gene ORFs fragment of EcoR I restriction endonuclease recognition sequence for two ends.
Fig. 3 is the SDS-PAGE electrophoretogram of H5 subtype avian influenza virus HA epitope recombinant expression protein.Wherein, swimming lane 1 is preparatory dsred protein MarkerIII, is followed successively by 94KD, 60KD, 45KD, 27KD and 18KD from top to bottom; Swimming lane 2 is not induced contrast for reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA, and swimming lane 3 is the inclusion body of reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA, and arrow indication place is destination protein.
Fig. 4 is the SDS-PAGE electrophoretogram of H5 subtype avian influenza virus HA gene ORFs recombinant expression protein.Wherein, swimming lane 1 is preparatory dsred protein MarkerIII, is followed successively by 94KD, 60KD, 45KD and 27KD from top to bottom; Swimming lane 2 is not induced contrast for reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF, and swimming lane 3 is the inclusion body of reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF, and arrow indication place is destination protein.
Fig. 5 is the Western-Blot analysis result of H5 subtype avian influenza virus HA epitope recombinant expression protein.Wherein, swimming lane 1 is preparatory dsred protein MarkerIII, is followed successively by 94KD, 60KD, 45KD, 27KD and 18KD from top to bottom; Swimming lane 2 is the reaction result of HA epitope recombinant expression protein and H5 subtype avian influenza virus positive serum; Swimming lane 3 is the negative chicken serum reaction result of HA epitope recombinant expression protein and SPF.
Fig. 6 is the side structure synoptic diagram of test strips of the present invention.Wherein, 1 is sample pad, and 2 is the collaurum pad, and 3 is nitrocellulose filter, and 4 is adsorptive pads, and 5 is base plate (PCV offset plate).
Fig. 7 is the vertical view of test strips of the present invention.Wherein, 1 is sample pad, and 2 is the collaurum pad, and 3 is nitrocellulose filter, and 4 is adsorptive pads, and 6 is detection line (T line), and 7 is nature controlling line (C line).
Fig. 8 goes up the vertical view behind the lower casing for test strips of the present invention assembling.Wherein, A is a sample well, and B is for detecting the hole, and T is a detection line, and C is a nature controlling line.
Fig. 9 is the testing result synoptic diagram of test strips of the present invention.Wherein, the positive result of A, the negative result of B, C and D are that the result is invalid; 6 is detection line (T line), and 7 is nature controlling line (C line).
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The source of the used serum of following embodiment is following:
H5 subtype avian influenza chicken positive serum:, be positive through the evaluation of blood clotting inhibition test method available from biotechnology development company of Harbin dimension section.
H7 subtype avian influenza chicken positive serum:, be positive through the evaluation of blood clotting inhibition test method available from biotechnology development company of Harbin dimension section.
H9 subtype avian influenza chicken positive serum:, be positive through the evaluation of blood clotting inhibition test method available from biotechnology development company of Harbin dimension section.
The positive chicken serum of infective rhinitis:, be positive through the evaluation of agar gel diffusion test method available from China Veterinery Drug Inspection Office.
The positive chicken serum of ewcastle disease: herd the development in science and technology center available from Beijing Kang Nongxing, identify through blood clotting inhibition test method and be positive.
The positive chicken serum of egg drop syndrome: herd the development in science and technology center available from Beijing Kang Nongxing, identify through the agar gel diffusion test method and be positive.
The positive chicken serum of infective bronchitis:, be positive through the evaluation of agar gel diffusion test method available from along vigorous biotechnology (Beijing) company limited.
The composition of the used solution of following embodiment is following:
Coating buffer: solvent is a water, and solute is Na
2CO
3And NaHCO
3, solute Na
2CO
3And NaHCO
3Concentration in coating buffer is respectively 1.59g/L and 2.93g/L, and pH value is 9.6.
The PBST damping fluid: solvent is a water, and solute is NaCl, KCl, KH
2PO
4, Na
2HPO
4And Tween-20, solute NaCl, KCl, KH
2PO
4And Na
2HPO
4Concentration in the PBST damping fluid is respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L, and the volumn concentration of Tween-20 in the PBST damping fluid is 0.05%, and pH value is 7.4.
The confining liquid first: solvent is the PBST damping fluid, and solute is a skim milk, and the concentration of solute skim milk in confining liquid is 50g/L.
The sample diluting liquid first: solvent is the PBST damping fluid, and solute is bovine serum albumin(BSA) (BSA), and the concentration of solute BSA in sample diluting liquid is 10g/L.
The TMB storage liquid: solvent is an absolute ethyl alcohol, and solute is tetramethyl benzidine (TMB), and the concentration of solute TMB in the TMB storage liquid is 2mg/mL, and 4 ℃ keep in Dark Place.
The tmb substrate dilution: solvent is a water, and solute is Na
2HPO
4And citric acid, solute Na
2HPO
4Be respectively 18.27g/L and 4.665g/L with the concentration of citric acid in the tmb substrate dilution, pH value is 5.5.
Substrate colour developing liquid: contain TMB storage liquid 0.5mL, tmb substrate dilution 10mL, H
2O
232 μ L.
Stop buffer: concentration is the aqueous sulfuric acid of 2M.
The preparation and the evaluation of embodiment 1, H5 subtype avian influenza virus reorganization HA albumen
1, the clone of H5 subtype avian influenza virus HA gene
Duck hepatic tissue to be accredited as H5N1 avian influenza virus A/Duck/GX/1/00 (H5N1) infection through normal experiment methods such as virus separation, hemagglutination-inhibition tests is the total RNA of sample extraction; Reverse transcription obtains cDNA; Be that primer carries out pcr amplification with 5 '-atggagaaaatagtgcttc-3 ' and 5 '-ttaaatgcaaattctgcactgt-3 ' again; Obtain the dna fragmentation that the size shown in the sequence table sequence 1 is 1704bp, this sequence is the ORFs (ORF) of H5N1 subtype avian influenza virus HA gene.
2, the rare codon analysis of H5 subtype avian influenza virus HA gene
Analyze rare codon occurrence number and the distribution situation in gene order of ORFs (its sequence is shown in sequence table sequence 1) and the HA antigen epitope genes (its sequence is shown in the 289th to the 1020th of sequence table sequence 1) of H5N1 subtype avian influenza virus HA gene.The result is shown in Fig. 1 and table 2.
Table 2.HA gene is in the rare codon analysis of expression in escherichia coli
Annotate: the e. coli codon frequency of utilization is low more, and foreign gene is beyond expression of words more.
The result shows; In 567 amino acid code of the ORFs amino acid sequence coded total length of H5N1 subtype avian influenza virus HA gene, frequency of utilization≤1% have 156, frequency of utilization≤0.28% have 23; Part rare codon close proximity, even adjacent appearance arranged side by side.Especially 3 frequencies of utilization that occur side by side in the 341st the-the 343rd position (Fig. 1) are merely 0.28% rare codon RRR; In 244 amino acid code of HA antigen epitope genes coding; One of frequency of utilization≤1% has 70; Frequency of utilization≤0.28% have 9, these rare codons distribute and compare with HA ORF gene codon, help the expression of destination protein in Escherichia coli relatively.Especially 3 frequencies of utilization having avoided (Fig. 1) on the 341st the-the 343rd position are merely 0.28% rare codon RRR, effectively reduce the probability of protein translation premature termination.
3, the acquisition of H5 subtype avian influenza virus HA epitope recombinant protein
1) construction of recombinant expression plasmid
Add the sequence " ctgatatcggatccgtc " (this sequence is the sequence of EcoR I restriction enzyme site one side of plasmid pET-32a (+)) of 17bp and the sequence " tgtcgacggagctcg " (this sequence is the sequence of the EcoR I restriction enzyme site opposite side of plasmid pET-32a (+)) of 17bp respectively at dna fragmentation two ends shown in the 289th to the 1020th of sequence table sequence 1; Obtain the dna fragmentation of 766bp, electrophoresis result is shown in the swimming lane 1 of Fig. 2; With EcoR I restriction enzyme with plasmid pET-32a (+) (Novagen company) linearization after; Dna fragmentation and linearizing plasmid pET-32a (+) (Novagen company) with this 766bp utilizes
recombinant clone kit (L00339 again; Nanjing Genscript Biotechnology Co., Ltd.) connects; Transform Rosetta-gami B (DE3) (Novagen company) again; Extract the positive colony plasmid; Through sequence verification; Obtained the recombinant expression plasmid of dna fragmentation shown in the 289th to the 1020th with the EcoR I site catenation sequence table sequence 1 at plasmid pET-32a (+) MCS place, with this recombinant expression plasmid called after pET-32a (+)-HA.The albumen that 244 amino acid shown in dna fragmentation code sequence shown in the 289th to the 1020th of the sequence table sequence 1 tabulation sequence 2 are formed (being the i.e. albumen formed of 97-340 amino acids of dash area among Fig. 1).
Add the sequence " ctgatatcggatccgtc " of 17bp and the sequence " tgtcgacggagctcg " of 17bp respectively at the dna fragmentation two ends of 1704bp shown in the sequence table sequence 1, the dna fragmentation electrophoresis result that obtains 1738bp is shown in the swimming lane 3 of Fig. 2; With EcoR I restriction enzyme with plasmid pET-32a (+) (Novagen company) linearization after; Dna fragmentation and linearizing plasmid pET-32a (+) (Novagen company) with this 1738bp utilizes
recombinant clone kit (L00339 again; Nanjing Genscript Biotechnology Co., Ltd.) connects; Transform Rosetta-gami B (DE3) (Novagen company) again; Extract the positive colony plasmid; Through sequence verification; Obtained recombinant expression plasmid, with this recombinant expression plasmid called after pET-32a (+)-HAORF with dna fragmentation shown in the EcoR I site catenation sequence table sequence 1 at plasmid pET-32a (+) MCS place.The albumen of forming by 567 amino acid that dna fragmentation shown in the sequence table sequence 1 coding is shown in Figure 1.
2) abduction delivering of recombinant protein
PET-32a (+)-HA is changed among the bacterial strain Rosetta-gami B (DE3) (Novagen company), obtain containing reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA of recombinant expression plasmid pET-32a (+)-HA.
PET-32a (+)-HAORF is changed among the bacterial strain Rosetta-gami B (DE3) (Novagen company), obtain containing reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF of recombinant expression plasmid pET-32a (+)-HAORF.
Should recombinate bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA and Rosetta-gami B (DE3) cultivates respectively as follows, collects bacterial sediment:
Be inoculated into 5mL and contain the LB fluid nutrient medium of 50 μ g/mL ampicillins, 37 ℃ of overnight incubation are inoculated into 500mL by 10% inoculum concentration again and contain in the LB fluid nutrient medium of 50 μ g/mL ampicillins; 37 ℃, 150 rev/mins joltings are cultured to exponential phase; With the OD600 light absorption value of ultraviolet spectrophotometer mensuration bacterium liquid, when light absorption value reached any one value between 0.5～0.8, adding final concentration was isopropyl-β-D-sulfo-galactopyranoside (IPTG) derivant of 1mmol/L; Continuation is 37 ℃, 150 rev/mins jolting overnight incubation; Obtain bacteria suspension, the centrifugal 10min of 2500 * g collects bacterial sediment.
3) purifying of recombinant protein
With step 2) reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA that obtains and the bacterial sediment of Rosetta-gami B (DE3)/pET-32a (+)-HAORF handle respectively as follows: (solvent is a water, and solute is the NaCl of 8g/L, the KCl of 0.2g/L, the Na of 1.44g/L with the PBS solution of 10mL
2HPO
4KH with 0.24g/L
2PO
4, using HCl to transfer pH is 7.4) and resuspended, add the lysozyme that final concentration is 100 μ g/mL (the green skies, Shanghai Bioisystech Co., Ltd) simultaneously, carry out ultrasonic treatment on ice, the centrifugal collection inclusion body deposition of 10000 * g.
Getting the inclusion body deposition of reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA and carry out the SDS-PAGE evaluation, is contrast with reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA that does not carry out the IPTG abduction delivering, and the result is as shown in Figure 3; Getting the inclusion body deposition of reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF and carry out the SDS-PAGE evaluation, is contrast with reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF that does not carry out the IPTG abduction delivering, and the result is as shown in Figure 4.
It is the fusion (called after HA epitope recombinant protein) of 48.1KD that the destination protein that bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA that recombinates among Fig. 3 is expressed and 6 histidine-tagged proteins of pET-32a (+) carrier form molecular weight; Further the gel scanning analysis shows that the destination protein of this 48.1KD accounts for 35% of bacterial protein; It is 84.7KD (called after HA recombinant protein) that the destination protein that bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF that recombinates among Fig. 4 is expressed and 6 histidine-tagged proteins of pET-32a (+) carrier form molecular weight; Further the gel scanning analysis shows; The destination protein of this 84.7KD accounts for 8% of bacterial protein, explains that the prokaryotic expression amount of HA epitope recombinant protein that the HA antigen epitope genes is carried out recombinant expressed acquisition is apparently higher than the HA recombinant protein that the HA full length gene is carried out recombinant expressed acquisition.
Utilize QIAexpress Ni-NTA Fast Sart (German QIAGEN company; Cat.306000) the 48.1KD albumen to 6 histidine marks in reorganization bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA inclusion body deposition carries out purifying, obtains HA epitope recombinant protein.
4) activity of recombinant protein detects
By conventional method, the lauryl sodium sulfate-polyacrylamide gel (SDS-PAGE) of preparation 12%, the HA epitope recombinant protein of getting 10 μ L step 3) purifying carries out electrophoresis on SDS-PAGE, carry out Western-Blot then and analyze, and the result is as shown in Figure 5.The result shows that HA epitope recombinant protein can the hybridization colour developing take place with H5 subtype avian influenza virus positive serum, the hybridization reaction band of 48.1kD occurs, and does not have any specific hybrid reaction zone with the negative chicken serum of SPF.
The specific detection of embodiment 2, H5 subtype avian influenza virus HA epitope recombinant protein
With in embodiment 1 step 2 3) the HA epitope recombinant protein of purifying is antigen; Adopt indirect ELISA method (indirect ELISA) that the following chicken serum of identifying through normal experiment methods such as hemagglutination-inhibition test, agar gel diffusion tests (serum to be checked) is detected: H5 subtype avian influenza chicken positive serum (positive control), SPF chicken serum (negative control), H7 subtype avian influenza chicken positive serum (serum 1 to be checked), H9 subtype avian influenza chicken positive serum (serum 2 to be checked), the positive chicken serum (serum 3 to be checked) of infective rhinitis, the positive chicken serum (serum 4 to be checked) of ewcastle disease, the positive chicken serum (serum 5 to be checked) of egg drop syndrome, the positive chicken serum (serum 6 to be checked) of infective bronchitis, concrete steps are following:
1, the dilution of antigen
Concentration with the HA epitope recombinant protein of BCA determination of protein concentration kit (the green skies, Shanghai Bioisystech Co., Ltd P0012) measures in embodiment 1 step 3 3) purifying is diluted to 15 μ g/ μ L with coating buffer with purifying protein, as envelope antigen.
2, antigen encapsulates
15 μ g/ μ L envelope antigens are joined elisa plate by the amount in 100 μ L/ holes.Behind 37 ℃ of effect 1h, 4 ℃ act on 2h again, and the PBST damping fluid is washed plate 3 times, the last drying.Add the confining liquid first sealing 40min in 100 μ L/ holes, the PBST damping fluid is washed plate 3 times, dries subsequent use.
3, the adding of serum to be checked
With 100 times of serum dilutions to be checked, the amount of pressing 100 μ L/ holes adds elisa plate with the sample diluting liquid first, 3 repetitions of each sample, and 37 ℃ of effect 40min, the PBST damping fluid is washed plate 3 times.
4, the adding of ELIAS secondary antibody
With the sample diluting liquid first with the goat-anti chicken IgG of horseradish peroxidase-labeled two anti-(KPL company, 14-24-06) amount by 100 μ L/ holes adds each reacting hole after 5000 times of the dilutions, 37 ℃ of effect 40min, the PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
5, colour developing
Amount by 100 μ L/ holes adds substrate colour developing liquid to each reacting hole, and 37 ℃ of effect 5min add 50 μ l stop buffers.
6, the result detects
ELISA Plate is placed ELIASA, measure the OD value of 450nm.Calculate positive decision content according to formula X+3STD, the negative control sample OD of the X in the formula
450Mean value, the negative control sample variance of STD.OD when serum to be checked
450During mean value>=positive decision content, judge that serum to be checked is H5 subtype avian influenza virus positive serum, the result is as shown in table 3.
Indirect ELISA testing result (the OD of table 3.H5 subtype avian influenza virus HA epitope recombinant protein
450)
Learn that according to last table calculating X is 0.0750, STD is 0.006, and positive decision content X+3STD is 0.093.
The result of table 3 shows that the specificity of HA epitope recombinant protein is fine, the antiserum generation specific binding reaction that can infect with the H5 subtype avian influenza virus.
The sensitivity of embodiment 3, H5 subtype avian influenza virus HA epitope recombinant protein detects
One, the sensitivity of H5 subtype avian influenza virus HA epitope recombinant protein
With in embodiment 1 step 3 3) the HA epitope recombinant protein of purifying is antigen; Method according to embodiment 2 steps 1 is diluted to 20 μ g/ μ L, 15 μ g/ μ L, 10 μ g/ μ L, 5 μ g/ μ L, 2 μ g/ μ L, 1 μ g/ μ L, 0.5 μ g/ μ L, 0.25 μ g/ μ L respectively with antigen; Antigen coated method by step 2 among the embodiment 2 encapsulates elisa plate, and each dilutability repeats 3 times.The H5 subtype avian influenza chicken positive serum (positive control) and the SPF chicken serum (negative control) that will pass through the evaluation of hemagglutination-inhibition test method dilute 100 times respectively with the sample diluting liquid first, press the sensitivity of embodiment 2 step 3-6 time-and-motion study envelope antigens.The result is as shown in table 4, and numerical value is the mean value of 3 repetitions in the table.
Sensitivity testing result (the OD of table 4.H5 subtype avian influenza virus HA epitope recombinant protein
450)
Annotate: sample OD
450Value is positive during greater than positive decision content, and is negative during less than positive decision content.
Table 4 is the result show, the H5 subtype avian influenza virus HA epitope recombinant protein of 0.25 μ g/ μ L can detect the positive serum of 100 times of dilutions.
Two, H5 subtype avian influenza virus HA epitope recombinant protein detects sero-fast sensitivity
To pass through the positive chicken serum (positive control) of hemagglutination-inhibition test method evaluation H5 subtype avian influenza and be accredited as negative SPF chicken serum (negative control) and carry out 50,100,200,300,400,500,600,700,800,900 and 1000 times of dilutions with the sample diluting liquid first respectively; With concentration is that the HA epitope recombinant protein of 15 μ g/ μ L is an envelope antigen; Method according to embodiment 2 detects respectively above-mentioned diluted feminine gender and positive; The sample of each concentration repeats 3 times, and the result is as shown in table 5.
The indirect ELISA sensitivity testing result (OD of table 5.H5 subtype avian influenza virus reorganization HA albumen
450)
The result of table 5 shows, is envelope antigen with the H5 subtype avian influenza virus HA epitope recombinant protein of 15 μ g/ μ L, and the indirect ELISA detection sensitivity of the chicken antiserum that the H5 subtype avian influenza virus is infected reaches 1: 800.
The accuracy rate of embodiment 4, H5 subtype avian influenza virus HA epitope recombinant protein detects
One, H5 subtype avian influenza virus HA epitope recombinant protein monitoring chicken crowd's accuracy rate
With in embodiment 1 step 3 3) the H5 subtype avian influenza virus HA epitope recombinant protein of purifying is antigen; Method according to following steps is monitored the chicken crowd; With the negative contrast of SPF chicken serum; With the positive contrast of H5 subtype avian influenza chicken positive serum, each sample repeats for 3 times, and the result representes with mean value:
1, the collection of blood serum sample to be checked
By conventional method, with the syringe of 2mL specification at random from the chicken crowd's to be checked chicken wings radicular vein 1mL that takes a blood sample, separation of serum, subsequent use in 4 ℃ of preservations.
2, the dilution of antigen
Method is with the step 1 of embodiment 2.
3, antigen encapsulates
Method is with the step 2 of embodiment 2.
4, the adding of serum to be checked
Method is with the step 3 of embodiment 2.
5, the adding of ELIAS secondary antibody
With the sample diluting liquid first with the goat-anti chicken IgG of horseradish peroxidase-labeled two anti-(KPL company, 14-24-06) amount by 100 μ L/ holes adds each reacting hole after 5000 times of the dilutions, 37 ℃ of effect 40min, the PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
6, colour developing
Method is with the step 5 of embodiment 2.
7, the result judges
Method is with the step 6 of embodiment 2.
The result: in 52 parts of chicken serum samples, being the positive sample of H5 subtype avian influenza virus has 47 parts, and being the negative sample of H5 subtype avian influenza virus has 5 parts, and testing result is shown in table 6 and table 7; With above-mentioned 52 parts of chicken serum samples is bird flu H5 antibody assay kit (the Avian Influenza H5 Antibody Test Kit of antigen in order to the H5 subtype avian influenza virus simultaneously; 06-40979-02; Idex ASA) detects; The result is the positive sample of H5 subtype avian influenza virus has 49 parts, and being the negative sample of H5 subtype avian influenza virus has 3 parts.
The result shows: be that antigen is compared with the H5 subtype avian influenza virus, be that the coincidence rate that antigen uses the ELISA method to detect the H5 subtype avian influenza virus antibody in the chicken serum is 96.15% with H5 subtype avian influenza virus HA epitope recombinant protein of the present invention.
Table 6.H5 subtype avian influenza virus HA epitope recombinant protein is to chicken crowd's ELISA testing result (OD
450)
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A | 0.095 | 0.120 | 0.179 | 0.142 | 0.217 | 0.127 | 0.165 |
| B | 0.098 | 0.122 | 0.342 | 0.213 | 0.188 | 0.171 | 0.274 |
| C | 0.221 | 0.177 | 0.257 | 0.282 | 0.301 | 0.097 | 0.189 |
| D | 0.246 | 0.277 | 0.126 | 0.159 | 0.199 | 0.143 | 0.150 |
| E | 0.149 | 0.197 | 0.124 | 0.257 | 0.176 | 0.122 | 0.239 |
| F | 0.164 | 0.112 | 0.200 | 0.219 | 0.083 | 0.274 | 0.304 |
| G | 0.219 | 0.122 | 0.092 | 0.300 | 0.252 | 0.079 | 0.111 |
| H | 0.237 | 0.119 | 0.108 | 0.158 | 0.130 | 0.112 | 0.097 |
Annotate: the negative results of comparison of A1 and B1, the positive results of comparison of C1 and D1, all the other are chicken serum sample result to be measured; Learn that according to negative control result calculating in the table X is 0.0965, STD is 0.002121, and positive decision content X+3STD is 0.1014, sample 0D
450Value is positive during greater than positive decision content 0.1014, and is negative during less than positive decision content 0.1014.
Table 7.H5 subtype avian influenza virus HA epitope recombinant protein is to chicken crowd's ELISA testing result (the moon/sun)
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| B | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| C | Positive control | Positive | Positive | Positive | Positive | Negative | Positive |
| D | Positive control | Positive | Positive | Positive | Positive | Positive | Positive |
| E | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
| F | Positive | Positive | Positive | Positive | Negative | Positive | Positive |
| G | Positive | Positive | Negative | Positive | Positive | Negative | Positive |
| H | Positive | Positive | Positive | Positive | Positive | Positive | Negative |
Two, H5 subtype avian influenza virus HA epitope recombinant protein monitoring duck crowd's accuracy rate
With in embodiment 1 step 3 3) the H5 subtype avian influenza virus HA epitope recombinant protein of purifying is antigen; Method according to following steps is monitored the duck crowd; Negative control is non-immune health duck serum; It is negative to prove the H5 subtype avian influenza through the blood clotting inhibition test, and positive control is H5N1 hypotype recombinant fowl influenza virus inactivated vaccine (H5N1Re_5 strain) (YEBIO Bioengineering Co., Ltd of Qingdao) immune duck serum, identifies through the blood clotting inhibition test and is positive; Each sample repeats for 3 times, and the result representes with mean value:
1, the collection of blood serum sample to be checked
By conventional method, with the syringe of 2mL specification from the duck crowd's to be measured duck wing radicular vein 1mL that takes a blood sample, separation of serum, subsequent use in 4 ℃ of preservations.
2, the dilution of antigen
Method is with the step 1 of embodiment 2.
3, antigen encapsulates
Method is with the step 2 of embodiment 2.
4, the adding of serum to be checked
Method is with the step 3 of embodiment 2.
5, the adding of ELIAS secondary antibody
With the sample diluting liquid first with the anti-duck IgG of the rabbit of horseradish peroxidase-labeled two anti-(KPL company, 04-25-06) amount by 100 μ L/ holes adds each reacting hole after 1000 times of the dilutions, 37 ℃ of effect 40min, the PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
6, colour developing
Method is with the step 5 of embodiment 2.
7, the result judges
Method is with the step 6 of embodiment 2.
Result: in 52 parts of duck blood serum samples, be 37 parts of the H5 subtype avian influenza virus positive; Be negative 15 parts of H5 subtype avian influenza virus, the result is shown in table 8 and table 9; Use avian influenza virus H5 hypotype hemagglutination-inhibition test antigen (biotechnology development company of Harbin dimension section) to carry out hemagglutination-inhibition test simultaneously above-mentioned duck blood serum sample; The result is 41 parts of the H5 subtype avian influenza virus positive, is 11 parts of H5 subtype avian influenza virus feminine gender.
The above results shows, compares with hemagglutination-inhibition test, and be that antigen detects the H5 subtype avian influenza virus antibody in the duck serum with the ELISA method with H5 subtype avian influenza virus reorganization HA albumen of the present invention, coincidence rate is 92.30%.
Table 8.H5 subtype avian influenza virus HA epitope recombinant protein is to duck crowd's ELISA testing result (OD
450)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| A | 0.614 | 0.097 | 0.095 | 0.095 | 0.094 | 0.088 | 0.315 |
| B | 0.673 | 0.435 | 0.376 | 0.394 | 0.278 | 0.263 | 0.298 |
| C | 0.081 | 0.106 | 0.073 | 0.089 | 0.571 | 0.182 | 0.071 |
| D | 0.075 | 0.132 | 0.144 | 0.160 | 0.114 | 0.207 | 0.175 |
| E | 0.123 | 0.064 | 0.059 | 0.125 | 0.047 | 0.254 | 0.085 |
| F | 0.081 | 0.071 | 0.173 | 0.086 | 0.093 | 0.071 | 0.106 |
| G | 0.154 | 0.300 | 0.297 | 0.341 | 0.281 | 0.214 | 0.059 |
| H | 0.193 | 0.070 | 0.259 | 0.055 | 0.166 | 0.154 | 0.258 |
Annotate: the positive results of comparison of A1 and B1, the negative results of comparison of C1 and D1 learns that according to this table calculating the X of negative control is 0.078, and STD is 0.0042426, and positive decision content X+3STD is 0.0907279; Sample OD
450Value is positive during greater than positive decision content 0.0907279, and is negative during less than positive decision content 0.0907279.
Table 9.H5 subtype avian influenza virus HA epitope recombinant protein is to duck crowd's ELISA testing result (the moon/sun)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| A | Positive control | Positive | Positive | Positive | Positive | Negative | Positive |
| B | Positive control | Positive | Positive | Positive | Positive | Positive | Positive |
| C | Negative control | Positive | Negative | Negative | Positive | Positive | Negative |
| D | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| E | Positive | Negative | Negative | Positive | Negative | Positive | Negative |
| F | Negative | Negative | Positive | Negative | Positive | Negative | Positive |
| G | Positive | Positive | Positive | Positive | Positive | Positive | Negative |
| H | Positive | Negative | Positive | Negative | Positive | Positive | Positive |
The HA epitope colloidal gold fast detecting test paper strip of embodiment 5, H5 subtype avian influenza virus antibody
One, the preparation of reagent
1, PBS encapsulates damping fluid: solvent is a ultrapure water, and solute is NaCl, KCl, KH
2PO
4And Na
2HPO
4, solute NaCl, KCl, KH
2PO
4And Na
2HPO
4Concentration be respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L, pH value is 7.2.
2, confining liquid second: solvent is that PBS encapsulates damping fluid, and solute is bovine serum albumin(BSA) (BSA), NaN
3, polyvinylpyrrolidone and sucrose, solute bovine serum albumin(BSA) (BSA), NaN
3, polyvinylpyrrolidone and concentration of sucrose be respectively 20g/L, 0.5g/L, 3g/L and 25g/L, 0.22 μ m filtering with microporous membrane, 4 ℃ of preservations are subsequent use.
3, the NaCl WS of sample diluting liquid second: 80g/L.
4,1% chlorauric acid solution: solvent is a ultrapure water, and solute is a gold chloride, and the concentration of solute gold chloride is 10g/L, and 4 ℃ of preservations are subsequent use.
5,1% citric acid three sodium solution: solvent is a ultrapure water, and solute is a trisodium citrate, and the concentration of solute trisodium citrate is 10g/L, and 0.22 μ M membrane filtration is joined existing usefulness at present.
6,0.1M solution of potassium carbonate: solvent is a ultrapure water, and solute is a sal tartari, and the concentration of solute sal tartari is 13.8g/L, 0.22 μ M membrane filtration, and 4 ℃ of preservations are subsequent use.
7,2%PEG-2000 solution: solvent is a ultrapure water, and solute is PEG-2000, and the concentration of solute PEG-2000 is 20g/L, 0.22 μ M membrane filtration, and 4 ℃ of preservations are subsequent use.
8, mark cleansing solution: solvent is that PBS encapsulates damping fluid, and solute is BSA and NaN
3, solute BSA and NaN
3Concentration be respectively 20g/L and 0.5g/L, 0.22 μ M membrane filtration, 4 ℃ of preservations are subsequent use.
9, the preparation of collaurum:
1% gold chloride that in the 999.9mL ultrapure water, adds 100 μ L; Magnetic agitation is heated to 100 ℃; Add the 13mL1% trisodium citrate, be heated to the liquid continued that takes on a red color and boil 7～10min, stop heating; Volume is supplied to 1000mL with ultrapure water in the cooling back, and gold ion aggregates into the colloid gold particle of 40nm.
10, the preparation of the anti-chicken igg antibody of colloid gold label:
With the 0.1M solution of potassium carbonate pH of collaurum is adjusted to 8.2, adds anti-chicken IgG, magnetic agitation 30min by the amount of 5～10 μ g antibody/mL collaurums; The adding final concentration is 1% BSA, leaves standstill 1h, 4 ℃ of centrifugal 20min of 10000 * g; Abandon supernatant; Deposition is with mark cleansing solution washing 2 times, and with the resuspended deposition of mark cleansing solution of 1/10th initial collaurum volumes, 4 ℃ of preservations are subsequent use.
Two, the preparation of test strips
1, the preparation of sample pad
With sample pad (BT50, thickness 0.52mm, water absorbing capacity 53mg/cm
2, Shanghai gold mark bio tech ltd) be soaked in the confining liquid second and take out behind the 30min, and place 37 ℃ of dry for standby.
2, the preparation of collaurum pad
With gold pad (SB08, thickness 0.33mm, water absorbing capacity 63.1mg/cm
2Shanghai gold mark bio tech ltd) is soaked in the confining liquid second and takes out behind the 30min; Placing 37 ℃ of oven dry, is even the spraying on gold mark pad of the anti-chicken IgG of rabbit (particle diameter is 30nm) of 5 μ g/mL gold mark with concentration with BIODOT XYZ specking system (XYZ3200).
3, the cellulose nitrate chromatographic film encapsulates
With BCA determination of protein concentration kit (the green skies, Shanghai Bioisystech Co., Ltd; P0012) measure in embodiment 1 step 3 3) concentration of the HA epitope recombinant protein solution of purifying; With PBS encapsulate damping fluid with this purifying protein solution dilution to 1.0mg/mL; The purifying protein solution that will dilute with BIODOT XYZ specking system (XYZ3200) the cellulose nitrate chromatographic film (NC-a101 Millipore 135, the aperture is 8 μ m, 2.5cm * 30cm; Backing is arranged, and Millipore) the front end line is as detection line (being designated as the T line) on test strips; Using BIODOT XYZ specking system (XYZ3200) simultaneously is the anti-rabbit igg antibody (ICP9801 of 5 μ g/mL with concentration; Nanning City's blue light bio tech ltd) line is as nature controlling line (being designated as the C line) on test strips in NC cellulose nitrate chromatographic film rear end, and the vertical range of nature controlling line and detection line is 4mm.
4, the assembling of test strips
Sample pad, collaurum pad, nitrocellulose filter and adsorptive pads (CH27, thickness 0.69mm, absorption speed 65.8s/4cm, water absorbing capacity 62.5mg/cm that step 1-3 is prepared
2Shanghai gold mark bio tech ltd) pressing Fig. 6 is connected with sequential cascade shown in Figure 7; Cutting into width is little of 4mm, and sticks on the PCV offset plate, and making detection line is 1mm with the vertical range of an end margin that is connected nitrocellulose filter of collaurum pad.Add upper and lower plastic box cover, be assembled into quick detection test paper bar easy to use, as shown in Figure 8.Only need during use from the sample well (12mm * 10mm) drip sample, that leans on detection line one end then through detecting the change color observations of hole detection line and nature controlling line.
Three, the specific detection of test strips
With H5 subtype avian influenza chicken positive serum (positive control), SPF chicken serum (negative control), H7 subtype avian influenza chicken positive serum, H9 subtype avian influenza chicken positive serum, the positive chicken serum of infective rhinitis, the positive chicken serum of ewcastle disease, the positive chicken serum of egg drop syndrome and the positive chicken serum of infectiousness property bronchitis is testing sample; Detect with the test strips of the step 2 preparation method according to following steps, each sample repeats for 3 times:
1, detection method
With 10 times of sample diluting liquid second dilutions, promptly 10 μ L testing samples add 90 μ L sample diluting liquid second, mix, and are added drop-wise in the sample well of test strips with testing sample, let alone freely to spread along test strips observations in the 5min.
2, the result judges
As shown in Figure 9, when two aubergine reaction zones appearred respectively in T line and C line position, the result was positive; At the reactionless band of T line position and when a mauve reaction zone appearred in the C line position, the result was negative;
A mauve reaction zone occurs at the T line position, and show reactionless the taking out of of C line position, perhaps show at T line and all reactionless the taking out of of C line position, then the result is invalid.
3, the testing result of testing sample
Through the detection of test strips, each repeats H5 subtype avian influenza chicken positive serum to occur two aubergine reaction zones simultaneously at T line and C line position, and the result is judged to be the positive; SPF chicken serum, H7 subtype avian influenza chicken positive serum, H9 subtype avian influenza chicken positive serum, the positive chicken serum of infective rhinitis, the positive chicken serum of ewcastle disease, the positive chicken serum of egg drop syndrome, the positive chicken serum of infectiousness property bronchitis respectively repeat only an aubergine reaction zone to occur at the C line position, and the result is judged to be feminine gender.
Four, the sensitivity of test strips detects
With sample diluting liquid second H5 subtype avian influenza chicken positive serum is diluted 10 times, 20 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times respectively; Detect with the test strips of the step 2 preparation method according to following steps, each dilute concentration repeats 3 times:
1, detection method
Method is with 1 in embodiment 5 step 3.
2, the result judges
Method is with 2 in embodiment 5 step 3.
3, the testing result of testing sample
The result: the H5 subtype avian influenza chicken positive serum of sample diluting liquid second dilution, its 10 times, 20 times, 50 times, 100 times, 200 times, 300 times each repetition of serum dilution two aubergine reaction zones occur simultaneously at T line and C line position, and the result is judged to be the positive; Each repeats 400 times, 500 times serum dilute sample only an aubergine reaction zone to occur at the C line position, and the result is judged to be feminine gender.The sensitivity of this test strips of presentation of results is 1: 300.
The HA epitope colloidal gold fast detecting test paper strip of embodiment 6, H5 subtype avian influenza virus antibody
One, the preparation of reagent
Identical with the step 1 among the embodiment 5.
Two, the preparation of test strips
1, the preparation of sample pad
With sample pad (BT50, thickness 0.52mm, water absorbing capacity 53mg/cm
2, Shanghai gold mark bio tech ltd) be soaked in the confining liquid second and take out behind the 30min, and place 37 ℃ of dry for standby.
2, the preparation of collaurum pad
With gold pad (SB08, thickness 0.33mm, water absorbing capacity 63.1mg/cm
2Shanghai gold mark bio tech ltd) is soaked in the confining liquid second and takes out behind the 30min; Placing 37 ℃ of oven dry, is even the spraying on gold mark pad of the anti-chicken IgG of rabbit (particle diameter is 30nm) of 10 μ g/mL gold mark with concentration with BIODOT XYZ specking system (XYZ3200).
3, the cellulose nitrate chromatographic film encapsulates
With BCA determination of protein concentration kit (the green skies, Shanghai Bioisystech Co., Ltd; P0012) measure in embodiment 1 step 3 3) concentration of the HA epitope recombinant protein solution of purifying; With PBS encapsulate damping fluid with this purifying protein solution dilution to 1.5mg/mL; The purifying protein solution that will dilute with BIODOT XYZ specking system (XYZ3200) the cellulose nitrate chromatographic film (NC-a101 Millipore 135, the aperture is 8 μ m, 2.5cm * 30em; Backing is arranged, and Millipore) the front end line is as detection line (being designated as the T line) on test strips; Using BIODOT XYZ specking system (XYZ3200) simultaneously is the anti-rabbit igg antibody (ICP9801 of 10 μ g/mL with concentration; Nanning City's blue light bio tech ltd) line is as nature controlling line (being designated as the C line) on test strips in NC cellulose nitrate chromatographic film rear end, and the vertical range of nature controlling line and detection line is 7mm.
4, the assembling of test strips
Sample pad, collaurum pad, nitrocellulose filter and adsorptive pads (CH27, thickness 0.69mm, absorption speed 65.8s/4cm, water absorbing capacity 62.5mg/cm that step 1-3 is prepared
2Shanghai gold mark bio tech ltd) pressing Fig. 6 is connected with sequential cascade shown in Figure 7; Cutting into width is little of 4mm, and sticks on the PCV offset plate, and making detection line is 0.5mm with the vertical range of an end margin that is connected nitrocellulose filter of collaurum pad.Add upper and lower plastic box cover, be assembled into quick detection test paper bar easy to use, as shown in Figure 8.Only need during use from the sample well (12mm * 10mm) drip sample, that leans on detection line one end then through detecting the change color observations of hole detection line and nature controlling line.
Three, the specific detection of test strips
With H5 subtype avian influenza chicken positive serum (positive control), SPF chicken serum (negative control), H7 subtype avian influenza chicken positive serum, H9 subtype avian influenza chicken positive serum, the positive chicken serum of infective rhinitis, the positive chicken serum of ewcastle disease, the positive chicken serum of egg drop syndrome and the positive chicken serum of infectiousness property bronchitis is testing sample; Detect with the test strips of the step 2 preparation method according to following steps, each sample repeats for 3 times:
1, detection method
1 identical with step 3 among the embodiment 5.
2, the result judges
2 identical with step 3 among the embodiment 5.
3, the testing result of testing sample
3 identical with step 3 among the embodiment 5.
Four, the sensitivity of test strips detects
With sample diluting liquid second H5 subtype avian influenza chicken positive serum is diluted 10 times, 20 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times respectively, three method detects set by step, and each dilute concentration repeats 3 times.
1, detection method
Method is with 1 in embodiment 5 step 3.
2, the result judges
Method is with 2 in embodiment 5 step 3.
3, the testing result of testing sample
3 identical with in the step 4 among the embodiment 5.
The application of the HA epitope colloidal gold fast detecting test paper strip of embodiment 7, H5 subtype avian influenza virus antibody
By conventional method, to 50 the 5 monthly age commercial chickens of from live-bird market, inspecting by random samples at random, vein is gathered the 0.5mL whole blood and is prepared serum; Getting 10-20 μ L serum splashes in the sample well of the test strips that step 2 prepares among the embodiment 5; Add 50-100 μ L sample diluting liquid second again, behind the 1min from detecting the hole observations, with the positive contrast of H5 subtype avian influenza chicken positive serum; 3 repetitions are done in the negative contrast of SPF chicken serum, each sample to be checked.If T line and C line all demonstrate the aubergine reaction zone, chicken then to be detected is positive for the H5 subtype avian influenza infects; If have only the C line to demonstrate the aubergine reaction zone, the T line does not occur, and chicken then to be detected is negative for the H5 subtype avian influenza infects.
Result: have 44 detection chickens respectively to repeat to be the H5 subtype avian influenza and infect positive; 6 are detected chicken and respectively repeat to be H5 subtype avian influenza infection feminine gender; Positive control H5 subtype avian influenza chicken positive serum is positive for the H5 subtype avian influenza infects; Negative control SPF chicken serum is negative for the H5 subtype avian influenza infects, and this result is identical with normal experiment method qualification results such as hemagglutination-inhibition test, ELISA.
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