CN105353125A - A portable rapid test strip for syphilis for household use - Google Patents
A portable rapid test strip for syphilis for household use Download PDFInfo
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- CN105353125A CN105353125A CN201510779735.3A CN201510779735A CN105353125A CN 105353125 A CN105353125 A CN 105353125A CN 201510779735 A CN201510779735 A CN 201510779735A CN 105353125 A CN105353125 A CN 105353125A
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- syphilis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
A portable rapid test strip for syphilis for household use is disclosed. Syphilis antigens TP17 and TP47 are adopted to construct an escherichia coli expression vector. Through codon optimization, carrying of purification tags, construction of the syphilis antigen expression vector and addition of a molecular chaperone signal peptide having a molecular weight of 12 KD, a target gene can be expressed efficiently in vitro and the exogenous target gene in a form of a soluble protein is secreted into periplasm, and therefore subsequent purification of target antigens is convenient. Through codon preference of the escherichia coli, the two antigen genes of the syphilis are optimized and artificially synthesized, efficient expression of the syphilis antigens in the escherichia coli can be achieved, and soluble expression can be achieved through controlling expression conditions, thus avoiding tedious and inefficient denaturation and renaturation processes of inclusion bodies. In terms of clone strategies, the C ends of the antigens carry the purification tags, and therefore product purification steps are simplified. The test strip is rapid, simple, economical, easily popularized, easily available in materials, and not restricted by experiment conditions.
Description
Technical field
The invention belongs to medical science, particularly relate to a kind of portable family syphilis Rapid detection test strip.
Background technology
At present, in vitro culture syphilopathy substance does not still realize.Therefore, with laboratory, the diagnosis of syphilis is mainly concentrated on to the detection of Serological Characterization clinically.Although also can by directly determining that pathogen is diagnosed, but existing technology mainly utilizes dark-field microscope directly to observe sample, such operation has stronger subjectivity, even if sophisticated tester, also may occur false negative result; PCR method also directly can measure the existence of pathogen, but the somewhat expensive of the method, not easily as the conventional sense of blood station and hospital.As can be seen here, syphilitic's Serologic detection is most important in controller used in syphilis diagnosis.Serodiagnosis comprises special and non-specific two kinds of methods, distinct therebetween.Non-specific controller used in syphilis diagnosis method mainly contains the tests such as VDRL, USR, RPR and TRUST, they have identical antigenic component, i.e. a kind of Venereal Disease Research Laboratory antigen (VDRL) found by U.S. Pangborn etc., the heart extracted from bovine cardiac intends fat, add cholesterol and lecithin in right amount to improve susceptibility, this antigen of usual title is that the heart intends fat antigen, can efficient diagnosis neurolues.But the impact that the specificity of these class methods is reacted by biological false positive reaction and some physiological, namely the sensitivity of the method depends on the stage residing for the course of disease.Therefore, non-specific controller used in syphilis diagnosis method can only be limited to routine screening.Specificity syphilis antibody detection method has FTA-ABS method (FluorescentTreponemalAntibodyAbsorption, syphilis fluorescence antibody absorption method), TPHA method (TreponemaPallidumHemagglutinationAssay), TPPA method (TreponemaPallidumParticleAgglutinationAssay),
syphilisG/SyphilisM method etc.Specificity method is usually used in the confirmation of non-specific method positive sample, and TPHA/TPPA method is also usually used in the confirmation of primary dcreening operation result.The process of the anti-syphilis antibody of specificity and disease does not have correlativity, even if can carry all the life through treatment yet.In specificity syphilis antibody detection method, FTA-ABS has hypersensitivity, especially for early syphilis, but a certain proportion of false positive results (0.35%) can occur when HIV, autoimmune disease and pregnancy.FTA-ABS method is all responsive to the syphilis in each stage, but evaluates and have subjectivity, sometimes more difficult and complicated, is not suitable for preliminary examination, is only applicable to confirmation.Other a little species specificity syphilis antibody detection methods also have same problem, because traditional controller used in syphilis diagnosis Measures compare is loaded down with trivial details, consuming time longer, not easily promote.
Application number be 02137626.3 patent discloses a kind of syphilis spirochete antibody labeled diagnosis reagent for Diagnosis of Syphilis spirochaete infection.It comprises the antigen with antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 0.1 ~ 10 μ g/ml is coated in ELISA Plate, ELIAS secondary antibody (IgG, IgM): combine and solubilizer containing 1: 20 ~ 1: 100K ELIAS secondary antibody dilute solution and developer, and the developer in described developer combination is TMB propanesulfonate.Developer combination can comprise the combination of TMB propanesulfonate and hydrogen phosphide cumene or derivatives thereof or the combination of TMB propanesulfonate and hydroxycarbamide.This syphilis spirochete antibody labeled diagnosis reagent, on the antigen basis of application antigenic synthetic peptide, gene engineering expression, employ a kind of TMB salt of water-soluble fabulous, stable performance, coordinate appropriate solubilizer, stabilizing agent, make syphilis spirochete antibody labeled diagnosis reagent obtain better stability.Its high specific, high sensitivity, easy and simple to handle, stable reagent, make the robotization of Lues Assay energy, the batch screening of blood source, standardized management be guaranteed.
The shortcoming of said method is main is that cardinal principle detects sample with enzyme linked immunoassay, needs microplate reader, hatches the large-scale instruments such as incubator, is not suitable for family and uses.And complicated operation, consuming time longer, be unfavorable for that primary dcreening operation detects fast.
Application number be 201010215044.8 patent discloses a kind of microspironema pallidum based on flow microsphere carrier technique (TP) antibody test kit and preparation and determination methods method thereof, belong to immunoassay medical diagnostic techniqu field.Specifically comprise and use TP recombinant antigen bag by high dimeric molecule microballoon, close blank binding site with bovine serum albumin(BSA), make specificity T P probe-Gao dimeric molecule microballoon; Catch TP antibody with sample Dual culture to be measured, wash the unconjugated TP antibody of centrifugal removing, then add fluorescently-labeled anti-human igg or IgM antibody; Use the fluorescence intensity of flow cytomery microballoon, qualitative or quantitative test is carried out to tested antibodies.This method has highly sensitive, high specificity, good stability advantage, and can carry out trace, multivalence analysis to sample.
The shortcoming of said method needs to use flow cytometer this expensive large-scale instrument, and this instrument is less in the configuration of blood station and basic hospital, and the maintenance cost of this instrument is higher, inapplicable popularization, and cannot carry out family and use and detect.
Summary of the invention
The object of the present invention is to provide a kind of portable family syphilis Rapid detection test strip, be intended to solve traditional controller used in syphilis diagnosis Measures compare loaded down with trivial details, consuming time longer, costly, the problem not easily promoted.
The present invention realizes like this, a kind of portable family syphilis Rapid detection test strip comprises sample pad, pad, nitrocellulose filter and absorption layer, Treponema pallidum antigen is fixed on nitrocellulose filter as capture antigen, and colloid gold label reagent is adsorbed on pad.
Further, build coli expression carrier with Treponema pallidum antigen TP17 and TP47, expression, the Purification and Characterization method of Treponema pallidum antigen are:
Utilize the inclined preferendum engineer of e. coli codon and synthetic gene, BamHI and NotI site is contained at the gene two ends of synthesis, and expression vector is pETDuet-1, and this expression vector contains T7 promoter, under the induction of IPTG, foreign gene is transcriptional expression under the effect of t7 rna polymerase;
The C end of described expression vector is containing HisTag sequence, and the foreign protein of expression is purified by metal chelate affinity chromatography method, when purpose of design gene, inserts the molecular chaperones peptide chain of 12KD size, is secreted in periplasmic by the foreign protein of expression;
The foreign gene of synthesis is cut link by enzyme to be inserted in carrier pETDuet-1, in transformation of E. coli BL21, by IPTG abduction delivering, antigen is expressed in bacteria periplasm with soluble form, collect bacterium, ultrasonication, supernatant is by metal-chelating and sieve chromatography, and the albumen of purifying is by SDS-PAGE, Western-blot qualification.
Further, the preparation method of described portable family syphilis Rapid detection test strip comprises:
The design of step one, TP genes of interest and clone;
By US National Biotechnology Information center website, search the whole genome sequence of microspironema pallidum TP-17 and TP-47;
CDNA sequence in synthesis genes of interest open reading frame, and synthetic sequence clone is entered pMD18-TSimple carrier, when carrying out sequent synthesis, respectively at 5 ' end of aim sequence and the restriction enzyme site of 3 ' end insertion BamHI and NotI, and utilize these two kinds of restriction enzymes to be sheared from pMD18-TSimple carrier by genes of interest, carry out agarose gel electrophoresis qualification, reclaim electrophoretic band;
After the object band reclaimed is passed through dephosphorylation ferment treatment, with the pETDuet-1 carrier through same restriction enzyme and dephosphorylation ferment treatment under T4 ligase existent condition, 16 DEG C of connections are spent the night; The carrier connected electricity is forwarded in the competent cell of Escherichia coli DH10B bacterial classification, coats containing in 50 μ g/ml ammonia benzyl antibiotic LB solid culture plate, 37 DEG C of overnight incubation; Within second day, picking monoclonal is inoculated into containing in the antibiotic 5mlLB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm, concussion overnight incubation; Within 3rd day, utilize little plasmid extraction kit of taking out to extract plasmid in bacterium liquid, carry out BamHI and NotI double digestion and to go forward side by side row agarose gel electrophoresis qualification; The positive plasmid electricity with object band is forwarded in the competent cell of e. coli bl21 bacterial classification, repeat above operation, coated plate, choose after clone carries out Liquid Culture, identified by BamHI and NotI double digestion and agarose gel electrophoresis, the plasmid containing object band or bacterium liquid are checked order;
The expression and purification of step 2, destination protein;
The bacterium liquid of the plasmid containing correct genes of interest TP17 and TP47 sequence being seeded to respectively 50ml contains in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, in 250rpm, and 37 DEG C of overnight incubation; Subsequently the bacterium liquid of two bottles of 50ml incubated overnight is inoculated into 1L to contain in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm shakes cultivation;
As the A of bacterium liquid
600when OD value is 0.2 ~ 0.4, incubator temperature is adjusted to 22 DEG C, still 250rpm shakes cultivation 2.5 ~ 3h; As the A of bacterium liquid
600when OD value is 0.9 ~ 1.0, adding IPTG derivant to final concentration is 1mmol/L, and incubator temperature is adjusted to 18 DEG C, and 250rpm shakes overnight incubation;
Within second day, collect bacterium liquid, 4000g, centrifugal 20min, collect bacterial sediment, weigh, record thalline weight;
Then add according to 1g thalline weight the resuspended bacterium of phosphate buffer that 5ml concentration is 0.1M;
Be placed on ice by the bacterium liquid after resuspended, carry out ultrasonication, ultrasonic process 10s, suspend 20s, after effect 10min, in 12000g, centrifugal 25min, collects supernatant; The supernatant collected is continued at 12000g, and centrifugal 25min, regathers supernatant; Again repeat aforesaid operations, then by the supernatant the collected filter membrane by 0.45 μm, detect the protein concentration in solution with BCA kit, record result;
Select the NiSepharose of GE company
tMthe pillar of 6FastFlow filler and XK16/20, carry out affinitive layer purification, after dress post, the 0.1M sodium phosphate buffer of 5 times of column volumes is utilized to balance, when the value of A280 is tending towards straight line, zeroing, by above-mentioned slightly carry after bacterial supernatant solution upper prop, after completion of the sample, first wash affinity column with the 0.1M sodium phosphate buffer of 2 times of column volumes; When the destination protein of purifying is TP-17, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 25mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 150mM imidazoles; When the destination protein of purifying is TP-47, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 30mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 100mM imidazoles;
Collect the destination protein of wash-out, measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content, other foreign proteins removed by the molecular sieve being S200 by filler, measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content;
The preparation of step 3, collaurum;
According to trisodium citrate reduction method, colloid gold particle is fired into the gold grain that diameter is about 40nm, detailed process is as follows: joined by the ultrapure water of 100ml after 0.22 μm of filtering with microporous membrane in the 200ml triangular flask after silicidation, put into clean rotor, be placed in sealing on magnetic stirring apparatus and be heated to liquid boiling.Then, when liquid does not splash, rotor speed is adjusted to maximum;
Add 1% chlorauric acid solution in this case, add the citric acid three sodium solution of 0.85mL1% subsequently fast, continue to add thermal agitation, treat that solution colour is transformed into claret gradually by colourless, black, purple, after continuing to add thermal agitation 10min, stop heating;
Cool under room temperature, 4 DEG C of sealings save backup, and utilize colloid gold particle size, shape, uniformity coefficient and dispersion degree prepared by transmission electron microscope observing after 24h, use ultraviolet spectrophotometer to measure absorption peak and the OD value of collaurum;
The mark of step 4, albumin A;
The operating process be combined with albumin A by colloid gold particle is as follows: first get the vial and each one of lesser trochanter handled well, be placed on magnetic stirring apparatus, the colloidal gold solution baked by 10ml adds wherein, uses K
2cO
3solution ph is adjusted to 8.5, stirs; Then the ratio that the colloidal gold solution baked with every 500 μ L joins 6 μ g albumin As adds albumin A, stirs 20min;
Add the bovine serum albumin(BSA) V solution that concentration is 10% subsequently, make final concentration be 1%, mix after stirring 20min;
Last with the centrifugal 15min of 9000rpm/min, suck supernatant, adding final concentration is the sucrose of 10% and the abundant dissolution precipitation of aqueous trehalose of 2.5%, the solution for standby of formation;
The assembling of step 5, test strips and drying;
First be prepare nitrocellulose filter, get nitrocellulose filter one, cut into the rectangular of 2.5cm × 5cm, 1:1 is by antigen TP-17 and TP-47 mixed in equal amounts, then be respectively the mixed solution of 1mg/mL to final concentration by two kinds of antigen diluents with C/T dilution, and with C/T dilution, sheep anti-mouse igg be diluted to 2mg/mL;
Draw in two shower nozzles of film instrument by the antigen diluted and the two anti-metal sprayings that add respectively, detection line and nature controlling line discharge rate are all set to 1 μ L/cm;
After being fixed the position of nitrocellulose filter, carry out spray film;
After the nitrocellulose filter sprayed being placed in 37 DEG C of oven dryings, preserve in 4 DEG C of sealings;
The manufacturing process of gold mark pad is as follows:
First getting glass fibre element film accuflowG, be cut to the rectangular of 2cm × 5cm, by the amount of 6 μ L/cm, use metal spraying to draw film instrument metal spraying, then by having sprayed after the element of the glass fibre after gold film is placed in 37 DEG C of oven dryings, preserving in 4 DEG C of sealings;
Prepare sample pad:
First one whole glass fibre element film is cut into the rectangular of 1.5cm × 5cm, then use 15mM, the PBS of pH7.4 fully soaks, and after being finally placed in 37 DEG C of oven dryings, preserves in 4 DEG C of sealings;
Finally nitrocellulose filter, gold mark pad and sample pad are assembled, make colloidal gold strip;
Assembling process is as follows: when temperature is 22 ~ 24 DEG C, during humidity <40%, assemble test strips; First the diaphragm in the middle part of PCV plate is taken off, the nitrocellulose filter prepared is affixed in the middle part of plate, make its nature controlling line near adsorptive pads one end; Then be affixed on PCV plate by slightly overlapping to pad edge and nitrocellulose filter detection line one end; Sample pad and gold being marked to pad successively subsequently is slightly overlappingly affixed on PCV plate; Finally adsorptive pads is affixed on another end of PCV plate, then the test strips assembled is cut into wide rectangular of 0.4cm with test strips cutting machine, preserve in 4 DEG C of sealings;
Step 6, detection and result judge;
According to antigen-antibody immune response, the syphilis polyclonal antibody in 20 μ L rabbit sources is joined in 80 μ L dilutions, drips after mixing in test strips sample pad; Solution, will along test strips to the swimming of adsorptive pads direction under the draw of test strips adsorptive pads.When the antibody-solutions after diluting arrives gold-marking binding pad, syphilis antibody can be combined by the gold mark protein A complexes on gold-marking binding pad, forms the compound of golden mark-albumin A-syphilis antibody and swimming forward together; When solution arrives detection line T, the hybrid antigen being fixed on TP17 and TP47 on detection line T can catch the gold mark-albumin A-syphilis antibody compound still in solution with antigen binding site, after the Treponema pallidum antigen of mixing is combined with the compound of GOLD FROM PLATING SOLUTION mark-albumin A-syphilis antibody, the redness of collaurum can be shown.Syphilis antibody all has the characteristic of reacting with Treponema pallidum antigen, and therefore this patent adopts indirect method to detect the syphilis antibody in measuring samples; In sample, syphilis antibody is more, and the antigen so on detection line T is combined will be more, and namely on detection line T, the interception of gold mark-albumin A-syphilis antibody is more, manifests redness darker; Otherwise syphilis antibody is fewer in sample, on detection line T, the interception of gold mark-albumin A-syphilis antibody will be fewer, and namely detection line T can show light red or not develop the color; But no matter in sample, syphilis antibody is mostly few, and excessive gold mark-protein A complexes all can be combined and aobvious redness by the sheep anti-mouse igg on nature controlling line C.If nature controlling line C does not develop the color, namely judge that this result is invalid;
The sensitivity of step 7, colloidal gold strip and the detection of blood sample;
Commercialization is diluted to for the rabbit source polyclonal antibody that the initial concentration of microspironema pallidum is 100 μ g/ml the antibody standard solution that concentration is 0,0.1,0.2,0.4,0.8,1.6,3.2,6.4 μ g/ml;
Then respectively get 20 μ l antibody standard solution and add 80 μ l dilutions, the component proportion of this dilution is: the PBS of 15mM, PH7.4,100ml; Bovine serum albumin(BSA) V, 1g; Tween-20,0.5ml; Sodium azide, 0.01g; Sucrose, 1g; Drip after mixing in test strips well;
After room temperature places 10min, observe the colour developing situation of ELISA test strip line T;
Nature controlling line C should unify aobvious red, if without colour developing, namely this result is invalid.
Effective result of the present invention:
The present invention builds coli expression carrier with Treponema pallidum antigen TP17 and TP47, by the optimization of codon, and carrying of purification tag, and the control of expression condition can realize the solution expression with high efficiency of Treponema pallidum antigen;
In the structure of Treponema pallidum antigen expression vector, add the molecular chaperones signal peptide of 12KD, can external efficient expression genes of interest, and make external source genes of interest be secreted in periplasmic with soluble protein, avoid the sex change renaturation process of inclusion body complexity, simplify the purge process of Treponema pallidum antigen;
Two antigens are wrapped simultaneously are used to detect, optimize ratio between the two simultaneously by simultaneously expression and purification two Treponema pallidum antigen, can improve the Sensitivity and Specificity of Lues Assay in each in period;
The dot immune gold filtration assay adopted, fast and convenient, accurate, have high degree of specificity and hypersensitivity, visual result is reliable, consuming time short, only within ten minutes, can result be seen, the high expression of Treponema pallidum antigen in Escherichia coli, and higher productive rate and the recovery, greatly reduce the cost of detection kit, and reagent and amount of samples are few, without the need to instrument and equipment, simplify loaded down with trivial details normal operation.
Accompanying drawing explanation
Fig. 1 is preparation method's process flow diagram of the portable household formula syphilis Rapid detection test strip that the embodiment of the present invention provides;
Fig. 2 is the plasmid pETDuet-TP17 that provides of the embodiment of the present invention and pETDuet-TP47 double digestion agarose gel electrophoresis figure;
Fig. 3 is the SDS-PAGE electrophoretogram of TP17 and the TP47 recombinant protein of the IPTG induction Bacillus coli expression that the embodiment of the present invention provides;
Fig. 4 is the SDS-PAGE electrophoretogram of the purifying different phase destination protein that the embodiment of the present invention provides.
Embodiment
For summary of the invention of the present invention, Characteristic can be understood further, hereby exemplify following examples, and coordinate accompanying drawing to be described in detail as follows.
As shown in Figure 1, the present invention realizes like this, a kind of portable household formula syphilis Rapid detection test strip comprises sample pad, pad, nitrocellulose filter and absorption layer, and Treponema pallidum antigen is fixed on nitrocellulose filter as capture antigen, and colloid gold label reagent is adsorbed on pad.
Further, build coli expression carrier with Treponema pallidum antigen TP17 and TP47, expression, the Purification and Characterization method of Treponema pallidum antigen are:
Utilize the inclined preferendum engineer of e. coli codon and synthetic gene, BamHI and NotI site is contained at the gene two ends of synthesis, and expression vector is pETDuet-1, and this expression vector contains T7 promoter, under the induction of IPTG, foreign gene is transcriptional expression under the effect of t7 rna polymerase;
The C end of described expression vector is containing HisTag sequence, and the foreign protein of expression is purified by metal chelate affinity chromatography method, when purpose of design gene, inserts the molecular chaperones peptide chain of 12KD size, is secreted in periplasmic by the foreign protein of expression;
The foreign gene of synthesis is cut link by enzyme to be inserted in carrier pETDuet-1, in transformation of E. coli BL21, by IPTG abduction delivering, antigen is expressed in bacteria periplasm with soluble form, collect bacterium, ultrasonication, supernatant is by metal-chelating and sieve chromatography, and the albumen of purifying is by SDS-PAGE, Western-blot qualification.
Further, the preparation method of described portable household formula syphilis Rapid detection test strip comprises:
The design of S101, TP genes of interest and clone;
The expression and purification of S102, destination protein;
The preparation of S103, collaurum;
The mark of S104, collaurum albumin A;
The assembling of S105, test strips and drying;
S106, detection and result judge;
The sensitivity of S107, colloidal gold strip and the detection of blood sample.
Concrete steps are:
The design of S101, TP genes of interest and clone;
By the website at US National Biotechnology Information center (NCBI), search the whole genome sequence of microspironema pallidum TP-17 and TP-47.Entrust the cDNA sequence in Shanghai Invitrogen Corp. synthesis genes of interest open reading frame, and synthetic sequence clone is entered pMD18-TSimple carrier.When carrying out sequent synthesis, respectively at 5 ' end of aim sequence and the restriction enzyme site of 3 ' end insertion BamHI and NotI, and utilizing these two kinds of restriction enzymes to be sheared from pMD18-TSimple carrier by genes of interest, carrying out agarose gel electrophoresis qualification.The electrophoretic band of the genes of interest identical with expection size is reclaimed according to the operation steps of glue recovery kit.After the object band reclaimed is passed through dephosphorylation ferment treatment, with the pETDuet-1 carrier through same restriction enzyme and dephosphorylation ferment treatment under T4 ligase existent condition, 16 DEG C of connections are spent the night.The carrier connected electricity is forwarded in the competent cell of Escherichia coli DH10B bacterial classification, coats containing in 50 μ g/ml ammonia benzyl antibiotic LB solid culture plate, 37 DEG C of overnight incubation.Within second day, picking monoclonal is inoculated into containing in the antibiotic 5mlLB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm, concussion overnight incubation.Within 3rd day, utilize little plasmid extraction kit of taking out to extract plasmid in bacterium liquid, carry out BamHI and NotI double digestion and to go forward side by side row agarose gel electrophoresis qualification.The positive plasmid electricity with object band is forwarded in the competent cell of e. coli bl21 bacterial classification, repeats above operation, coated plate, choose after clone carries out Liquid Culture, still identified by BamHI and NotI double digestion and agarose gel electrophoresis.Plasmid containing object band or Jun Yesong Huada Gene Research Center, Beijing are checked order, sequencing result is consistent with the sequence of expection genes of interest.The double digestion of TP-17 and TP-47 prokaryotic expression carrier identifies electrophoretogram as shown in Figure 2, in Fig. 2, and M:DNAMarker; The double digestion result of 1: plasmid pETDuet-TP17; The double digestion result of 2: plasmid pETDuet-TP47.
The expression and purification of S102, destination protein;
The bacterium liquid of the plasmid containing correct genes of interest TP17 and TP47 sequence being seeded to respectively 50ml contains in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, in 250rpm, and 37 DEG C of overnight incubation.Subsequently the bacterium liquid of two bottles of 50ml incubated overnight is inoculated into 1L to contain in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm shakes cultivation.As the A of bacterium liquid
600(about cultivate about 1h) when OD value is 0.2 ~ 0.4, incubator temperature is adjusted to 22 DEG C, still 250rpm shakes cultivation 2.5 ~ 3h.As the A of bacterium liquid
600when OD value is 0.9 ~ 1.0, adding IPTG derivant to final concentration is 1mmol/L, and incubator temperature is adjusted to 18 DEG C, and 250rpm shakes overnight incubation.Within second day, collect bacterium liquid, 4000g, centrifugal 20min, collect bacterial sediment, weigh, record thalline weight.Then add according to 1g thalline weight the resuspended bacterium of phosphate buffer that 5ml concentration is 0.1M.The SDS-PAGE electrophoretic band of the recombinant protein produced after IPTG induction as shown in Figure 3.In Fig. 3, the bacterium liquid that 1:IPTG does not induce; 2:IPTG induction produces the bacterium liquid of TP17 recombinant protein; 3:IPTG induction produces the bacterium liquid of TP47 recombinant protein; M: albumen Mark.
Be placed on ice by the bacterium liquid after resuspended, carry out ultrasonication, ultrasonic process 10s, suspend 20s, after effect 10min, in 12000g, centrifugal 25min, collects supernatant.The supernatant collected is continued at 12000g, and centrifugal 25min, regathers supernatant.Again repeat aforesaid operations, then by the supernatant the collected filter membrane by 0.45 μm, detect the protein concentration in solution with BCA kit, record result.Solution for standby, so that the purifying of follow-up destination protein.Because of on pETDuet-1 carrier with histidine-tagged, can Ni be utilized
2+affinity column carries out purifying to destination protein.Select the NiSepharose of GE company
tMthe pillar of 6FastFlow filler and XK16/20, carries out affinitive layer purification.After dress post, the 0.1M sodium phosphate buffer of 5 times of column volumes is utilized to balance, when the value of A280 is tending towards straight line, zeroing, by above-mentioned slightly carry after bacterial supernatant solution upper prop, after completion of the sample, first wash affinity column with the 0.1M sodium phosphate buffer of 2 times of column volumes.When the destination protein of purifying is TP-17, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 25mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 150mM imidazoles; When the destination protein of purifying is TP-47, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 30mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 100mM imidazoles.Collect the destination protein of wash-out, measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content.After affinity column purifying, still containing other foreign proteins a small amount of in solution, but its molecular weight and destination protein differ greatly, therefore we remove other foreign proteins by the molecular sieve that filler is S200, obtain highly purified destination protein, still measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content.For the preparation of follow-up colloidal gold kit lays the foundation.The SDS-PAGE electrophoresis pattern of destination protein TP-17 and TP-47 expression and each purification step afterproduct as shown in Figure 4.In Fig. 4, the SDS-PAGE electrophoretogram of A: target protein TP-17 different purification phase product.1 not purified total bacterial protein; 2, the bacterium liquid supernatant after ultrasonic process; 3, Ni
2+destination protein after affinity column purifying; 4, the destination protein after molecular sieve purification; M, albumen Mark.The SDS-PAGE electrophoretogram of B: target protein TP-47 different purification phase product.1 not purified total bacterial protein; 2, the bacterium liquid supernatant after ultrasonic process; 3, Ni
2+destination protein after affinity column purifying; 4, the destination protein after molecular sieve purification; M, albumen Mark.
The preparation of step 3, collaurum;
In the process preparing collaurum, involved all vessel all need to carry out strict process.First by vessel, in potassium bichromate solution, (concentrated sulphuric acid 200mL, potassium dichromate 120g, distilled water 1L) soaks 24h, then take out and drain, clean with tap water, repeatedly rinse three times finally by distilled water, semitight is dried, and carries out silicidation after to be dried.Silicification method is as follows: first fill in dried vessel by careful for the chloroformic solution containing 5% dichlorodimethylsilane, avoid producing bubble, after leaving standstill 3min, reclaim silication liquid, subsequently vessel distilled water good for silication is rinsed 2 ~ 3 times, semiclosed drying for standby.According to trisodium citrate reduction method, colloid gold particle is fired into the gold grain that diameter is about 40nm.Detailed process is as follows: the ultrapure water of 100ml after 0.22 μm of filtering with microporous membrane is joined (200ml) in the triangular flask after silicidation, puts into clean rotor, is placed in sealing on magnetic stirring apparatus and is heated to liquid boiling.Then, when liquid does not splash, rotor speed is adjusted to maximum.Add 1% chlorauric acid solution in this case, add the citric acid three sodium solution of 0.85mL1% subsequently fast, continue to add thermal agitation, treat that solution colour is transformed into claret gradually by colourless, black, purple, after continuing to add thermal agitation 10min, stop heating.Cool under room temperature, 4 DEG C of sealings save backup, and utilize colloid gold particle size, shape, uniformity coefficient and dispersion degree prepared by transmission electron microscope observing after 24h, use ultraviolet spectrophotometer to measure absorption peak and the OD value of collaurum.The colloid gold particle visual inspection of preparation is claret, and light transmission is good, without precipitation and other suspensions.Ultraviolet determination maximum absorption band is that 530 ~ 532nm, OD value is about 1.Its even particle size of electron microscopic observation, without assembling, favorable dispersibility, particle diameter, at about 40nm, can carry out follow-up test.
The mark of S104, albumin A;
Albumin A is marked on collaurum, first need the best combination pH determining collaurum and albumin A.Concrete operations are as follows: get 10 1.5mL centrifuge tubes, and often pipe adds the colloidal gold solution that 500 μ L bake respectively, adds the solution of potassium carbonate of different volumes subsequently in each pipe.By high precision PH test paper, the pH value in every pipe is adjusted to 7.0,7.5,8.0,8.2,8.4,8.5,8.7,9.0,9.5 and 10.0, then in every pipe, adds equal protein A respectively, the mixed instrument that spirals mixes 20min.Add the NaCl solution of 150 μ L10% in the most backward each pipe, mixing 20min, after leaving standstill 3h, utilizes ultraviolet spectrophotometer to measure absorption peak and the OD value of often pipe solution.Found that, when pH value is 8.5, its absorption peak is 532nm, OD value is 1.0, with the maximum absorption band of collaurum and OD value closest, so pH value is the optimum pH that albumin A combines.Next needs the optimal dose determining that albumin A is combined with collaurum.Method is as follows: get 10 1.5mL centrifuge tubes, often pipe adds the colloidal gold solution that 500 μ L bake respectively, solution ph is transferred to 8.5, in every pipe, add 1 μ g, 2 μ g subsequently, albumin A that 3 μ g, 4 μ g, 5 μ g, 6 μ g, 7 μ g, 8 μ g, 9 μ g, 10 μ g handle well, the mixed instrument that spirals mixes 20min.Then in each pipe, add the NaCl solution of 150 μ L10%, mixing 20min, utilizes ultraviolet spectrophotometer to measure absorption peak and the OD value of often pipe solution.Found that, when the amount of albumin A is 6 μ g, its absorption peak is 532nm, OD value is 1.0, with the maximum absorption band of collaurum and OD value closest, so the best combination amount that amount is albumin A.The operating process be combined with albumin A by colloid gold particle is as follows: first get the vial and each one of lesser trochanter handled well, be placed on magnetic stirring apparatus, the colloidal gold solution baked by 10ml adds wherein, with K2CO3, solution ph is adjusted to 8.5, stirs.Then add the albumin A of optimal dose, stir 20min.Add bovine serum albumin(BSA) V (10%) solution subsequently, make final concentration be 1%, mix after stirring 20min.Last with the centrifugal 15min of 9000rpm/min, suck supernatant, adding final concentration is the sucrose of 10% and the abundant dissolution precipitation of aqueous trehalose of 2.5%, the solution for standby of formation.
The assembling of S105, test strips and drying;
The assembling of colloidal gold colloidal gold detection test paper strip comprises following operation: be first prepare nitrocellulose filter, get nitrocellulose filter one, cut into the rectangular of 2.5cm × 5cm, 1:1 is by antigen TP-17 and TP-47 mixed in equal amounts, then be respectively the mixed solution of 1mg/mL to final concentration by two kinds of antigen diluents with C/T dilution, and with C/T dilution, sheep anti-mouse igg be diluted to 2mg/mL.Draw in two shower nozzles of film instrument by the antigen diluted and the two anti-metal sprayings that add respectively, detection line and nature controlling line discharge rate are all set to 1 μ L/cm.After being fixed the position of nitrocellulose filter, carry out spray film.After the nitrocellulose filter sprayed being placed in 37 DEG C of oven dryings, preserve in 4 DEG C of sealings.Next is the making of gold mark pad, and detailed process is as follows: first get glass fibre element film accuflowG, be cut to the rectangular of 2cm × 5cm, by the amount of 6 μ L/cm, use metal spraying to draw film instrument metal spraying, then by after having sprayed the element of the glass fibre after gold film being placed in 37 DEG C of oven dryings, preserve in 4 DEG C of sealings.
Then sample pad is prepared, specific as follows: first one whole glass fibre element film is cut into the rectangular of 1.5cm × 5cm, then use 15mM, the PBS of pH7.4 fully soaks, and after being finally placed in 37 DEG C of oven dryings, preserves in 4 DEG C of sealings.Finally nitrocellulose filter, gold mark pad and sample pad are assembled, make colloidal gold strip.Assembling process is as follows: when temperature is 22 ~ 24 DEG C, during humidity <40%, assemble test strips.First the diaphragm in the middle part of PCV plate is taken off, the nitrocellulose filter prepared is affixed in the middle part of plate, make its nature controlling line near adsorptive pads one end.Then pad edge is affixed on PCV plate with nitrocellulose filter detection line one end is slightly overlapping.Successively sample pad is marked with gold subsequently and pad slightly overlapping being affixed on PCV plate.Finally adsorptive pads is affixed on another end of PCV plate, then the test strips assembled is cut into wide rectangular of 0.4cm with test strips cutting machine, preserve in 4 DEG C of sealings.
Step 6, detection and result judge
The ELISA test strip principle of preparation is mainly according to antigen-antibody immune response.The syphilis polyclonal antibody in 20 μ L rabbit sources is joined in 80 μ L dilutions, drips after mixing in test strips sample pad.Solution, will along test strips to the swimming of adsorptive pads direction under the draw of test strips adsorptive pads.When the antibody-solutions after diluting arrives gold-marking binding pad, syphilis antibody can be combined by the gold mark protein A complexes on gold-marking binding pad, forms the compound of golden mark-albumin A-syphilis antibody and swimming forward together.When solution arrives detection line T, the hybrid antigen being fixed on TP17 and TP47 on detection line T can catch the gold mark-albumin A-syphilis antibody compound still in solution with antigen binding site, after the Treponema pallidum antigen of mixing is combined with the compound of GOLD FROM PLATING SOLUTION mark-albumin A-syphilis antibody, the redness of collaurum can be shown.Syphilis antibody all has the characteristic of reacting with Treponema pallidum antigen, adopts indirect method to detect the syphilis antibody in measuring samples.In sample, syphilis antibody is more, and the antigen so on detection line T is combined will be more, and namely on detection line T, the interception of gold mark-albumin A-syphilis antibody is more, manifests redness darker; Otherwise syphilis antibody is fewer in sample, on detection line T, the interception of gold mark-albumin A-syphilis antibody will be fewer, and namely detection line T can show light red or not develop the color.But no matter in sample, syphilis antibody is mostly few, and excessive gold mark-protein A complexes all can be combined and aobvious redness by the sheep anti-mouse igg on nature controlling line C.If nature controlling line C does not develop the color, namely judge that this result is invalid.
The sensitivity of step 7, colloidal gold strip and the detection of blood sample
Commercialization is diluted to for the rabbit source polyclonal antibody (initial concentration is 100 μ g/ml) of microspironema pallidum the antibody standard solution that concentration is 0,0.1,0.2,0.4,0.8,1.6,3.2,6.4 μ g/ml.Then respectively get 20 μ l antibody standard solution and add 80 μ l dilution [PBS (15mMPH7.4), 100ml; Bovine serum albumin(BSA) V, 1g; Tween-20,0.5ml; Sodium azide, 0.01g; Sucrose, 1g] drip in test strips well after mixing.After room temperature places 10min, observe the colour developing situation of ELISA test strip line T.Nature controlling line C should unify aobvious red, if without colour developing, namely this result is invalid.
Choose the negative serum sample of 3 kinds of separate sources, and the serum specimen choosing the separate sources of 6 kinds of confirmation positives carries out detecting (TPPA method confirmation), find that the detection line T of negative blood sample does not all develop the color, and confirm the redness of detection line T showed different in positive blood sample, 2 are wherein had to be very by force G6,1 is G5, also has 2 intensity to be G2 or G3, have one more weak for G1 or G2.
The expression vector used in the embodiment of the present invention is business-like, and the current expression vector kind for source, Escherichia coli inside and outside gene is a lot, and other workers can select the research that other expression vectors carry out this invention;
His purification tag in the embodiment of the present invention, can change other purification tags into, also can realize the purifying of destination protein.
Beneficial effect of the present invention:
The present invention builds coli expression carrier with Treponema pallidum antigen TP17 and TP47, by the optimization of codon, and carrying of purification tag, and the control of expression condition can realize the solution expression with high efficiency of Treponema pallidum antigen;
In the structure of Treponema pallidum antigen expression vector, add the molecular chaperones signal peptide of 12KD, can external efficient expression genes of interest, and make external source genes of interest be secreted in periplasmic with soluble protein, avoid the sex change renaturation process of inclusion body complexity, simplify the purge process of Treponema pallidum antigen;
Two antigens are wrapped simultaneously are used to detect, optimize ratio between the two simultaneously by expression and purification two Treponema pallidum antigen simultaneously, can improve the Sensitivity and Specificity of early syphilis and late syphilis detection;
The dot immune gold filtration assay adopted, fast and convenient, accurate, have high degree of specificity and hypersensitivity, visual result is reliable, consuming time short, only within ten minutes, can result be seen, the high expression of Treponema pallidum antigen in Escherichia coli, and higher productive rate and the recovery, greatly reduce the cost of detection kit, and reagent and amount of samples are few, without the need to instrument and equipment, simplify loaded down with trivial details normal operation.
The above is only to preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, every according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent variations and modification, all belong in the scope of technical solution of the present invention.
Claims (10)
1. a portable family syphilis Rapid detection test strip, it is characterized in that, described portable family syphilis Rapid detection test strip comprises sample pad, gold conjugation pad, NC nitrocellulose filter, absorption layer and PVC offset plate, Treponema pallidum antigen is fixed on nitrocellulose filter as capture antigen, and colloid gold label reagent is adsorbed on pad.
2. portable family syphilis Rapid detection test strip as claimed in claim 1, is characterized in that, build coli expression carrier with Treponema pallidum antigen TP17 and TP47, expression, the Purification and Characterization method of Treponema pallidum antigen are:
Utilize the inclined preferendum engineer of e. coli codon and synthetic gene, BamHI and NotI site is contained at the gene two ends of synthesis, and expression vector is pETDuet-1, and this expression vector contains T7 promoter, under the induction of IPTG, foreign gene is transcriptional expression under the effect of t7 rna polymerase;
The C end of described expression vector is containing HisTag sequence, the foreign protein of expressing is purified by metal chelate affinity chromatography method, when purpose of design gene, insert the expressing gene of the molecular chaperones peptide chain of 12KD size, be convenient to the foreign protein of expression to be secreted in periplasmic;
The foreign gene of synthesis is cut link by enzyme to be inserted in carrier pETDuet-1, and in transformation of E. coli BL21, by IPTG abduction delivering, antigen is expressed in bacteria periplasm with soluble form, and collect bacterium, ultrasonication, supernatant passes through Ni
2+metal-chelating and sieve chromatography, the albumen of purifying is by SDS-PAGE, Western-blot qualification.
3. portable family syphilis Rapid detection test strip as claimed in claim 1, is characterized in that, the preparation method of described portable family syphilis Rapid detection test strip comprises:
The design of step one, TP genes of interest and clone;
The expression and purification of step 2, destination protein;
The preparation of step 3, collaurum;
The mark of step 4, collaurum albumin A;
The assembling of step 5, test strips and drying;
Step 6, detection and result judge;
The sensitivity of step 7, colloidal gold strip and the detection of blood sample.
4. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the design of TP genes of interest and the concrete steps of clone are:
Step one, by US National Biotechnology Information center website, search the whole genome sequence of microspironema pallidum TP-17 and TP-47;
CDNA sequence in step 2, synthesis genes of interest open reading frame, and synthetic sequence clone is entered pMD18-TSimple carrier, when carrying out sequent synthesis, respectively at 5 ' end of aim sequence and the restriction enzyme site of 3 ' end insertion BamHI and NotI, and utilize these two kinds of restriction enzymes to be sheared from pMD18-TSimple carrier by genes of interest, carry out agarose gel electrophoresis qualification, reclaim electrophoretic band;
Step 3, by the object band that reclaims by after dephosphorylation ferment treatment, with the pETDuet-1 carrier through same restriction enzyme and dephosphorylation ferment treatment under T4 ligase existent condition, 16 DEG C of connections are spent the night; The carrier connected electricity is forwarded in the competent cell of Escherichia coli DH10B bacterial classification, coats containing in 50 μ g/ml ammonia benzyl antibiotic LB solid culture plate, 37 DEG C of overnight incubation; Within second day, picking monoclonal is inoculated into containing in the antibiotic 5mlLB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm, concussion overnight incubation; Within 3rd day, utilize little plasmid extraction kit of taking out to extract plasmid in bacterium liquid, carry out BamHI and NotI double digestion and to go forward side by side row agarose gel electrophoresis qualification; The positive plasmid electricity with object band is forwarded in the competent cell of e. coli bl21 bacterial classification; repeat above operation; coated plate; choose after clone carries out Liquid Culture; identified by BamHI and NotI double digestion and agarose gel electrophoresis, the plasmid containing object band or bacterium liquid are checked order.
5. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the concrete steps of the expression and purification of destination protein are:
Step one, the bacterium liquid of the plasmid containing correct genes of interest TP17 and TP47 sequence is seeded to 50ml respectively contains in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, in 250rpm, 37 DEG C of overnight incubation; Subsequently the bacterium liquid of two bottles of 50ml incubated overnight is inoculated into 1L to contain in the antibiotic LB fluid nutrient medium of 50 μ g/ml ammonia benzyl, 37 DEG C, 250rpm shakes cultivation;
Step 2, A when bacterium liquid
600when OD value is 0.2 ~ 0.4, incubator temperature is adjusted to 22 DEG C, still 250rpm shakes cultivation 2.5 ~ 3h; As the A of bacterium liquid
600when OD value is 0.9 ~ 1.0, adding IPTG derivant to final concentration is 1mmol/L, and incubator temperature is adjusted to 18 DEG C, and 250rpm shakes overnight incubation;
Step 3, second day collection bacterium liquid, 4000g, centrifugal 20min, collect bacterial sediment, weigh, record thalline weight;
Step 4, add according to 1g thalline weight the resuspended bacterium of phosphate buffer that 5ml concentration is 0.1M;
Step 5, be placed on ice by the bacterium liquid after resuspended, carry out ultrasonication, ultrasonic process 10s, suspend 20s, after effect 10min, in 12000g, centrifugal 25min, collects supernatant; The supernatant collected is continued at 12000g, and centrifugal 25min, regathers supernatant; Again repeat aforesaid operations, then by the supernatant the collected filter membrane by 0.45 μm, detect the protein concentration in solution with BCA kit, record result;
Step 6, select the NiSepharose of GE company
tMthe pillar of 6FastFlow filler and XK16/20, carry out affinitive layer purification, after dress post, the 0.1M sodium phosphate buffer of 5 times of column volumes is utilized to balance, when the value of A280 is tending towards straight line, zeroing, by above-mentioned slightly carry after bacterial supernatant solution upper prop, after completion of the sample, first wash affinity column with the 0.1M sodium phosphate buffer of 2 times of column volumes; When the destination protein of purifying is TP-17, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 25mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 150mM imidazoles; When the destination protein of purifying is TP-47, wash post with the 0.1M sodium radio-phosphate,P-32 solution containing 30mM imidazoles, with the 0.1M sodium radio-phosphate,P-32 solution wash-out destination protein containing 100mM imidazoles;
The destination protein of step 7, collection wash-out, measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content, other foreign proteins removed by the molecular sieve being S200 by filler, measured the concentration of destination protein in eluent by BCA detection kit, and calculate its content.
6. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the concrete steps of the preparation of collaurum are:
Step one, foundation trisodium citrate reduction method, colloid gold particle is fired into the gold grain that diameter is about 40nm, detailed process is as follows: joined by the ultrapure water of 100ml after 0.22 μm of filtering with microporous membrane in the 200ml triangular flask after silicidation, put into clean rotor, be placed in sealing on magnetic stirring apparatus and be heated to liquid boiling.Then, when liquid does not splash, rotor speed is adjusted to maximum;
Step 2, add 1% chlorauric acid solution in this case, add the citric acid three sodium solution of 0.85mL1% subsequently fast, continue to add thermal agitation, treat that solution colour is transformed into claret gradually by colourless, black, purple, after continuing to add thermal agitation 10min, stop heating;
Cool under step 3, room temperature, 4 DEG C of sealings save backup, and utilize colloid gold particle size, shape, uniformity coefficient and dispersion degree prepared by transmission electron microscope observing after 24h, use ultraviolet spectrophotometer to measure absorption peak and the OD value of collaurum.
7. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the concrete steps of the mark of albumin A are:
Step one, first get the vial handled well and each one of lesser trochanter, be placed on magnetic stirring apparatus, the colloidal gold solution baked by 10ml adds wherein, uses K
2cO
3solution ph is adjusted to 8.5, stirs; Then the ratio that the colloidal gold solution baked with every 500 μ L joins 6 μ g albumin As adds albumin A, stirs 20min;
Step 2, add the bovine serum albumin(BSA) V solution that concentration is 10%, make final concentration be 1%, mix after stirring 20min;
Step 3, with the centrifugal 15min of 9000rpm/min, suck supernatant, adding final concentration is the sucrose of 10% and the abundant dissolution precipitation of aqueous trehalose of 2.5%, the solution for standby of formation.
8. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the assembling of test strips and the concrete steps of drying are:
Step one, prepare nitrocellulose filter, get nitrocellulose filter one, cut into the rectangular of 2.5cm × 5cm, 1:1 is by antigen TP-17 and TP-47 mixed in equal amounts, then be respectively the mixed solution of 1mg/mL to final concentration by two kinds of antigen diluents with C/T dilution, and with C/T dilution, sheep anti-mouse igg be diluted to 2mg/mL;
Step 2, draw in two shower nozzles of film instrument by the antigen diluted and the two anti-metal sprayings that add respectively, detection line and nature controlling line discharge rate are all set to 1 μ L/cm;
Step 3, the position of nitrocellulose filter is fixed after, carry out spray film;
Step 4, the nitrocellulose filter sprayed is placed in 37 DEG C of oven dryings after, in 4 DEG C sealing preserve;
Step 5, making gold mark pad: first get glass fibre element film accuflowG, be cut to the rectangular of 2cm × 5cm, by the amount of 6 μ L/cm, use metal spraying to draw film instrument metal spraying, then by after having sprayed the element of the glass fibre after gold film being placed in 37 DEG C of oven dryings, preserve in 4 DEG C of sealings;
Step 6, preparation sample pad:
First one whole glass fibre element film is cut into the rectangular of 1.5cm × 5cm, then use 15mM, the PBS of pH7.4 fully soaks, and after being finally placed in 37 DEG C of oven dryings, preserves in 4 DEG C of sealings;
Step 7, finally by nitrocellulose filter, gold mark pad and sample pad assemble, make colloidal gold strip;
Assembling process is as follows: when temperature is 22 ~ 24 DEG C, during humidity <40%, assemble test strips; First the diaphragm in the middle part of PCV plate is taken off, the nitrocellulose filter prepared is affixed in the middle part of plate, make its nature controlling line near adsorptive pads one end; Then be affixed on PCV plate by slightly overlapping to pad edge and nitrocellulose filter detection line one end; Sample pad and gold being marked to pad successively subsequently is slightly overlappingly affixed on PCV plate; Finally adsorptive pads is affixed on another end of PCV plate, then the test strips assembled is cut into wide rectangular of 0.4cm with test strips cutting machine, preserve in 4 DEG C of sealings.
9. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, is characterized in that, detect and the concrete grammar that judges of result as:
According to antigen-antibody immune response, the syphilis polyclonal antibody in 20 μ L rabbit sources is joined in 80 μ L dilutions, drips after mixing in test strips sample pad, if nature controlling line C does not develop the color, namely judge that this result is invalid.
10. the preparation method of portable family syphilis Rapid detection test strip as claimed in claim 3, it is characterized in that, the concrete steps of the sensitivity of colloidal gold strip and the detection of blood sample are:
Step one, commercialization is diluted to for the rabbit source polyclonal antibody that the initial concentration of microspironema pallidum is 100 μ g/ml the antibody standard solution that concentration is 0,0.1,0.2,0.4,0.8,1.6,3.2,6.4 μ g/ml;
Step 2, respectively get 20 μ l antibody standard solution and add 80 μ l dilutions, the component proportion of this dilution is: the PBS of 15mM, PH7.4,100ml; Bovine serum albumin(BSA) V, 1g; Tween-20,0.5ml; Sodium azide, 0.01g; Sucrose, 1g; Drip after mixing in test strips well;
After step 3, room temperature place 10min, observe the colour developing situation of ELISA test strip line T; Nature controlling line C should unify aobvious red, if without colour developing, namely this result is invalid.
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| CN201510779735.3A CN105353125A (en) | 2015-11-13 | 2015-11-13 | A portable rapid test strip for syphilis for household use |
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| CN201510779735.3A Pending CN105353125A (en) | 2015-11-13 | 2015-11-13 | A portable rapid test strip for syphilis for household use |
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| CN111638331A (en) * | 2020-06-15 | 2020-09-08 | 郑州方欣生物科技有限责任公司 | Detection kit for treponema pallidum antibody in urine, preparation method and use method |
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