CN102288754A - Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen - Google Patents
Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen Download PDFInfo
- Publication number
- CN102288754A CN102288754A CN2010105023853A CN201010502385A CN102288754A CN 102288754 A CN102288754 A CN 102288754A CN 2010105023853 A CN2010105023853 A CN 2010105023853A CN 201010502385 A CN201010502385 A CN 201010502385A CN 102288754 A CN102288754 A CN 102288754A
- Authority
- CN
- China
- Prior art keywords
- liquid
- pentastome
- agkistrodon acutus
- colour developing
- weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kit adopting a specific polyclonal antibody for detection of an agkistrodon acutus tongue-shaped worm circulating antigen, which comprises a rabbit anti-Aag-IgG, an enzyme-labeled antibody (HRP-Aag-IgG), a nitrocellulose membrane, 1 percent of PBST (polybuthylenesuccinate) solution of bovine serum albumin, PBST washing liquid, substrate developing solution, positive and negative control samples and a polyethylene reaction plate. The invention also provides a preparation method of the kit adopting the specific polyclonal antibody for detection of the agkistrodon acutus tongue-shaped worm circulating antigen. Since the polyclonal antibody is adopted to determine the circulating antigen, early diagnosis and prognostic judgment can be facilitated. When in-vivo polypides die, the circulating antigen can disappear rapidly, and the kit can be applied in efficacy assessment. Moreover, the kit has high sensitivity and high specificity, and rapid diagnosis, drug screening and efficacy assessment of the agkistrodon acutus linguatulosis can be achieved.
Description
Technical field:
The invention belongs to field of biological detection, relate in particular to a kind of kit and preparation method thereof, is that a kind of specificity that adopts resists the kit that detects agkistrodon acutus pentastome circulating antigen more specifically.
Background technology:
Pentastomiasis is animal derived Amphixenosis.About 118 kinds of the known pentastome in the whole world, the pentastome of parasitic human body mainly contains 6 and belongs to 10 kinds, is respectively sawtooth pentastome (Linguatula serrata), armillifer armillatus (Armillifer armillatus), agkistrodon acutus ligule worm (A.aggkistrodontis), armillifer moniliformis (A.moniliformis), serpent ligule worm (A.grandis), the lizard tiger relies sharp linguatulid (Raillietiella hemidactyli), Cincinnati Lay pendant linguatulid (Leiperia cincinnalis), a rattle snake hole linguatulid (Porocephalus crotali), a plucked instrument skin linguatulid (P.Sebekia) and hole, a Taiwan linguatulid (P.taiwana) etc.The pentastome that China puts down in writing parasitic human body has sawtooth pentastome, agkistrodon acutus ligule worm and hole, a Taiwan linguatulid.In recent years, all once find to have the report of many cases agkistrodon acutus ligule insect infection human body in China Taiwan, Zhejiang, Guangxi.Although pentastome case number is less, but because the existence of plague area is arranged, the feed custom that people are different, for natural conditions have been set up in the infection of this disease, the people can become the intermediate host of agkistrodon acutus ligule worm, mainly be owing to people drunk the water source that infected property worm's ovum pollutes or drink raw snake blood, snake gall infects.Show as internal organs such as parasitic human body mesenterium of larva and liver,spleen,kidney, cause the damage of many internal organs diffusivity, particularly important internal organs such as liver, spleen endanger very great.Add the expansion in pentastomiasis epidemic-stricken area over nearly 20 years, the new pathogenic kind discovery and the increase of fatal case make the diagnosis of this rare parasitic disease of pentastome propose new challenge.
Summary of the invention:
The object of the present invention is to provide a kind of specificity that adopts to resist the kit that detects agkistrodon acutus pentastome circulating antigen, how the anti-kit that detects agkistrodon acutus pentastome circulating antigen will solve the method that can't detect the existing disease infection of agkistrodon acutus pentastomiasis in the prior art to described this employing specificity more.
The invention provides a kind of specificity that adopts and resist the kit that detects agkistrodon acutus pentastome circulating antigen more, it is characterized in that: the anti-agkistrodon acutus pentastome larva polyclonal antibody that comprises the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, horseradish peroxidase-labeled.
Further, described kit also comprises PBST solution, PBST cleansing solution, substrate colour developing liquid, the positive and the negative control sample composition and the tygon reaction plate of cellulose nitrate film, 1% bSA.
Further, the PBST solution of described 1% bSA is made up of bSA and PBST cleansing solution, and the weight ratio of described bSA and PBST cleansing solution is: 1: 99.
Further, described PBST cleansing solution is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl, Tween-20 and water forms, in the process of preparation PBST cleansing solution, the step of a base fluid of preparation earlier, described base fluid is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described sodium chloride in described base fluid is 0.8%, described KH
2PO
4Percentage by weight at described base fluid is 0.02%, described Na
2HPO
4Percentage by weight in described base fluid is 0.29%, and the percentage by weight of described KCl in described base fluid is 0.02%, and surplus is a water, after preparing base fluid, add Tween-20 again, add the 0.5ml Tween-20 among every 100ml, the pH value of described PBST cleansing solution is 7.4.
Further, described substrate colour developing liquid is made up of colour developing liquid A liquid, colour developing liquid B liquid and colour developing liquid C liquid, and described colour developing liquid A liquid is by the adjacent aniline of 3-3-diamido, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of the adjacent aniline of described 3-3-diamido in colour developing liquid A liquid is 0.1%, the percentage by weight of described sodium chloride in colour developing liquid A liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid A liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid A liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid A liquid is 0.02%, and surplus is the water in the colour developing liquid A liquid, and described colour developing liquid B liquid is by 4-chloro-1-naphthols, methyl alcohol, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described 4-chloro-1-naphthols in colour developing liquid B liquid is 0.05%, the percentage by weight of described methyl alcohol in colour developing liquid B liquid is 20%, and the percentage by weight of described sodium chloride in colour developing liquid B liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid B liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid B liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid B liquid is 0.02%, and surplus is the water in the colour developing liquid B liquid; Described colour developing liquid C liquid is the aqueous solution of hydrogen peroxide, and the weight percent concentration of described hydrogen peroxide in aqueous solution is 30%.
Further, described substrate demonstration liquid is made up of first reagent and second reagent, and first reagent is 4-chloro-1-naphthols reagent, and second reagent is 3,3 '-diaminobenzidine reagent.
The present invention also provides above-mentioned a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen more, the step that comprises an anti-agkistrodon acutus pentastome larva soluble antigen of preparation, the step of the anti-agkistrodon acutus pentastome of a preparation rabbit larva polyclonal antibody, the step of the anti-agkistrodon acutus pentastome larva polyclonal antibody of a horseradish peroxidase-labeled.
Further, in the step of an anti-agkistrodon acutus pentastome larva soluble antigen of preparation, get larva, grind the back Ultrasonic Pulverization, preparation larva soluble antigen from infecting 3-5 month mouse web portion separation of agkistrodon acutus pentastome worm's ovum.
Further, in the step of the anti-agkistrodon acutus pentastome of a preparation rabbit larva polyclonal antibody, to mix with isopyknic Freund's complete adjuvant with agkistrodon acutus pentastome larva soluble antigen, 3 immunity are carried out to rabbit in fully emulsified back, collect rabbit anteserum, extract also how anti-purifying is.
Further, in the step of anti-agkistrodon acutus pentastome larva polyclonal antibody of a preparation horseradish peroxidase-labeled, adopt ELISA method or double-diffusion process to detect tiring of the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, with periodates labelling method mark enzyme.
Further, described positive control is agkistrodon acutus pentastomiasis patients serum, and described negative control sample is a normal human serum.
It is of the present invention that a kind of how anti-adopt the specificity principle that detects the kit of agkistrodon acutus pentastome circulating antigen be that (rabbit resist-Aag-IgG) puts on cellulose nitrate film with the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, be that specific antibody is attached to upward formation insolubilized antibody of solid phase carrier (cellulose nitrate film), add serum to be checked, many corresponding antigens anti-and in the serum to be checked combine and form immune complex, the anti-agkistrodon acutus pentastome polyclonal antibody (HRP-Aag-IgG) that adds enzyme labeling after the washing, the antibody of enzyme labeling (HRP-Aag-IgG) combines with antigen in the immune complex and forms enzyme labelled antibody-antigen-insolubilized antibody compound, add the substrate colour developing, judge antigenic content.
Agkistrodon acutus pentastome larval antigens immunizing rabbit has obtained anti-agkistrodon acutus pentastome larva polyclonal antibody (Aag-IgG), with many anti-circulating antigen in the mice serum that detect, confirm can detect in mouse the 2nd week after infection the circulating antigen in its body, can be used as the foundation that detects existing disease infection, efficacy assessment, reaches drug screening.
The present invention measures circulating antigen with polyclonal antibody (Aag-IgG) and has the following advantages: (1) can measure antigen before antibody occurs, help making early diagnosis.(2) content of circulating antigen is directly proportional with host entozoa quantity, helps the judgement of prognosis.(3) after polypide death in the body, circulating antigen rapidly disappears, and can be used for efficacy assessment.
The polyclonal antibody of the anti-agkistrodon acutus pentastome of application specific of the present invention larva, development dot-ELISA method detects agkistrodon acutus pentastome circulating antigen kit.This kit is easy and simple to handle, quick; Susceptibility height, high specificity, good reproducibility.The present invention is fit to auxiliary diagnosis, efficacy assessment and the drug screening that departments such as hospital laboratory, disease prevention and control center, She Kang center, private clinic are used for the clinical existing disease infection of agkistrodon acutus pentastomiasis, has very high practical value and considerable economy and social benefit.
How the anti-kit that detects agkistrodon acutus pentastome circulating antigen is used agkistrodon acutus pentastome larval antigens immunizing rabbit acquisition polyclonal antibody to employing specificity of the present invention, detect the dot-ELISA (dot-ELISA) of circulating antigen (CAg) in patient's serum, can be used as the foundation that detects existing disease infection and efficacy assessment, dot-ELISA method susceptibility height, high specificity that the present invention sets up can be realized quick diagnosis, drug screening and the efficacy assessment of agkistrodon acutus pentastomiasis.
Description of drawings:
Fig. 1 adopts a kind of specificity that adopts of the present invention to resist the kit that detects agkistrodon acutus pentastome circulating antigen to detect the schematic diagram of agkistrodon acutus pentastomiasis more.
Embodiment:
Reagent and material source: 1% bSA (purchasing white Shanghai institute of Biological Products).
Embodiment adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen for 1 one kinds more
The invention provides a kind of adopt the how anti-kit that detects agkistrodon acutus pentastome circulating antigen of specificity comprise the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody (rabbit is anti--Aag-IgG), PBST solution, PBST cleansing solution, substrate colour developing liquid, the positive and the negative control sample of the anti-agkistrodon acutus pentastome polyclonal antibody (HRP-Aag-IgG) of horseradish peroxidase-labeled, cellulose nitrate film, 1% bSA (BSA) form and 96 hole tygon reaction plates.
Further, the PBST solution of described 1% bSA is made up of bSA and PBST cleansing solution, and the weight ratio of described bSA and PBST cleansing solution is: 1: 99.
Further, described PBST cleansing solution is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl, Tween-20 and water forms, and in the process of preparation PBST cleansing solution, comprises a step of preparation base fluid earlier, described base fluid is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described sodium chloride in described base fluid is 0.8%, described KH
2PO
4Percentage by weight at described base fluid is 0.02%, described Na
2HPO
4Percentage by weight in described base fluid is 0.29%, and the percentage by weight of described KCl in described base fluid is 0.02%, and surplus is a water, after preparing base fluid, add Tween-20 again, add the 0.5ml Tween-20 among every 100ml, the pH value of described PBST cleansing solution is 7.4.
Further, described substrate colour developing liquid is made up of colour developing liquid A liquid, colour developing liquid B liquid and colour developing liquid C liquid, and described colour developing liquid A liquid is by the adjacent aniline of 3-3-diamido, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of the adjacent aniline of described 3-3-diamido in colour developing liquid A liquid is 0.1%, the percentage by weight of described sodium chloride in colour developing liquid A liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid A liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid A liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid A liquid is 0.02%, and surplus is the water in the colour developing liquid A liquid, and described colour developing liquid B liquid is by 4-chloro-1-naphthols, methyl alcohol, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described 4-chloro-1-naphthols in colour developing liquid B liquid is 0.05%, the percentage by weight of described methyl alcohol in colour developing liquid B liquid is 20%, and the percentage by weight of described sodium chloride in colour developing liquid B liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid B liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid B liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid B liquid is 0.02%, and surplus is the water in the colour developing liquid B liquid; Described colour developing liquid C liquid is the aqueous solution of hydrogen peroxide, and the weight percent concentration of described hydrogen peroxide in aqueous solution is 30%.Concrete, when adopting kit to detect, get A liquid+B liquid mixed in equal amounts after, add C liquid 40ul in per 100 milliliters of mixed liquors and mix the back and use.
Further, described substrate demonstration liquid is made up of first reagent and second reagent, and first reagent is 4-chloro-1-naphthols reagent, and second reagent is 3,3 '-diaminobenzidine reagent.4-chloro-1-naphthols reagent and 3 during use, 3 '-diaminobenzidine reagent mixed in equal amounts is used.
Further, described positive control is agkistrodon acutus pentastomiasis patients serum, and described negative control sample is a normal human serum.
Embodiment 2
The present invention also provides a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen more, the step that comprises an anti-agkistrodon acutus pentastome larva soluble antigen of preparation, preparation rabbit is anti--step of Aag-IgG, the step of the antibody of an enzyme labeling (HRP-Aag-IgG) also comprises the step of a PBST solution of placing cellulose nitrate film, 1% bSA (BSA) in kit, PBST cleansing solution, substrate colour developing liquid, positive control, negative control sample, tygon reaction plate.
Further, in the step of a preparation agkistrodon acutus pentastome larva soluble antigen, get larva, preparation larva soluble antigen from infecting 3-5 month mouse web portion separation of agkistrodon acutus pentastome worm's ovum.
Further, preparation rabbit anti--step of Aag-IgG in, mix with isopyknic Freund's complete adjuvant with agkistrodon acutus pentastome larva soluble antigen (AagA), fully emulsifiedly then rabbit is carried out 3 immunity, collect rabbit anteserum, purifying how anti-(rabbit is anti--Aag-IgG).
Further, in the step of anti-agkistrodon acutus pentastome larva polyclonal antibody of a preparation horseradish peroxidase-labeled, adopt ELISA method or double-diffusion process to detect tiring of the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, with periodates labelling method mark enzyme (the HRP-rabbit is anti--Aag-IgG).
Concrete:
1. the collection step of the anti-agkistrodon acutus pentastome children's polyclonal antibody of the preparation of agkistrodon acutus pentastome larva soluble antigen and rabbit (Aag-IgG) is as follows:
Get larva from infecting the separation of 4 months mouse web portion of agkistrodon acutus pentastome worm's ovum, preparation larva soluble antigen, recording protein content is 7.58mg/ml.
100 μ g mix with isopyknic Freund's complete adjuvant with agkistrodon acutus pentastome larva soluble antigen (AagA), the multiple spot immunity is carried on the back to rabbit in fully emulsified back, use AagA antigen and equal-volume incomplete Freund booster immunization once after 4 weeks, dosage is the same.Carry out three immunity altogether.The antibody horizontal that the ELISA method records after the rabbit immunity is 1: 320000.Adopt the salting out method purifying how anti-(rabbit is anti--Aag-IgG), it is 1: 80000 that ELISA records many anti-tiring, double-diffusion process is 1: 128, purify through saturated sulfuric acid amine, with DEAE-cellulose (DEAE) cellulose ion-exchange column chromatography purifying, obtain the polyclonal antibody (Aag-IgG) of anti-agkistrodon acutus pentastome larva.
(1) preparation of saturated ammonium sulfate solution: 500g ammonium sulfate adds in the 500ml distilled water, is heated to dissolving fully, ambient temperature overnight, and the crystallization of separating out is let alone to stay in the bottle.Face with before getting required amount, transfer pH to 7.8 with 2mol/L NaOH.
(2) saltout: (rabbit resists-PW-IgG) moves in the small beaker, under agitation, drips saturated ammonium sulfate solution 5.0ml to draw the 10ml rabbit anteserum; Continue slowly to stir 30min; The centrifugal 15min of 10000r/min; Abandoning supernatant, sediment suspends with 1/3 saturation degree ammonium sulfate, and beating action 30min is centrifugal with method; Repeat back 1～2 time; Sediment is dissolved in 1.5ml PBS (0.01mol/L pH7.2) or the Tris-HCl damping fluid.
(3) desalination: the sample of will saltouing is crossed Sephadex G-50 chromatographic column, with PBS or Tris-HCl damping fluid as equilibrium liquid and eluent, flow velocity 1ml/min.First protein peak is the antibody-solutions of desalination.Dialysis is in 2%NaHCO3 with bag filter, boil 10min in the 1mmol/L EDTA solution, clean the bag filter surfaces externally and internally, boil bag filter 10min with distilled water again with distilled water, be chilled to room temperature and can use (also can be in 0.2mol/L EDTA solution, 4 ℃ of preservations are standby).The sample of will saltouing is packed in the bag filter, to the PBS of 50～100 times of volumes or Tris-HCl damping fluid dialysis (4 ℃) 12～24 hours, change dislysate therebetween 5 times, with Nai Shi reagent (mercuric iodixde 11.5g, potassium iodide 8g, adding distil water 50ml is after waiting to dissolve, add 20%NaOH 50ml again) detect, till extracellular fluid dialysis does not have yellow formation.
(4) mensuration of protein content: (Pr) (mg/ml)=(1.45 * OD280-0.74 * OD260) * extension rate; Or (Pr)=OD280 * extension rate/3
2. the anti-agkistrodon acutus pentastome children's polyclonal antibody of enzyme labeling rabbit (HRP-Aag-IgG) preparation
Adopt periodates labelling method mark enzyme (the HRP-rabbit is anti--PW-IgG).The marker enzyme of described label be horseradish peroxidase (horseradish peroxidase, HRP).The common method of horseradish peroxidase (HRP) labelled antibody is the sodium periodate method.Its principle is that the glycosyl of HRP is oxidized to aldehyde radical with sodium periodate, and this aldehyde radical combines with IgG is amino after the adding IgG antibody, forms SchiffShi alkali.For aldehyde radical and the amino generation of himself the albumen coupling that prevents sugar among the HRP, amino with the dinitrofluorobenzene blocking-up earlier with the sodium periodate oxidation.Oxidation reaction is used sodium borohydride stabilized SchiffShi alkali finally.Its operation steps is as follows:
1. 5mg HRP is dissolved in the 0.5ml 0.1mol/L NaHCO3 solution; Add 0.5ml10mmol/L NaIO4 solution, mixing covers tight bottle stopper, room temperature lucifuge effect 2 hours;
2. add 0.75ml 10.1mol/L Na2CO3 mixing;
3. add 0.75ml rabbit anteserum (how anti-), mixing;
4. take by weighing Sephadex G25 dry powder 0.3g, add in the 5ml syringe urceolus of an end opening pad glass wool; Subsequently above-mentioned cross-linking agent is moved into syringe jacket; Lid is tight, and room temperature effect (lucifuge) was spent the night in 3 hours or 4 ℃;
5. with a little PBS cross-linking agent is all washed out, collect eluate, add the freshly prepared 5mg/ml NaBH4 of 1/20V volume solution, mixing, room temperature effect 30min; Add 3/20VNaBH4 solution again, mixing, room temperature effect 1 hour (or 4 ℃ spend the night);
6. (2.5 * 50cm) chromatographic purifyings are in charge of the collection first peak cross-linking agent to be crossed Sephadex G200 or Sepharose 6B;
7. enzyme conjugates Quality Identification: the mensuration of enzymatic activity and antibody activity, use the enzymatic activity that ELISA method, immunodiffusion, DAB-H2O2 chromogenic reaction are measured enzyme conjugates, antibody activity and tire, specificity;
8. the preservation of HRP antibody conjugates: after adding equivalent glycerine, packing-20 ℃ is deposited in a small amount, prevents multigelation; Or 4 ℃ of preservations of adding equivalent 60% glycerine; Should not add NaN3 or phenol is anticorrosion, otherwise can influence enzymatic activity, can also freeze-drying preserve in case of necessity, make protective agent with BSA or skim milk.
Embodiment 3
A kind of operation steps following (principle is as shown in Figure 1) that adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen of the present invention more:
A) with rabbit anti--Aag-IgG is diluted to 200 μ g/ml, draws 1 μ l antibody on cellulose nitrate film with sample applicator, the room temperature bag was by 10 minutes.
B) sealing: will wrap the PBST solution that is immersed 1% bSA (BSA) by good diaphragm.37 ℃, 15 minutes.
C) washing: the film of sealing is washed 3 times with cleansing solution, and each 3 minutes airings are " examining film soon ".
D) tested antigen: " examining film soon " cut by grid, and light is put into 96 hole tygon reaction plate hole towards last, and every hole adds the serum 100 μ l of tested serum, establishes blank (PBS), feminine gender and positive contrast simultaneously.37℃1h。
E) washing is with (3)
F) enzyme labeling HRP-Aag-IgG: after enzyme labelled antibody dilution in 1: 100, every hole adds 100 μ l, 37 ℃ of 1h.
G) washing is with (3)
H) substrate: get substrate colour developing liquid A liquid 5ml, substrate colour developing liquid B liquid 5ml, substrate colour developing liquid C liquid 4ul, mix each reacting hole of back and add 100ul, room temperature lucifuge 10min, distilled water cessation reaction.)
Criterion: have or not or shade according to chromogenic reaction, carry out qualitative or sxemiquantitative; Spot presents yellow blue color spot point and is judged as the positive, and it is then negative color not occur.
+++spot is deep yellow blue look
++ spot is Huang Lanse
+ spot is yellowish blue look
-there is not a visible spot
Claims (11)
1. one kind is adopted specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen more, it is characterized in that: the anti-agkistrodon acutus pentastome larva polyclonal antibody that comprises the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, horseradish peroxidase-labeled.
2. a kind of specificity that adopts as claimed in claim 1 resists the kit that detects agkistrodon acutus pentastome circulating antigen more, it is characterized in that: the PBST solution, PBST cleansing solution, substrate colour developing liquid, the positive and the negative control sample that also comprise cellulose nitrate film, 1% bSA are formed and the tygon reaction plate.
3. specificity resists the kit that detects agkistrodon acutus pentastome circulating antigen more according to claim 1, it is characterized in that: the PBST solution of described 1% bSA is made up of bSA and PBST cleansing solution, and the weight ratio of described bSA and PBST cleansing solution is: 1: 99.
4. as the anti-kit that detects agkistrodon acutus pentastome circulating antigen how of specificity as described in claim 1 or 3, it is characterized in that: described PBST cleansing solution is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl, Tween-20 and water forms, in the process of preparation PBST cleansing solution, the step of a base fluid of preparation earlier, described base fluid is by sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described sodium chloride in described base fluid is 0.8%, described KH
2PO
4Percentage by weight at described base fluid is 0.02%, described Na
2HPO
4Percentage by weight in described base fluid is 0.29%, and the percentage by weight of described KCl in described base fluid is 0.02%, and surplus is a water, after preparing base fluid, add Tween-20 again, add the 0.5ml Tween-20 among every 100ml, the pH value of described PBST cleansing solution is 7.4.
5. as the anti-kit that detects agkistrodon acutus pentastome circulating antigen how of specificity as described in the claim 2, it is characterized in that: the substrate colour developing liquid in the described kit is made up of colour developing liquid A liquid, colour developing liquid B liquid and colour developing liquid C liquid, and described colour developing liquid A liquid is by the adjacent aniline of 3-3-diamido, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of the adjacent aniline of described 3-3-diamido in colour developing liquid A liquid is 0.1%, the percentage by weight of described sodium chloride in colour developing liquid A liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid A liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid A liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid A liquid is 0.02%, and surplus is the water in the colour developing liquid A liquid, and described colour developing liquid B liquid is by 4-chloro-1-naphthols, methyl alcohol, sodium chloride, KH
2PO
4, Na
2HPO
4, KCl and water forms, the percentage by weight of described 4-chloro-1-naphthols in colour developing liquid B liquid is 0.05%, the percentage by weight of described methyl alcohol in colour developing liquid B liquid is 20%, and the percentage by weight of described sodium chloride in colour developing liquid B liquid is 0.8%, described KH
2PO
4Percentage by weight in colour developing liquid B liquid is 0.02%, described Na
2HPO
4Percentage by weight in colour developing liquid B liquid is 0.29%, and the percentage by weight of described KCl in colour developing liquid B liquid is 0.02%, and surplus is the water in the colour developing liquid B liquid; Described colour developing liquid C liquid is the aqueous solution of hydrogen peroxide, and the weight percent concentration of described hydrogen peroxide in aqueous solution is 30%.
6. specificity as claimed in claim 2 resists the preparation method of the kit that detects agkistrodon acutus pentastome circulating antigen more, it is characterized in that: described substrate demonstration liquid is made up of first reagent and second reagent, first reagent is 4-chloro-1-naphthols reagent, second reagent is 3,3 '-diaminobenzidine reagent.
7. the described specificity of claim 1 resists the preparation method of the kit that detects agkistrodon acutus pentastome circulating antigen more, it is characterized in that: the step that comprises an anti-agkistrodon acutus pentastome larva soluble antigen of preparation, the step of the anti-agkistrodon acutus pentastome of a preparation rabbit larva polyclonal antibody, the step of the anti-agkistrodon acutus pentastome larva polyclonal antibody of a horseradish peroxidase-labeled.
8. a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen as claimed in claim 7 more, it is characterized in that: in the step of an anti-agkistrodon acutus pentastome larva soluble antigen of preparation, get larva from infecting 3-5 month mouse web portion separation of agkistrodon acutus pentastome worm's ovum, grind the back Ultrasonic Pulverization, preparation larva soluble antigen.
9. a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen as claimed in claim 7 more, it is characterized in that: in the step of the anti-agkistrodon acutus pentastome of a preparation rabbit larva polyclonal antibody, mix with isopyknic Freund's complete adjuvant with agkistrodon acutus pentastome larva soluble antigen, 3 immunity are carried out to rabbit in fully emulsified back, collect rabbit anteserum, extract also how anti-purifying is.
10. a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen as claimed in claim 7 more, it is characterized in that: in the step of the anti-agkistrodon acutus pentastome larva polyclonal antibody of a horseradish peroxidase-labeled, adopt ELISA method or double-diffusion process to detect tiring of the anti-agkistrodon acutus pentastome of rabbit larva polyclonal antibody, with periodates labelling method mark enzyme.
11. a kind of preparation method who adopts specificity to resist the kit that detects agkistrodon acutus pentastome circulating antigen as claimed in claim 7 more, it is characterized in that: described positive control is agkistrodon acutus pentastomiasis patients serum, and described negative control sample is a normal human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105023853A CN102288754A (en) | 2010-10-09 | 2010-10-09 | Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105023853A CN102288754A (en) | 2010-10-09 | 2010-10-09 | Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102288754A true CN102288754A (en) | 2011-12-21 |
Family
ID=45335344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105023853A Pending CN102288754A (en) | 2010-10-09 | 2010-10-09 | Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102288754A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033630A (en) * | 2012-12-20 | 2013-04-10 | 中国海洋大学 | Kit for qualitative detection of lipovitellin of cyprinid fish and application thereof |
| CN103323448A (en) * | 2013-01-20 | 2013-09-25 | 温州医学院 | Horseradish peroxidase black developer, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5843981A (en) * | 1994-04-12 | 1998-12-01 | Miller; Jeffery D. | Method for killing dust mites and preventing associated allergies |
| WO2007133093A2 (en) * | 2006-05-16 | 2007-11-22 | Biozymatic Sus | Method for decomposing organic waste |
-
2010
- 2010-10-09 CN CN2010105023853A patent/CN102288754A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5843981A (en) * | 1994-04-12 | 1998-12-01 | Miller; Jeffery D. | Method for killing dust mites and preventing associated allergies |
| WO2007133093A2 (en) * | 2006-05-16 | 2007-11-22 | Biozymatic Sus | Method for decomposing organic waste |
| WO2007133093A3 (en) * | 2006-05-16 | 2008-04-10 | Biozymatic Sus | Method for decomposing organic waste |
Non-Patent Citations (3)
| Title |
|---|
| SHAO-HONG CHEN ET AL: "Multi-host Model-Based Identification of Armillifer agkistrodontis (Pentastomida), a New Zoonotic Parasite from China", 《PLOS NEGLECTED TROPICAL DLSEASES》, vol. 4, no. 4, 6 April 2010 (2010-04-06) * |
| 沈玉娟等: "尖吻蝮蛇舌状虫粗抗原诊断价值的初步研究", 《国际医学寄生虫病杂志》, vol. 37, no. 1, 31 January 2010 (2010-01-31) * |
| 陈韶红等: "卫氏并殖吸虫感染循环抗原检测方法的建立与应用", 《国寄生虫学与寄生虫病杂志》, vol. 25, no. 6, 31 December 2007 (2007-12-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033630A (en) * | 2012-12-20 | 2013-04-10 | 中国海洋大学 | Kit for qualitative detection of lipovitellin of cyprinid fish and application thereof |
| CN103033630B (en) * | 2012-12-20 | 2014-10-22 | 中国海洋大学 | Kit for qualitative detection of lipovitellin of cyprinid fish and application thereof |
| CN103323448A (en) * | 2013-01-20 | 2013-09-25 | 温州医学院 | Horseradish peroxidase black developer, and preparation method and application thereof |
| CN103323448B (en) * | 2013-01-20 | 2018-01-16 | 温州医科大学 | A kind of horseradish peroxidase black developer and preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111733141B (en) | Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application | |
| CN211148669U (en) | S-antigen-based novel coronavirus 2019-nCoV antibody rapid detection kit | |
| Li et al. | Development of a convenient immunochromatographic strip for the diagnosis of infection with Japanese encephalitis virus in swine | |
| WO2008014709A1 (en) | Analysis device for biologicla sample | |
| CN103048459B (en) | Immune detection reagent for detecting respiratory syncytial virus | |
| CN102279269A (en) | Preparation method of cystatin C detection kit | |
| CN102731615B (en) | Detection reagent and detection method for PRRSV | |
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN102830229B (en) | Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof | |
| CN104142399B (en) | A kind ofly utilize colloidal gold immunochromatographimethod technology quantitatively test strips detecting Serum Pepsinogen and its preparation method and application | |
| CN105675866B (en) | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| CN109900902A (en) | A kind of porcine pseudorabies virus gB blocks ELISA antibody assay kit and its application | |
| CN1877331A (en) | Foot-and-mouth disease virus detecting test paper tape and its preparation method and using method | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN101592660A (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit | |
| CN102288754A (en) | Kit adopting specific polyclonal antibody for detection of agkistrodon acutus tongue-shaped worm circulating antigen | |
| CN103364546B (en) | A kind of kit and method detecting Furaxone metabolite | |
| CN102749446A (en) | Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody | |
| CN102980997B (en) | EB virus capsid antigen IgM antibody colloidal gold method detection reagent and preparation method thereof | |
| CN108761091A (en) | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof | |
| CN102279259A (en) | Diagnostic reagent kit for paragonimiasis detection and preparation method thereof | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody | |
| CN101498726A (en) | Colloidal gold test paper card for detecting enrofloxacin medicament residue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C05 | Deemed withdrawal (patent law before 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111221 |