CN101315368B - ELISA reagent kit for on-site detection - Google Patents
ELISA reagent kit for on-site detection Download PDFInfo
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- CN101315368B CN101315368B CN 200810063583 CN200810063583A CN101315368B CN 101315368 B CN101315368 B CN 101315368B CN 200810063583 CN200810063583 CN 200810063583 CN 200810063583 A CN200810063583 A CN 200810063583A CN 101315368 B CN101315368 B CN 101315368B
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Abstract
The invention discloses an ELISA kit used for on-site detection. The kit comprises a kit body; an antibody detection plate, a sample dilution reagent, positive/negative contrast, a cleaning reagent, an enzyme conjugate reagent, a substrate reagent, a stopping reagent, a disposable dropper and a cover plate membrane are arranged in the kit body; the substrate reagent is composed of a substrate, a substrate diluent and an indicator II; the substrate diluent is a concentrated solution; the sample dilution reagent, the cleaning reagent, the enzyme conjugate reagent, the stopping reagent, the substrate and the indicator II are all solid. The ELISA kit has the advantages of great portability, convenient use and high safety. Furthermore, the requirement of the on-site detection can be met.
Description
Technical field
The present invention relates to a kind of ELISA kit for Site Detection.
Background technology
Enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) is a kind of detection technique that the immunological response with the catalytic reaction of enzyme and antigen-antibody combines.Because the height selectivity of antigen, antibody response, the catalytic efficiency of enzyme is very high in addition, has indirectly amplified immunoreactive result, therefore can make assay method reach very high specificity and sensitivity.In recent years, ELISA with its sensitivity, accurately, the characteristics such as good reproducibility, no radioactivity pollute, be widely used in the various aspects such as virus, bacterium, hormone, medicament residue and detect.
But the ELISA kit that uses at present is liquid because of reagent such as antibody, enzyme labeling things, thereby need to hide in refrigerator and cooled and preserve, and also needs to adopt the cooling measures such as ice bag, insulation can in case the kit inefficacy in the mailing process; And usually need before use this human of user experiment reagent commonly used to prepare some damping fluids temporarily; The ELISA reagent of alkali phosphatase enzyme mark lacks suitable indicator at present at present, and operating process is made mistakes easily; It is highly basic (3mol/l NaOH) with stop buffer usually, has corrosivity; Process of the test needs the pipettor of purchasing price costliness, thereby causes the ELISA kit only to be confined to be used by the testing staff that the ELISA operating experience is arranged in laboratory with good conditionsi.
In sum, existing ELISA kit exists following deficiency:
1, contains the plurality of liquid reagent such as antibody-solutions, enzyme labeling thing solution, Sample Dilution damping fluid (concentrate) because of kit, make kit need stored refrigerated, cause carrying, posting inconvenience; And kit repeatedly rising again in use make the susceptible to of tiring of antibody and enzyme, repeatedly easily causes damage in the process of draw solution and pollute, sometimes because having a power failure or breakdown of refrigerator can cause all kits to lose efficacy.
2, biological safety is poor: the Quality Control sample of plant virus detection kit commonly used is to make by positive or negative sample extraction thing is directly carried out freeze drying at present.Although virus easily is inactivated under the drying condition in normal temperature, if carry out vacuum drying after freezing, then can make viral long-term surviving again.
3, ELISA is the very meticulous test of a kind of operation requirements, the ELISA reagent of alkali phosphatase enzyme mark lacks suitable indicator at present at present, operating process is made mistakes easily, especially in TSA-ELISA (tri-anti sandwich type) kit, contain and detect antibody and two kinds of valuable reagent of colourless trace of enzyme mark anti-antibody, if distinctive mark in addition significantly not, easier making mistakes.
4, the 3mol/L NaOH of commonly using at present stops the alkaline phosphatase enzyme reaction, and the result can only keep a few hours, and NaOH has corrosivity.
Summary of the invention
The above-mentioned technical matters that the present invention will solve also provides a kind of ELISA kit for Site Detection, and this ELISA kit carries, uses more convenient, and security is good, and can satisfy the Site Detection demand.
In order to solve the problems of the technologies described above, the invention provides a kind of ELISA kit for Site Detection, comprise box body, be provided with antibody test plate, Sample Dilution reagent, male/female contrast, washing reagent, enzyme conjugates reagent, substrate reagent, termination reagent, disposable dropper and cover plate film in box body, substrate reagent is comprised of substrate, substrate thinning agent and indicator II; The substrate thinning agent is concentrate, and Sample Dilution reagent, washing reagent, enzyme conjugates reagent, termination reagent, substrate and indicator II are solid-state.
Improvement as the ELISA kit for Site Detection of the present invention: the male/female contrast is the Quality Control sample through virus/germ deactivation, and used inactivator is beta-propiolactone, and inactivator is 1: 1000～1: 6000 with the volume of sample ratio.
Further improvement as the ELISA kit for Site Detection of the present invention: kit is the double antibodies sandwich type, and enzyme conjugates reagent contains enzyme labelled antibody and enzyme conjugates thinning agent, adds indicator I in the enzyme conjugates thinning agent.Perhaps kit is the tri-anti sandwich type, and enzyme conjugates reagent contains detection antibody, enzyme mark anti-antibody and enzyme conjugates thinning agent; With indicator I nothing to do with albumen generation hydrophobic binding, will detect again antibody or enzyme mark anti-antibody and be kept in conjunction with the indicator I after processing with above-mentioned first.
Further improvement as the ELISA kit for Site Detection of the present invention: the enzyme conjugates thinning agent is diluted to conventional enzyme conjugates dilution buffer liquid when reality is used, the concentration of indicator I in enzyme conjugates dilution buffer liquid is 0.01mg/mL～0.1mg/mL, and indicator I is that methyl orange, methyl red, phenol red, bromcresol green, bromine thymol blue, tea phenol are green, acid chrome blue K or methylene blue.
Further improvement as the ELISA kit for Site Detection of the present invention: the substrate thinning agent is diluted to conventional substrate dilution when reality is used, indicator II is malachite green, add before use in the substrate dilution, indicator II concentration in the substrate dilution is 0.05mg/mL～0.06mg/mL.
Further improvement as the ELISA kit for Site Detection of the present invention: antibody alkali phosphatase enzyme mark, stop reagent and be diluted to stop buffer when reality is used, described stop buffer is the aqueous solution that contains 0.5～2.0mol/L sodium carbonate and 0.1g/L～5g/L sodium sulphite.
Further improvement as the ELISA kit for Site Detection of the present invention: the cover plate film is that specification is the waterproof shading pressure sensitive membrane of 8cm * 12cm.
Further improvement as the ELISA kit for Site Detection of the present invention: disposable dropper comprises 1～3mL disposable plastic scale dropper and the little dropper of 0.20～0.50mL disposable plastic scale.Can divide 2～4 bags of packings, be used for moving liquid, piping and druming solution makes agent dissolves, and increases the anti-seismic performance of kit.
Key of the present invention is: preserving efficiently with convenient and practical solid-state form on the basis of detecting antibody and enzyme conjugates, again other assembly and the method for kit are carried out improvement and bring new ideas, preserve and have high sensitivity, a stable testing result purpose but make whole ELISA kit reach normal temperature.Because being, antibody and enzyme have bioactive macromolecular substances, its complex structure, many factors such as high/low temperature, contact under certain condition with indicator, plastics etc. and all may make its activity decreased or forfeiture, thus existing technology the user on hand all with liquid, stored refrigerated.Be in 94103930.7 patents at application number, antiserum directly is added on the filter paper have other irrelevant albumen or other material to exist in the antiserum, it is relatively less that antibody titer is reduced, and the antiserum relative low price; But with the high-purity antibody of modern biotechnology preparation, directly can cause that with the filter paper preservation antibody titer reduces and the redissolution effect is relatively poor, differences between batches are large.Therefore, the inventor has obtained Site Detection ELISA kit of the present invention on the basis through great many of experiments.
ELISA kit for Site Detection of the present invention has following characteristics:
In the box except the substrate thinning agent is concentrate, other reagent all provides with solid-state form, make kit of the present invention at normal temperatures the long period deposit, increased the tolerance range of kit to temperature.
2. the male/female contrast prevents from causing because of carrying of Quality Control sample the propagation of virus/germ for the Quality Control sample through virus/germ deactivation.Therefore not only increase biological safety, can also keep preferably the antigenicity of positive control.
3. prevent that mistake from occuring: in the enzyme conjugates thinning agent of double antibodies sandwich ELISA kit, added indicator I.In the detection antibody of tri-anti sandwich ELISA type kit or enzyme mark anti-antibody, effectively overcome the hydrophobic effect between antibody and the indicator I, successfully added indicator I.Found 8 kinds do not affect detect effect indicator I as the enzyme conjugates indicator, indicator I can select that methyl orange, methyl red, phenol red, bromcresol green, bromine thymol blue, tea phenol are green, acid chrome blue K or methylene blue.
4. prevented from because between indicator I and antibody (detecting antibody or enzyme mark anti-antibody) hydrophobic binding occuring antibody titer being reduced.
5. a kind of suitable substrate indicator (being indicator II)------malachite green and using method have been found.
6. invent a kind of new alkaline phosphatase stop buffer------and contained the aqueous solution of 0.5～2.0mol/L sodium carbonate and 0.1g/L～5g/L sodium sulphite.
7. replace hatching at present the wet box of use with repeatedly used waterproof shading pressure sensitive membrane.
8. provide disposable dropper to replace expensive pipettor in the kit, thereby further reach the target of Site Detection.And the anti-seismic performance of increase kit.
In the present invention, except Sample Dilution reagent and washing reagent existed with the form of pack, remaining every kind reagent left in the corresponding reagent bottle separately.All reagent bottles are all with sign pasting and knowledge waterline.The sign pasting sign content contains the simple and easy code of reagent and reagent name, and the simple and easy code of reagent forms for numeral or by numeral and letter, and reagent is taken from small to large by code digit; Identical numeral, the reagent of the code of different letters uses simultaneously.So design is intended to guide the user of kit to operate in an orderly manner, prevents that disorder from causing the failure of an experiment with misuse reagent.Knowing waterline (namely adding the water scale mark) as to add water for convenient to bottle, when waterline stops to add water when knowing waterline, namely finishes constant volume process in the original reagent bottle.Sample Dilution reagent and washing reagent (with the pack of solid form, to reduce kit weight, prolonging storage life) utilize the pure water of modal 500～600mL and body as reagent solvent and container.
Adopt the ELISA kit for Site Detection of the present invention, can make ELISA test also can carry out Site Detection leaving in the situation of fixing laboratory facility condition.ELISA kit of the present invention can be divided into following 2 kinds: double antibodies sandwich ELISA kit, tri-anti sandwich ELISA (TAS-ELISA) kit.At present, tri-anti sandwich ELISA (TAS-ELISA) method has primacy in specificity and sensitivity, but the operation of prior art is more loaded down with trivial details, and the chance of making mistakes is many, apply the present invention to the TAS-ELISA kit, the simplification degree and the error rate that detect operation reduce more obvious.
At the ELISA kit for Site Detection of the present invention, the male/female contrast is the male/female Quality Control sample through virus/germ deactivation; Make its forfeiture infection ability, and only keep antigenicity, preventing the propagation of pathogenic bacteria/virus, thereby improved the biological safety of kit.Add indicator at enzyme conjugates reagent and substrate reagent etc. in without color reagent, in order to reduce the chance of occurrence of mistake, can also stop to use hazardous chemical, thereby improved the stability of test.
During actual the use, the experiment equipments such as the cover plate film that provides in the kit and disposable dropper are provided the testing staff, are equipped with voluntarily scissors, pure water, cup, face tissue and simple and easy grinding or pulverizing tool again, just can be in the production base or the field finish test; And need not as existing ELISA kit, must in the laboratory, finish.Therefore, the present invention has the advantages such as applicability is strong, easy to operate, with low cost.
Below, the content of the ELISA kit for Site Detection of the present invention is done concrete statement:
1, during Sample Dilution reagent is packed a pack into, is saved with solid-state form.The method for making of this Sample Dilution reagent is as follows:
After in basic dilution, can adding a certain amount of antiseptic and inhibiting bacteria function agent, form behind concentrated the curing again.During actual the use, only need all reagent in the packing is dissolved in one bottle of pure water; Can form water-soluble Sample Dilution reagent.
The basis dilution is character and experiment purpose and decide (this is prior art) per sample, and the antiseptic and inhibiting bacteria function agent can be selected penicillin, streptomysin or Sodium azide etc.
But the Sample Dilution reagent normal temperature among the present invention was preserved more than 12 months, if in cleaning condition lower seal packing, storage life is longer; But the use liquid stored refrigerated after the dilution was put more than 1 month.
2, antibody test plate, its preparation method is as follows:
According to conventional method: be 0.05mol/L～0.10mol/L with ionic strength, pH9.0～pH9.6 carbonate buffer solution dilution coated antibody, an amount of coated antibody solution is added on the solid phase carriers such as polystyrene ELISA Plate, Polyvinylchloride ELISA Plate or nitrocellulose filter, and 4 ℃ are spent the night.After adding phosphate buffer and washing plate, pat dry next day, (with the irrelevant protein solution sealing of coated liquid measure 150%～200% volume ratio) (this is prior art).Then, in silica gel drier, place and spend the night, add drying agent and pack.
Antibody test plate among the present invention can be stablized preservation more than 18 months.
3, male/female contrast:
Positive control is packed in the reagent bottle (this reagent bottle for preserve positive control special-purpose), is saved with solid-state form.The method for making of this positive control is as follows:
Get positive, add sample extract homogenate usefulness, the double-layer sterile filtered through gauze is got filtrate or centrifugal, gets supernatant; , through virus/germ deactivation, freeze drying, packing adds drying agent and preserves.Add water to before use the knowledge waterline, dissolving can be used.
In like manner, negative control is packed in the reagent bottle (this reagent bottle for preserve negative control special-purpose), is saved with solid-state form.The method for making of this negative control is as follows:
Get negative sample, add the homogenate of sample extract, for preventing the propagation of other unknown virus/germ, also need after deactivation the same positive control of its processing procedure.
4, washing reagent:
Washing reagent is packed in the pack, is saved with solid-state form.The method for making of this washing reagent is as follows:
It is the anhydrous reagent that mixed phosphate, Tween-20 etc. form, and the above-mentioned anhydrous reagent of water-soluble solution of specified amount before use namely can be made into pH value, lavation buffer solution that ionic strength is stable.
5, enzyme conjugates reagent:
Enzyme conjugates reagent can be divided into double antibodies sandwich ELISA type and tri-anti sandwich ELISA type.
Double antibodies sandwich ELISA type (DAS-ELISA) contains enzyme labelled antibody and enzyme conjugates thinning agent.
Tri-anti sandwich ELISA type (TAS-ELISA) contains detection antibody, enzyme mark anti-antibody and enzyme conjugates thinning agent.
Method for making is specific as follows:
The interim solid-state carrier of Dispersal risk: enzyme mark anti-antibody or detection antibody are attached to solid-state carrier and preserve, interim solid-state carrier can be selected absorbent cotton, filter paper, plastic containers or other solid material, but must make it just simply to adhere to enzyme labelled antibody through suitable processing, chemical reaction does not occur, and both can separate in solution.Preparation process: the filter paper small pieces that will be twisted into globular absorbent cotton, are cut into fixed size and shape in containing the albuminous enzyme labelled antibody dilution of 1g/L～5g/L 20 ± 5 ℃ soaked 2～4 hours, take out filter paper and in lavation buffer solution, embathe 4～6 times, dry in the shade; The little centrifuge tube of plastics is closed with conventional albumin solution or the turbid fluid-tight of skimmed milk power, clean 4～6 times with lavation buffer solution, dry in the shade.
Detect antibody and drip respectively on different interim solid-state carriers with enzyme mark anti-antibody solution (antiseptic that contains 0.2g/L～1g/L), in the container of silica-gel desiccant, be controlled at≤20 ℃, dry in the shade in 4 hours, put into the same container that thinning agent is housed.Thinning agent contains the antiseptic of indicator I that (by using the liquid volumescope) comprise 0.01～0.1g/L, 0.5～50g/L sealer, 1～4g/L, and all the other are 10～30g/L stabilizing agent and basic damping fluid, make solid-state, packing.Adding water to before use the scale solubilising reagent can use.Enzyme conjugates system is done the process lime light: the one, and the concentration of enzyme labelled antibody and detection antibody (being used for TAS-ELISA) should not be excessively low, can consider loss at the concentrate more than 200 times that uses liquid, otherwise loss must be counted; The 2nd, the amount that enzyme labelled antibody drips will make liquid be covered with whole carrier not overflow again, as on the treated middling speed qualitative filter paper sheet of 6mm*6mm, drip 6～10 microlitres/sheet at every turn and be advisable; The 3rd, for guarantee enzyme labelled antibody be controlled at≤20 ℃, inherent cleaning condition dried in the shade as early as possible in 4 hours, in case rotten or sex change.
ECI reagent preparation: make first concentrate and regulate pH value, packing (or again freeze drying after the packing) (namely is reduced into enzyme conjugates dilution buffer liquid) behind the thin up and contains 1g/L～5g/L haemocyanin, 20g/L spreading agent, 0.2g/L～5g/L antiseptic bacteriostatic agent and 0.01g/L～0.1g/L indicator in every liter of dilution.
Indicator I in the described antibody diluent (ECI) is that 0.01g/L～0.1g/L methyl orange, methyl red, phenol red, bromcresol green, bromine thymol blue, tea phenol are green, acid chrome blue K or methylene blue.Indicator I select to require: color is easily distinguished and is not affected testing result (avoid using the indicator that may affect testing result, such as brilliant green and dimethyl diaminophenazine chloride may cause false positive results or blank value is increased).Preparation process: take by weighing that methyl orange, phenol red, tea phenol are green, acid chrome blue K or methylene blue 0.10 gram add water 100mL dissolving; 0.10 gram methyl red, bromcresol green or bromine thymol blue add water to 100mL with 0.1～0.2mL 3mol/L NaOH dissolving, adding final concentration again in ECI reagent is the indicator of 0.01g/L～0.1g/L.
In TAS-ELISA, indicator I can also be placed on the same filter paper with detecting antibody, detect antibody and enzyme labelled antibody with convenient the differentiation.Concrete preparation method: dissolve in 2g/L～5g/L haemocyanin in the indicator solution of 1 grams per liter, get an amount of dropping and on little filter paper, dry in the shade first, drip again and detect antibody, dry in the shade.Should avoid the indicator of undressed high concentration directly to contact for a long time with the detection antibody of high concentration, hydrophobic effect occurs for both can make antibody titer reduce.
6, substrate reagent
Contain substrate sheet, substrate thinning agent and indicator II.
The substrate thinning agent refers to the basic reagent concentrate that (usually containing magnesium salts, synergistic agent), adding preservative agent was made, packing; Substrate uses tablet or pulvis.But substrate sheet, substrate thinning agent and indicator II packing or close put.
The present invention announces a kind of ELISA indicator---malachite green of alkali phosphatase enzyme mark simultaneously.Malachite green color change interval pH value 11.0 (green)～13.5 (colourless).Adding method: behind acetonitrile or acetic acid equal solvent dissolving malachite green crystal, drip on carrier (normally filter paper or container), dry in the shade, keep in Dark Place.
In using the ELISA course of reaction of alkali phosphatase enzyme mark, alkaline phosphatase enzymatic para-nitro-pheneye phosphate (p-nitropheny phosate, pNPP), the phosphoric acid p-nitrophenyl can be hydrolyzed into phosphate and p-nitrophenol under the effect of alkaline phosphatase.In certain concentration of substrate scope, the hydrolysate p-nitrophenol of phosphoric acid p-nitrophenyl can cause the rising of 405nm place absorbance, and its climbing speed is directly proportional with the alkaline phosphatase enzyme activity.
Alkaline phosphatase
Phosphoric acid p-nitrophenyl (colourless)+H
2O----------→ phosphate+p-nitrophenol (yellow)
Chromogenic substrate solution: pNPP is dissolved in the 0.1mol/L diethanolamine buffer (pH 9.8) that contains 1mmol/L MgCl with the concentration of 10mmol/L, can uses; But this solution is colourless, and easy leakage adds or mistake adds, and adds 0.05～0.06g/L malachite green and makes solution be blue-green.When stopping enzymatic reaction, add an amount of sodium carbonate-sulfite solution or 1～3mol/L sodium hydroxide solution, the light absorption of pNPP strengthens under the alkali condition, when PH＞11 can make the rapid inactivation of alkaline phosphatase, and make green malachite green be converted into the procrypsis malachite green, thereby same indicator plays indicative function simultaneously at interpolation substrate solution and stop buffer.
7, stopping reagent is solid, adds water to know the waterline use.
The present invention announces a kind of ELISA stop buffer prescription simultaneously: in the ELISA of alkali phosphatase enzyme mark kit, stopping reagent is 0.5～2.0mol/L sodium carbonate 0.1g/L～5g/L sodium sulfite solution.
In the ELISA test of alkali phosphatase enzyme mark, usually make the alkaline phosphatase enzyme deactivation with 3mol/L NaOH (PH 13.5～13.7) solution, stop enzymatic reaction, but NaOH is corrosive substance; Also there is report to use 0.5mol/L sodium carbonate liquor (PH 11.6～11.8) as stop buffer, but effect is not as the former, if add malachite green as indicator in the substrate dilution, the 0.5mol/L sodium carbonate liquor can't make malachite green be converted into rapidly the procrypsis malachite green.Evidence adds at 0.5～1.0mol/L sodium carbonate liquor and (adds 5～100mg) sulphite in every 10mL solution on a small quantity, not only security is higher than using NaOH, and sodium carbonate-sulfite solution can make the malachite green solution of higher concentration fade rapidly, and the effect, the stability that stop enzymatic reaction all obviously are better than sodium hydroxide solution.Add in every 10mL1.0mol/L sodium carbonate liquor the 10mg sodium sulphite as stop buffer and 3mol/L NaOH contrast test (sample number is as 12) result as: in 1 hour, sun/negative Quality Control sample absorbance value odds ratio the latter increases by 9.7%, and fluctuation range only is the latter's 32%; The blank group of sodium carbonate after four days-sodium sulphite absorbance value increases by 16.85%, and sun/negative Quality Control sample absorptance reduces by 20%, and NaOH group then blank absorbance value increases by 34.99%, and sun/negative Quality Control reduces by 37% than absorbance value; The former makes the malachite green fading rate also faster than the latter simultaneously.As seen sodium carbonate-sodium sulphite stop buffer all is being better than the NaOH stop buffer aspect experiment effect and the security, and the sodium hydrate solid deliquescence that easily absorbs water between the reagent storage life, and (anhydrous) sodium carbonate-(anhydrous) sodium sulphite potpourri is relatively stable.
8, the cover plate film is not glued membrane of 8cm*12cm shading waterproof, can repeatedly use under wet environment.(8cm * 12cm) waterproof shading pressure sensitive membrane replaces wet box commonly used to hatch as the cover plate film in the designed, designed customization, this incubation method can be used in any ELISA test, the method is hatched the advantage of comparing with wet box: light weight not only, it is little to take volume, have certain shaded effect, and can avoid rocker or open that the box process shook ambassador's liquid splash and the pollution that causes; After hatching, the bottom of ELISA Plate is dry, need not the globule in the bottom of using the machine-readable front careful wiping ELISA Plate of microplate reader; It is more convenient to observe the colour developing degree.
9, disposable dropper comprises tens of of 1～3mL disposable plastic scale dropper number and 0.2～0.5mL disposable plastic scales.Make things convenient for Site Detection to use and increase kit transportation shock resistance used as moving the liquid instrument.
All reagent are sequentially put by use in box, and during use, each reagent was adding water to the knowledge waterline in several minutes in order before use, use after the dissolving.Certainly, Sample Dilution reagent and washing reagent are to use respectively the container of 500mL to carry out dissolving step.
The good effect that the present invention is useful is: but ELISA kit long period normal temperature is preserved, light, biological safety is high, and is simple to operate, easy to carry, and the mistake proofing that the kit R﹠D process also takes into full account design uses experimentation to be difficult for makeing mistakes, and need not buy chemical reagent and expensive pipettor, high specificity in conjunction with the ELISA test, high and the naked eyes observability as a result of susceptibility, thus make the ELISA kit be applicable to on-the-spot and the laboratories detection, be easy to apply on a large scale.
In order to prove the correctness of the inventive method, the inventor has made following example:
Example 1
This description of test virus can be survived at low temperatures
Get the plant extraction liquid that contains cymbidium mosaic virus, after liquid nitrogen freezes (196 ℃) and deposits 30 days, thaw, and this extract is spread upon on the wound of cymbidium mosaic virus heliophobous plant blade, after 3 weeks, be vaccinated other position of heliophobous plant and detected cymbidium mosaic virus, experiment shows that cymbidium mosaic virus frozenly still has an activity through-196 ℃, therefore is necessary the Quality Control sample is done suitable viral inactivation treatment.
Example 2
This experiment is that different ablation methods compare inactivation of virus (nucleic acid denaturation) effect and antigenicity loss (protein denaturation) degree.
Object of experiment: with inactivation of virus (nucleic acid denaturation), but need to keep its antigenicity.Test three kinds of method inactivation of virus cymbidium mosaic viruses, odontoglossum ring spot virus: 1. heat inactivation: 50 ℃, 56 ℃, 60 ℃, 62 ℃, 65 ℃, 68 ℃, 75 ℃, processed 30 minutes; 2. formalin-inactivated: 0.1% (volume ratio), 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 37 ℃ are processed 3. beta-propiolactone deactivation in 24 hours, 1: 1000,1: 2000,1: 4000,1: 6000,1: 8000, process 2 hours for 4 ℃.Inactivating efficacy: 1. more than 60 ℃, but heating in 30 minutes is complete inactivation virus all, but antigenicity also descends to some extent than before processing, more than 68 ℃, antigenicity disappears, 2. all but complete inactivation is viral for 0.2% above formaldehyde, but antigenicity also descends to some extent than before processing, and removing residue formaldehyde is removed trouble, 3. beta-propiolactone deactivation, 1: 1000～1: but 6000 equal complete inactivation viruses, antigenicity is also front without significant difference than processing, can better reach deactivation and keep antigenic effect with beta-propiolactone by contrast, and residual beta-propiolactone got final product complete hydrolysis in 2.5 hours 37 ℃ of water-baths.
Example 3,
Originally it is effective to experimental results show that sodium carbonate-sodium sulfite solution and traditional 3mol/L NaOH stop.
Add the 10mg sodium sulphite in every 10mL1.0mol/L sodium carbonate liquor and compare test (sample number is as 12) as stop buffer and 3mol/L NaOH, the result is: in 1 hour, sun/negative Quality Control sample absorbance value person increases by 9.7%, and fluctuation range only is the latter's 32%; The blank group of sodium carbonate after four days-sodium sulphite absorbance value increases by 16.85%, and sun/negative Quality Control sample absorptance reduces by 20%, and NaOH group then blank absorbance value increases by 34.99%, and sun/negative Quality Control reduces by 37% than absorbance value; The former makes the malachite green fading rate also faster than the latter simultaneously.As seen sodium carbonate-sodium sulphite stop buffer all is being better than the NaOH stop buffer aspect experiment effect and the security, and the sodium hydrate solid deliquescence that easily absorbs water between the reagent storage life, and (anhydrous) sodium carbonate-(anhydrous) sodium sulphite potpourri is relatively stable.
Embodiment
Embodiment 1
TAS-ELISA detects cymbidium mosaic virus (Cymbidium mosaic virus, be called for short CyMV) preparation and the use of Site Detection kit, contain in the kit 1. the antibody test plate, 2. Sample Dilution reagent, 3. washing reagent, 4. Quality Control thing (sun/negative control), 5. enzyme conjugates reagent (comprise and detect antibody, enzyme mark anti-antibody, thinning agent), 6. substrate reagent, 7. stop reagent, 8. some utility appliance.
1. concrete preparation process is as follows:
1.1 antibody test plate:
The coated damping fluid of preparation contains Na
2CO
31.59g/L, Na
2HCO
32.93g/L, NaN
30.2g/L regulate pH 9.6.Get coated antibody (the anti-cymbidium mosaic virus of rabbit, produced by U.S. agdia company) 50 microlitres, (be that per 50 μ L add 9.95mL and are coated with damping fluid with coated 1: 200 times of dilution of damping fluid, be mixed with 10mL and use liquid), on detachable type 96 hole high-affinity ELISA Plate (Canada produces), every reacting hole adds 100 microlitres, and 4 ℃ are spent the night.After adding the phosphate lavation buffer solution and washing plate 6 times, pat dry next day, places in silica gel drier and spend the night, and adds drying agent one bag, and with the sealing bag packing, sealing bag adds and is designated " 1. check-out console ", can preserve more than 12 months under the drying condition.
1.2 Sample Dilution reagent (GEB):
Kit include Sample Dilution reagent (GEB) one bag (about 27 ± 1g). contain Na among every bag GEB
2HPO
40.560 ± 0.005g, KH
2PO
40.100 ± 0.005g, NaCl 4.00g, KCl 0.10g, Na
2SO
30.65g, NaN
30.10g, egg powder 1.0 ± 0.2g, PVP K30 10 ± 1g, tween
-2010 ± 1g.
Preparation process divides 3 steps to finish:
1.2.1 prediction: accurately take by weighing Na with balance very much first
2HPO
40.560g, KH
2PO
40.100g, reach NaCl 4.00g, KCl 0.10g, Na
2SO
30.65g, NaN
30.10g, egg powder 1.0 ± 0.2g, PVP K30 10 ± 1g, Tween-20 10 ± 1g.With 500 milliliters of dissolved in purified water reagent, mix, with PH instrumentation amount pH value, suitably increase and decrease Na according to measured pH value
2HPO
4Or KH
2PO
4: preparation 0.10g/mL Na
2HPO
4With 0.02g/mL KH
2PO
4Solution is put into different burets respectively, when gained buffer solution ph＜7.40, suitably drips 0.10g/mL Na
2HPO
4Solution; When gained buffer solution ph＞7.40, suitably drip 0.02g/mL KH
2PO
4Solution is until PH adjusts in 7.38～7.42 scopes. and after this this batch amount of reagent accurately takes by weighing by adjustment amount.
1.2.2 packing: first packaging bag is after " peeling " on the balance, with tween
-20Directly be weighed in the packaging bag, accurately take by weighing other reagent (such as Na by adjustment amount again
2HPO
40.560 ± 0.005g namely is accurate to 0.005g; NaCl 4.00g namely is accurate to 0.01g), put into together packaging bag, vacuumize, sealing is preserved, and the reagent bag adds and is designated " 2. Sample Dilution reagent ", can preserve more than 18 months under the drying condition.
1.2.3 check: carry out totally the package encapsulation inspection and in proportion segmentation extract 2. number reagent bag and carry out PH and detect, when batch was less, the sampling observation number was no less than 5 bags, when Dang Jiashui is settled to 500 milliliters, PH answers scope between 7.36～7.44, actual measurement batch interior pH value fluctuation＜0.03; When adding different distilled water or pure water and be settled to 400～600 milliliters, actual measurement PH scope is between 7.32～7.46.
1.2.4 during actual the use, get first a container, 2. number reagent bag is carefully with 50～100 milliliters of dissolved in purified water; Add at last water and be settled to 500 milliliters, shake up; Namely get the working concentration of Sample Dilution damping fluid, this solution can be deposited 1 month at 2～6 ℃.
1.3 washing reagent (PBST):
Kit contains washing reagent one bag of 5 ± 0.05g, and its composition is: Na
2HPO
40.575 ± 0.005g, KH
2PO
40.100 ± 0.005g, KCl 0.10g, NaCl 4.0g, Tween-20 0.25 ± 0.05g.(prediction of PBST, packing, checking procedure are with the GEB preparation process), reagent bag add and are designated " 3. washing reagent ", can preserve more than 24 months under the drying condition.
Reality is dissolved the pure water of every bag washing reagent and 500 milliliters before use in container, get the lavation buffer solution (PBST) of PH7.35～7.45.
1.4 Quality Control thing (containing positive contrast and negative control):
Quality Control thing preparation process:
1.4.1 positive control: take by weighing 0.50 gram glucose, be dissolved in the above-mentioned Sample Dilution damping fluid of 100mL (GEB uses liquid), add the 5mL dimethyl sulfoxide (DMSO), mix, make extract → collection and contain CyMV orchid blade → add extract, with blade and extract weight: volume ratio (g/mL) homogenate on electric homogenate machine in 1: 10 → with double-layer sterile filtered through gauze → will filter fluid injection 3000rpm centrifugal 6 minutes → get supernatant, take supernatant: the beta-propiolactone volume ratio added inactivator (being that every 100mL supernatant adds 50 μ L beta-propiolactones) as 2000: 1, place inactivation of viruses → 37 ℃ water-bath in 120 minutes 2.5 hours for 4 ℃, hydrolysis beta-propiolactone → packing, every pipe 0.5mL is sub-packed in the 2.0mL freeze pipe → and-20 ℃ of pre-freezes spend the night →-46 ℃ of freeze dryings, seal preservation, freeze pipe adds and is designated " 4.-a positive control ".
Add water to before use the 0.5mL scale mark, make agent dissolves and mix and to use for several times with little suction pipe piping and druming.
1.4.2 negative control: first with used apparatus, with 70% alcohol-pickled or boiling water bath 30min, → collection does not contain CyMV orchid blade before the sampling, adds extract and grinds (subsequent preparation process is with the positive control preparation process).Freeze pipe adds and is designated " 4.-b negative control ".
1.5 enzyme conjugates reagent (contain the enzyme conjugates thinning agent, detect antibody and enzyme mark anti-antibody):
Detecting antibody is mouse-anti cymbidium mosaic virus (production of agdia company), enzyme mark anti-antibody is that alkaline phosphatase enzyme conjugates rabbit resists-the mouse-anti cymbidium mosaic virus, both are colourless liquid, the amount in per 1000 holes respectively is 0.525mL (the wherein loss reserve level of 0.025mL), is equivalent to per 100 hole 0.05mL.
1.5.1 preparation enzyme conjugates thinning agent (ECI): take by weighing Na
2HPO
40.620g, KH
2PO
40.100g, reach NaCl 4.00g, KCl 0.10g, NaN
30.10g, bovine serum albumin 1.0g, PVP K30 10+1g, Tween-20 0.25 ± 0.05g → with 80 milliliters of dissolved in purified water reagent, mix → use 0.10g/mL Na
2HPO
4Solution; Or 0.02g/mL KH
2PO
4Solution is regulated PH to 7.03 → be settled to 100mL, get 5 times of concentrate → tests of ECI: get the 2mL thin up to 10mL, namely getting enzyme conjugates dilution buffer liquid (PH 7.40～7.42) → every bottle of 2mL, to be sub-packed in total volume be 15mL, maximum scale is in the 10mL clear glass reagent bottle, and freeze drying is made and met loose shape solid.Get the enzyme conjugates thinning agent.Body adds and is designated " 5.-a enzyme conjugates thinning agent ", can preserve more than 12 months under the drying condition.
During actual the use, every part of enzyme conjugates thinning agent is added pure water is settled to the 10mL scale mark, dissolve fully to reagent for several times with suction pipe piping and druming, enzyme conjugates dilution buffer liquid.
In controllable temperature, dehumidifying, sterilize, prevent to finish under the clean environment of circulation of air the operation of following 1.5.2～1.5.4.
1.5.2 the interim solid-state carrier of Dispersal risk: in 10 milliliters of enzyme conjugates dilution buffer liquid, dissolve in 0.05 gram Bright brand skimmed milk power → the put into small pieces that two circle board middling speed qualitative filter papers are cut into 10mm*10mm, soaked 2 hours, during this time every 30 clocks stir once → take out filter paper in lavation buffer solution, embathe 2～3 times → spread out in clean glass slide, in exsiccator, dry in the shade, in exsiccator, save backup.Thereby obtain the interim solid-state carrier of antibody.
1.5.3 preparation contains indicator I and detects the filter paper of antibody: take by weighing in the methyl orange indicator solution that 0.02g BSA dissolves in 10 milliliter of 1 grams per liter, the filter paper of then ECI being processed immerses in the indicator solution, takes out, and the rear filter paper that dries in the shade is orange.Orange filter paper is separately lain on the clean slide, detection antibody at every filter paper minute 2 dropping 25 microlitres/sheets (is drawn antibody-solutions with pipettor, for the first time, add 12.5 microlitre antibody-solutions on the every filter paper, place a moment, about 5～10 minutes, behind liquid substantially dry on the filter paper, add again 12.5 microlitre antibody-solutions), 4～20 ℃ dry in the shade in exsiccator, get orange, and 2 slices/part pack, add and be designated " 5.-and the b antibody A ", under drying at room temperature, lucifuge condition, can preserve more than 12 months.
1.5.4 preparation contains the filter paper of enzyme mark anti-antibody: at the above-mentioned interim solid-state carrier that saves backup in exsiccator, divide the anti-antibody that drips the alkali phosphatase enzyme mark of 25 microlitres/sheets for 2 times, in exsiccator, dry in the shade, get white filter paper B, 2 slices/part pack, add and be designated " 5.-and c antibody B ", under drying at room temperature, lucifuge condition, can preserve more than 12 months.
1.5.5 when using, add respectively each 1 of orange A and white tablets B in per 5 milliliters of enzyme conjugates dilution buffer liquid, approximately enough the amount of 50 reacting holes.Soaked 5～10 minutes, and shook up and to use.
1.6 substrate reagent (containing substrate sheet, indicator II and substrate thinning agent):
1.6.1 substrate sheet: substrate is nitrophenyl phosphate, commercial substrate sheet 5mg/ sheet, and 2/box, store in the brown 1mL reagent bottle, add and be designated " 6.-a substrate ".Outsourcing black plastic film seals, keeps in Dark Place.Every available substrates diluted becomes 5mL, approximately enough the amount of 50 reacting holes.
1.6.2 the preparation of malachite green indicator sheet (indicator II): after taking by weighing the dissolving of 0.1g malachite green crystal → add 10mL acetonitrile (or 0.5mL acetic acid+0.5mL acetonitrile), get 10 μ L and inhale middling speed qualitative filter paper sheet in 6mm * 6mm) on, drying at room temperature under the lucifuge condition, make mazarine indicator sheet → 2 every part and store in the little sealed bag, add and be designated " 6.-the b indicator ".Wrap in the black plastic film with above-mentioned substrate sheet.Can preserve more than 12 months under the lucifuge condition
1.6.3 the preparation of substrate thinning agent: take by weighing MgCl6H
2O 0.1g, NaN
30.2g be dissolved in 10mL distilled water → add 97mL diethanolamine, 80mL distilled water stirs → regulates pH value to 10.03 → adding distil water with the 3.0mol/L hydrochloric acid solution to be settled to 200mL, namely make every bottle of 5 times of concentrate (20 ℃ for liquid) → 2mL and be sub-packed in the 10mL scale brown bottle → sealing, add and be designated " 6.-c substrate thinning agent ".Can preserve more than 12 months under the lucifuge condition.During use, add pure water to the 10mL scale mark, namely get the substrate dilution (using liquid to preserve 30 days at 2-6 ℃) of pH value 9.6～9.8.
1.6.4 during actual the use, the substrate thinning agent of 2mL is settled to 10mL (namely reverting to the concentration of substrate thinning agent stoste) with pure water, gets the PNP damping fluid; And then put into above-mentioned 2 substrate sheets and malachite green indicator sheet (indicator II), in the dark left standstill mixing 5～10 minutes.
1.7 termination reagent:
Take by weighing 1.20g Na
2CO
3, 10mg Na
2SO
3, store in the 10mL scale reagent bottle, add be designated that " 7. stop buffer reagent " is actual and use before, adding pure water to 10mL scale mark can use.
With above 1.2～1.7 described GEB, PBST, Quality Control thing, enzyme conjugates reagent, substrate reagent (except the substrate thinning agent) with stop reagent and pack together and add drying agent and preserve, substrate thinning agent brown bottle overcoat PE bubble bags is preserved.
1.8 utility appliance (containing waterproof membrane and disposable scale dropper):
Each kit contains 8 (1 bags of 2 of silver gray shading waterproof pressure sensitive membranes, 3mL disposable plastic scale dropper of the 8cm * 12cm of customization, be used for dilution reagent) and 0.2mL disposable plastic scale dropper 18～20 * 3 bags (being used for sample adding liquid), be put in respectively kit four sides, to increase the kit shock resistance.3mL disposable plastic scale dropper band is digital calibration, the scale nil sign on the little dropper of 0.2mL disposable plastic scale, and total volume is 0.25mL, and its first scale is 100 μ L, and the second scale is 200 μ L.
2 use procedures:
2.1 preliminary work
2.1.1 preparation sample extracting solution: get first a container, cut off 2. number reagent bag, GEB reagent is poured in the disposable water cup, divide with about 50～100mL pure water for several times residual reagent in the reagent bag to be washed water tumbler, be stirred to reagent with the 3mL dropper and dissolve fully; Mix; Add at last water and be settled to 500 milliliters, shake up; Get Sample Dilution damping fluid (GEB).
2.1.2 will be 4.-a positive control dried frozen aquatic products adds water (or above-mentioned GEB) before use to the 0.5mL scale mark, for several times reagent dissolved fully with little suction pipe piping and druming, gets positive quality control thing solution.
2.1.3 will be 4.-b negative control dried frozen aquatic products adds water (or above-mentioned GEB) before use to the 0.5mL scale mark, for several times reagent dissolved fully with little suction pipe piping and druming, gets negative Quality Control thing solution.
2.1.4 cut off 3. number reagent bag, the pure water of PBST reagent and 500 milliliters is dissolved to get cleansing solution (PH7.35～7.45), i.e. PBST damping fluid in container.
2.2 the extraction of sample
2.2.1 the extraction of sample
The plant tissue that----as far as possible selected symptom to occur is as sample, and usually with the test sample of blade as the ELISA experiment, other plant tissue also can be used for the ELISA experiment.
---add 10～30 times of GEB (plant that moisture is high adds 10 times of GEB such as iris, Bowring cattleya tissue, and the plant that moisture is lower adds 30 times of GEB such as orchid) in-sample and grind or homogenate, make sample solution to be measured.
Scissors and homogenate instrument are please thoroughly cleaned in----after every duplicate samples homogenate, in order to avoid cause the mutual pollution of sample room.
----each reacting hole needs the sample extraction dilution of 100 μ L, and preparation of samples is no less than 0.5mL solution to be measured and is beneficial to draw.
2.3 point sample and hatching
2.3.1 point sample
----in advance designs each testing sample, negative control and positive control reacting hole position separately.
----take apart be the packing of number check-out console 1., takes off enough reacting hole strips (other reacting hole strip is put back in the original packing bag, pulls on sealing and preserves).
----according to experimental design got 100 μ L (3, dropper the first scale is 100 μ L, the second scale is 200 μ L) sample extraction dilution with the little dropper of 0.2mL and added in the reacting hole of antibody test plate.
The negative Quality Control thing of----get 100 μ L solution adds in the respective reaction hole.
----get 100 μ L positive quality control thing solution add in the respective reaction hole.
2.3.2 hatch
----paste waterproof membrane at check-out console, and at waterproof membrane with palm applying light one time sealing reacting hole,
----pin check-out console at level table shaken check-out console 16～18 times gently with the circular motion form,
---hatched under-the room temperature 2 hours.
2.4 the preparation of enzyme labeling thing
----toward 5.-add pure water to the 10mL scale mark in a enzyme conjugates thinning agent bottle, blow and beat to reagent with dropper and dissolve fully, get the ECI damping fluid.
Detect----will contain antibody 5. orange-b filter paper A and enzyme labeling thing white 5.-c filter paper B is added in the above-mentioned ECI damping fluid, soaks 5～10 minutes, gets enzyme labeling thing dilution.
2.5 wash plate
After----hatch end, put at enzyme and carefully to take off waterproof membrane (not making fold, reusable 2～4 times), the reagent in the reacting hole is poured out, check-out console is tipped upside down on the thieving paper with residual liquid in the control dry hole.
----add 200～250 μ L the PBST damping fluid, capable of fast pouring afterwards, and on thieving paper with residual liquid in the control dry hole, repeat 6 times.
2.6 enzyme-added label dilution and hatching
----2.4 described enzyme labeling thing dilutions are shaken up;
----in each reacting hole adds 100 μ L enzyme labeling thing dilutions;
----at enzyme put on and sticked waterproof membrane, hatches under the room temperature 2 hours.
2.7PNP the preparation of substrate
Before----hatch finished 10～15 minutes, 6.-add pure water to the 10mL scale mark in the c 2mL substrate thinning agent bottle, shake up, get the PNP damping fluid.
----in the PNP damping fluid add PN 6.-a substrate sheet and b indicator sheet; In the dark left standstill 5～10 minutes, mixing gets the PNP substrate solution.
Attention: please don't directly contact PNP substrate sheet or substrate solution is exposed under the high light, directly contact or high light stimulation meeting cause background colour to disturb in the negative reaction hole.
2.8 wash plate, add the PNP substrate solution and hatch
2.8.1 wash plate
----with 2.5
2.8.2 add the PNP substrate solution
----in each reacting hole adds 100 μ L PNP substrate solutions (avoiding high light).
2.8.3 hatch
----at enzyme put on and sticked waterproof membrane, hatched 15～180 minutes in lucifuge, according to room temperature condition, obvious change color appears to positive quality control and till.
9 cessation reactions
----7. adding pure water to the 10mL scale mark in the stop buffer reagent bottle, shake up.
----when positive change color, hatch end after, in each reacting hole, add 100 μ L stop buffers.
10. result
----directly detects by an unaided eye or with microplate reader measurement result under the 405nm wavelength
Positive findings: obvious change color (being yellow) is arranged in the reacting hole.
Negative findings: do not have obvious change color in the reacting hole
Attention: only obtain positive findings in the positive quality control hole, when negative Quality Control hole change color did not occur, experimental result was only reliably.After adding stop buffer, stick the waterproof membrane result and in the dark can keep 2～3 days (as long as change color does not occur in negative Quality Control hole).
Embodiment 2
TAS-ELISA detects preparation and the use of CyMV Site Detection kit, below only states difference with embodiment 1: used carrier is the absorbent cotton bead; Detect antibody and do not contain indicator, add indicator I at enzyme mark anti-antibody; Used indicator I is acid chrome blue K; Indicator II preserves in little centrifuge tube.
1.5.2 the interim solid-state carrier of Dispersal risk: in 10 milliliters of enzyme conjugates dilution buffer liquid, dissolve in 0.05 gram Bright brand skimmed milk power → absorbent cotton the is twisted into bead of 3～4mm (mung bean size), soaked 2 hours, stirring once → embathe 4～6 times → control solid carbon dioxide every 30 clocks in lavation buffer solution during this time divides, spread out in clean glass slide, in exsiccator, dry in the shade, in exsiccator, save backup.Thereby obtain the interim solid-state carrier of antibody.
1.5.3 preparation contains the absorbent cotton bead that detects antibody: get above-mentioned interim solid-state carrier, each absorbent cotton bead drips the detection antibody of 25 microlitres, in exsiccator, dry in the shade, get white bead, 2 slices/part pack, add and be designated " 5.-b antibody A ", can preserve more than 12 months under dry, the lucifuge condition.
1.5.4 preparation contains the enzyme mark anti-antibody absorbent cotton bead of indicator I: take by weighing in the acid chrome blue K solution that 0.02g BSA dissolves in 10 milliliter of 1 grams per liter → each absorbent cotton bead drips in the 25 microlitre indicator solutions, be the enzyme mark anti-antibody antibody of purple → dropping 25 microlitres/sheet → in exsiccator, dry in the shade → add after drying in the shade and be designated " 5.-c antibody B ", can preserve more than 12 months under dry, the lucifuge condition.
1.5.5 when using, add respectively each 1 of white ball A and purple ball B in per 5 milliliters of enzyme conjugates dilution buffer liquid, soaked 5～10 minutes, with dropper piping and druming for several times, can use.
1.6.2 the preparation of malachite green indicator (indicator II): after taking by weighing the dissolving of 0.1g malachite green crystal → add 10mL acetonitrile, getting 10 μ L is put in the little centrifuge tube of 0.5mL, nitrogen dries up → 1 pipe/box under the lucifuge condition, add and be designated " 6.-the b indicator ".In the substrate sheet wraps in the black plastic film.Can preserve more than 12 months under the lucifuge condition.
Face the time spent, with a small amount of water or substrate dilution little centrifuge tube malachite green is dissolved, add in the lump mixing use in the substrate dilution with substrate.
Its use procedure is with embodiment 1.
Embodiment 3
DAS-ELISA detects preparation and the use of corium solani (Potato Leaf Roll Virus) (antibody is produced by agdia company) Site Detection kit.Below only state the difference with embodiment 1: 1, enzyme conjugates reagent only contains enzyme labelled antibody and enzyme conjugates thinning agent; 2, indicator is added in the enzyme conjugates thinning agent, and used indicator is methyl red.
1. prepare the enzyme conjugates thinning agent: take by weighing Na
2HPO
40.620g, KH
2PO
40.100g, reach NaCl 4.00g, KCl0.10g, NaN
30.10g, bovine serum albumin 1.0g, PVP K30 10 ± 1g, tween
-200.25 ± 0.05g → with 80 milliliters of dissolved in purified water reagent → adding 1g/L methyl red indicator 1mL, mix → use 0.10g/mL Na
2HPO
4Solution; Or 0.02g/mL KH
2PO
4Solution is regulated PH to 7.03 → be settled to 100mL, get 5 times of concentrate → tests of ECI: get the 2mL thin up to 10mL, namely getting enzyme conjugates dilution buffer liquid (PH 7.40～7.42) → every bottle of 2mL, to be sub-packed in total volume be 15mL, maximum scale is in the 10mL clear glass reagent bottle, and freeze drying is made and met loose shape solid.Get the enzyme conjugates thinning agent.Body adds and is designated " 5.-a enzyme conjugates thinning agent ", can preserve more than 12 months under the drying condition.During actual the use, with being settled to 10mL, solubilising reagent namely is mixed with enzyme conjugates dilution buffer liquid (ECI) with every part of enzyme conjugates thinning agent pure water.
2. Dispersal risk: first 1.5 milliliters of centrifuge tubes are spent the night with 1 milliliter of PBST sealing that contains the 2g/mL skimmed milk powder, with PBST clean 4～8 times → add 0.05 milliliter of enzyme labelled antibody → place 4 ℃ of airtight containers that are covered with silica-gel desiccant to place centrifuge tube to use enzyme labelled antibody fully to dry in the shade → centrifuge tube is sealed in 3～7 days, add sign.During use, add 1 ml of EC I and repeatedly blow and beat antibody with dropper and redissolve in ECI, and draw the antibody-solutions that redissolves and add in the enzyme conjugates thinning agent reagent bottle, shake up for subsequent use.
Using method is with reference to embodiment 1.
Embodiment 4
DAS-ELISA detects preparation and the use of odontoglossum ring spot virus (Odontoglossum ringspot virus is called for short ORSV) Site Detection kit.Test used antibody and produced by Dutch primediagnostic company, below only state difference with embodiment 1:
1, antibody test plate:
The dilution ratio of antibody is 1: 500 (20 μ L coated antibodies adds coated damping fluid and are diluted to 10mL).Check-out console adds 100 μ L with antibody sandwich → wash plate → every hole and contains the unnecessary binding site in 20～50g/L skimmed milk powder PBST capping hole → wash plate.
2. prepare the enzyme conjugates thinning agent: take by weighing Na
2HPO
40.620g, KH
2PO
40.100g, reach NaCl 4.00g, KCl0.10g, NaN
30.10g, bovine serum albumin 1.0g, PVP K30 10 ± 1g, Tween-20 0.25 ± 0.05g → with 80 milliliters of dissolved in purified water reagent → adding 1g/L methyl orange solution 1mL (or 1g/L methyl red/phenol red/bromcresol green/bromine thymol blue/acid chrome blue K/methylene blue 1mL, or the green 2mL of 1g/L tea phenol), mix → use 0.10g/mL Na
2HPO
4Solution; Or 0.02g/mL KH
2PO
4Solution is regulated PH to 7.03 → be settled to 100mL, get 5 times of concentrate → tests of ECI: get the 2mL thin up to 10mL, namely get in enzyme conjugates dilution buffer liquid (PH 7.40～the 7.42) → every bottle 2mL packing 10mL clear glass reagent bottle, orange ECI reagent is made in freeze drying.
3. preparation contains the filter paper of enzyme labelled antibody: two circle boards at a slow speed qualitative filter paper are cut into the small pieces of 6mm*6mm, soaked 2 hours with ECI, every 30 clocks stir once → lavation buffer solution embathe 2～3 times → dry in the shade, be placed on the clean slide → adding 10 microlitre antibody-solutions at every filter paper dries in the shade → 2 slices/part pack, add sign, under drying at room temperature, lucifuge condition, preserve.
During use, ECI reagent is added water to 10mL, solubilising reagent adds the filter paper that contains enzyme labelled antibody, soaks 5～10 minutes, shakes up and can use.
Using method is with reference to embodiment 1.
Embodiment 5, kit storage life of the present invention experiment:
Make cymbidium mosaic virus ELISA kit in January, 2007 by method of the present invention, antibody (agdia company, in July, 2006 production) term of life is in June, 2007, use room temperature, refrigeration, it is (original-pack to be saved in June, 2008 (press embodiment 1 described method operation) and control group under freezing+three kinds of conditions of refrigeration (wherein have three months-20 ℃ of preservations), 2～6 ℃ of cryopreserved liquid antibody term of life to 2008 year October, according to a conventional method operation) respectively to iris (sample number n=9), queen blue (n=2), Hymenocallis americana (n=11) and each one of male/female contrast detect, testing result: 4 groups all detect 8 samples and dye cymbidium mosaic virus is arranged, and absorbance value (OD) is without significant difference.As seen kit of the present invention obviously reduces the storage temperature conditional request, and the pot-life can reach more than 12 months.
Embodiment 6, kit of the present invention are estimated as a result with existing (stored refrigerated) kit, and accuracy compares
Gather the potato leaf of 13 different cultivars, the PSA corium solani ELISA kit (PSA: entire package type that uses respectively embodiment 3 described ELISA kits (using method is described with reference to embodiment 1) and existing agdia company to produce, include the ELISA reagent such as the check-out console, 0.15mL enzyme labeling thing of coated antibody, concentrated ECI damping fluid, 5 times of concentrated PNP substrate buffer solutions, 3MNaOH/ reaction stop solution, by its instructions operation, wet box is hatched) 13 samples are carried out the corium solani qualitative detection, the result is as follows:
Kit of the present invention detects 2 strong positive results, 3 weak positive findingses, 8 negative findingses; Negative control OD is respectively 0.096/0.098, and wherein the strong positive result No. 9 each hole OD of sample are respectively 1.764/1.801/1.786/1.779.
The agdia kit detects 2 strong positive results, 3 weak positive findingses, and 8 negative findingses, negative control OD are 0.104/0.097, wherein No. 9 sample OD are 1.668/1.832/1.794/1.812.Other sample OD exemplifies no longer one by one.
Two groups of testing results this shows that without significant difference kit of the present invention has identical accuracy with existing kit test result.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (6)
1. ELISA kit that is used for Site Detection, comprise box body, it is characterized in that: be provided with antibody test plate, Sample Dilution reagent, male/female contrast, washing reagent, enzyme conjugates reagent, substrate reagent, termination reagent, disposable dropper and cover plate film in box body, described substrate reagent is comprised of substrate, substrate thinning agent and indicator II; Described substrate thinning agent is concentrate, and described Sample Dilution reagent, washing reagent, enzyme conjugates reagent, termination reagent, substrate and indicator II are solid-state;
Described male/female contrast is the Quality Control sample through virus/germ deactivation, and used inactivator is beta-propiolactone, and inactivator is 1: 1000～1: 6000 with the volume of sample ratio;
Described kit is the double antibodies sandwich type, and described enzyme conjugates reagent contains enzyme labelled antibody and enzyme conjugates thinning agent, adds indicator I in the enzyme conjugates thinning agent;
Perhaps described kit is the tri-anti sandwich type, described enzyme conjugates reagent contains detection antibody, enzyme mark anti-antibody and enzyme conjugates thinning agent, make first indicator I nothing to do with albumen generation hydrophobic binding, will detect again antibody or enzyme mark anti-antibody and be kept in conjunction with the indicator I after processing with above-mentioned; Described irrelevant albumen is bovine serum albumin;
Described indicator I is that methyl orange, methyl red, phenol red, bromcresol green, bromine thymol blue, tea phenol are green, acid chrome blue K or methylene blue;
Described indicator II is malachite green.
2. the ELISA kit for Site Detection according to claim 1, it is characterized in that: the enzyme conjugates thinning agent is diluted to conventional enzyme conjugates dilution buffer liquid when reality is used, the concentration of described indicator I in enzyme conjugates dilution buffer liquid is 0.01mg/mL～0.1mg/mL.
3. the ELISA kit for Site Detection according to claim 2, it is characterized in that: the substrate thinning agent is diluted to conventional substrate dilution when reality is used, described indicator II adds in the substrate dilution before use, and indicator II concentration in the substrate dilution is 0.005mg/mL～0.06mg/mL.
4. the ELISA kit for Site Detection according to claim 3 is characterized in that: the antibody alkali phosphatase enzyme mark; Stop reagent and be diluted to stop buffer when reality is used, described stop buffer is the aqueous solution that contains 0.5～2.0mol/L sodium carbonate and 0.1g/L～5g/L sodium sulphite.
5. the ELISA kit for Site Detection according to claim 4, it is characterized in that: described cover plate film is that specification is the waterproof shading pressure sensitive membrane of 8cm * 12cm.
6. the ELISA kit for Site Detection according to claim 5, it is characterized in that: described disposable dropper comprises 1～3ml disposable plastic scale dropper and the little dropper of 0.20～0.50ml disposable plastic scale.
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| CN107941799A (en) * | 2017-12-20 | 2018-04-20 | 珠海科域生物工程股份有限公司 | A kind of fecal sample Test paper and preparation method thereof |
| CN112098654B (en) * | 2020-09-15 | 2023-07-18 | 武汉生之源生物科技股份有限公司 | Pepsinogen I/II assay kit and preparation method thereof |
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| CN2750319Y (en) * | 2004-11-15 | 2006-01-04 | 华中师范大学 | DNA-protein crosslinking quantitative determination kit |
| CN1877330A (en) * | 2005-06-10 | 2006-12-13 | 湖南景达基因有限公司 | Enzyme-linked immunologic diagnosis kit for core antigen of C type hepatitis virus and method for preparing same |
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| CN2694277Y (en) * | 2003-07-25 | 2005-04-20 | 北京望尔生物技术有限公司 | Streptomycin ELISA checking reagent case |
| CN2750319Y (en) * | 2004-11-15 | 2006-01-04 | 华中师范大学 | DNA-protein crosslinking quantitative determination kit |
| CN1877330A (en) * | 2005-06-10 | 2006-12-13 | 湖南景达基因有限公司 | Enzyme-linked immunologic diagnosis kit for core antigen of C type hepatitis virus and method for preparing same |
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