CN104569406B - A kind of Cordyceps enzyme-linked immunologic detecting kit and preparation method thereof - Google Patents
A kind of Cordyceps enzyme-linked immunologic detecting kit and preparation method thereof Download PDFInfo
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- CN104569406B CN104569406B CN201410804874.2A CN201410804874A CN104569406B CN 104569406 B CN104569406 B CN 104569406B CN 201410804874 A CN201410804874 A CN 201410804874A CN 104569406 B CN104569406 B CN 104569406B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention provides the preparation method of a kind of Cordyceps enzyme-linked immunologic detecting kit, comprise the following steps: S1, Cordyceps 70kDa glycoprotein extract and separate；S2, the preparation of monoclonal antibody；S3, the foundation of enzyme-linked immune detection method and assessment；S4, the assembling of test kit.Present invention also offers a kind of Cordyceps enzyme-linked immunologic detecting kit.The invention has the beneficial effects as follows: the authenticity of Cordyceps can be realized by Cordyceps enzyme-linked immunologic detecting kit.
Description
Technical field
The present invention relates to the qualification of Cordyceps, particularly relate to a kind of Cordyceps enzyme-linked immunologic detecting kit and system thereof
Preparation Method.
Background technology
" Chinese Pharmacopoeia " (version in 2010) regulation Cordyceps certified products is " section ergot fungus cordyceps sinensis bacterium
(Cordyceps sinensis (Berk.) Sacc.) colonizes in answering of the Stroma on Hepialidae insect larvae and larva corpse
Fit ".It has effect of the kidney invigorating lung benefiting, hemostasis and phlegm, with Radix Ginseng, Cornu Cervi Pantotrichum the most Chinese three big tonics.Cordyceps is raw
Long environment is special, resource scarcity, expensive, market occurs mix adulterant, adulterant in a large number, differentiates the most quickly and efficiently
The true and false, is to ensure that the matter of utmost importance that Cordyceps clinical drug safety effectively need to solve.Cordyceps adulterant specifically includes that (1)
Pretend to be after planting Cordyceps processing with it: nature is also distributed other Cordyceps substantial amounts of and is Cordyceps sinensis fungus and colonizes in various insecticide
The Stroma formed on body and the complex of polypide.It is reported, the whole world has now been found that Cordyceps kind more than 400, China it has been reported that
Cordyceps about 120 kinds, such as Cordyceps gunnii (Berk.) Berk., branch Cordyceps, Cordyceps liangshanensis Zang Hu et Liu, Cordyceps martialis Speg. etc..These " personation Cordycepses " are with Cordyceps gunnii (Berk.) Berk.
Fill puppet the most common.(2) weightening finish: insert bamboo let, tinsel, adheres to earth, injects, smears molten metal weightening finish, and saline soak increases
Heavily etc..(3) pressing mold " Cordyceps ": mainly add binding agent with starch, flour, Gypsum Fibrosum powder etc., meticulously press with Cordyceps mould
Dyeing processed forms.Part pressing mold " Cordyceps " is made the most true to nature, and layman is difficult to identification.In addition, it is grown on differently
The Cordyceps in district its price also difference is relatively big, is affected by the market propaganda and traditional view, and Tibetan area snow-coated plateau is given
Mystery flavour, generally believe " hide Cordyceps " performance optimal, praise highly Qinghai, the produced Cordyceps in producing region, Tibet, the market price is higher than " river
Cordyceps ".In China, Cordyceps is distributed mainly on Qinghai, Tibet, Sichuan, Gansu, Yunnan etc. and saves, wherein Sichuan, Tibet, green grass or young crops
Hai San saves and accounts for the overwhelming majority.
The authenticity present situation of Cordyceps: the discriminating of Cordyceps is only specified by existing " Chinese Pharmacopoeia " (version in 2010)
The check items such as source, properties and characteristics, assay, assay is with adenosine as index components, it is desirable to must not be less than containing adenosine
0.010%, working standard is far from the development and change meeting market today commodity.Its assay item index components gland
Glycosides specificity is not strong, and multiple Cordyceps adenosine content is more than certified products Cordyceps, is difficult to reflect sample to the assay of this composition
The true and false and degree best in quality.The research of Cordyceps chemical composition the most both at home and abroad is confined to a few species Cordyceps and winter worm
Summer grass to when variation analysis, little for differentiating the real and fake discrimination effect of Cordyceps.The nucleoside such as adenosine in Cordyceps
The content of class material in the past more uses thin layers of ultraviolet spectrophotometric or high effective liquid chromatography for measuring.Li Shao equality uses capillary tube
Electrophoresis method measures the content of 3 kinds of gradient elutions such as natural and Adenosine in Fermented Cordyceps, uridnine, guanosine, and becomes it
Change and investigated.Result proves: the nucleoside content in natural and Fermented Cordyceps is apparently higher than natural cordyceps, manually
In Cordyceps, the content difference of adenosine is notable, and height difference reaches 6 times.And the natural cordyceps of fresh collection is (as in Qinghai
The sample that cajaput, Huangzhong and Yunnan gather) in the content of adenosine etc. is extremely low even cannot be quantitative, and gather with the passing of time ucleosides and become
Divide content the highest.And the content of Adenosine in Fermented Cordyceps, guanosine and uridnine is without significant change.Prove the natural worm summer in winter
Grass can produce gradient elution during storing, and natural and Fermented Cordyceps Nucleosides has certain difference.
Adenosine is as the quality control index of Cordyceps, it is impossible to objectively respond the quality of natural cordyceps.Therefore, adenosine is in quality
Application in control is still worth research further.
The main composition of the technical barrier that the real and fake discrimination of Cordyceps always cannot be broken through, first Cordyceps
The trace element of cordycepin, Cordyceps polysaccharide, nucleosides material and Cordyceps, all kinds of scholars are unable to decide which is right.Secondly, Cordyceps
The most effective mode of discrimination method be that the Conventional wisdom used till today for a long time differentiates.Cause various pseudo-worms on market
The appearance of grass, has weightening finish, adulterates with bamboo let, and also have replaces Cordyceps with similar Cordyceps, allows people reflect especially
Not.
Therefore, the authenticity how realizing Cordyceps is those skilled in the art's problem demanding prompt solutions.
Summary of the invention
In order to solve the problems of the prior art, the invention provides a kind of Cordyceps enzyme-linked immunologic detecting kit and
Its preparation method.
The invention provides the preparation method of a kind of Cordyceps enzyme-linked immunologic detecting kit, comprise the following steps:
S1, Cordyceps 70kDa glycoprotein extract and separate；
S2, the preparation of monoclonal antibody；
S3, the foundation of enzyme-linked immune detection method and assessment；
S4, the assembling of test kit.
As a further improvement on the present invention, step S1 includes: by Liquid isoelectric focusing, protein electrophoresis, electroelution instrument
Cordyceps 70kDa glycoprotein glycoprotein is prepared, in this, as the mark in antigen, coating antigen and test kit Deng protein purification system
Quasi-product.
As a further improvement on the present invention, step S2 includes: by cell-fusion techniques, the preparation of antibody purification technology is anti-
Cordyceps 70kDa glycoprotein monoclonal antibody.
As a further improvement on the present invention, step S3 includes: set up Cordyceps enzyme-linked immune detection method and to it
Specificity, anti-matrix interference ability and repeatability are estimated.
As a further improvement on the present invention, step S4 includes: by the enzyme mark of pre-coated Cordyceps 70kDa glycoprotein
Plate, Cordyceps 70kDa glycoprotein standard solution, horseradish peroxidase-labeled Cordyceps 70kDa glycoprotein monoclonal anti
Body, nitrite ion and stop buffer are assembled into test kit.
Present invention also offers a kind of Cordyceps enzyme-linked immunologic detecting kit, by the winter as described in above-mentioned any one
The worm summer preparation method of grass enzyme-linked immunologic detecting kit is prepared from
The invention has the beneficial effects as follows: the true and false of Cordyceps can be realized by Cordyceps enzyme-linked immunologic detecting kit
Identify, anti-Cordyceps characteristic protein antibody for antigenic determinant heat-resisting, can quickly differentiate that the Cordyceps in sample is true
Puppet, applied widely, the beneficially guarantee of consumer legitimate right；Specificity is good, highly sensitive, and anti-matrix interference is effective,
Testing result accuracy is good, reproducible；The pre-treatment of sample is simple, detects simple to operate, and reagent is all with the form of working solution
There is provided, it is convenient to carry out the examination of great amount of samples.
Accompanying drawing explanation
Fig. 1 is Cordyceps 70kDa glycoprotein SDS-PAGE spectrum；
Fig. 2 is Cordyceps enzyme linked immunosorbent detection canonical plotting.
Detailed description of the invention
The present invention is further described for explanation and detailed description of the invention below in conjunction with the accompanying drawings.
The preparation of embodiment one target antigen.
After taking Cordyceps fruit cursive script part liquid nitrogen condition milling, take sample 10g, under ice bath, use 0.1M PBS buffering
Liquid (pH7.4, containing 0.5% mercaptoethanol, 0.1%Tween80) 100ml supersound extraction 12h.Centrifuging and taking supernatant, uses 60% to satisfy
With degree ammonium sulfate precipitation.Molten precipitate gone to by 8M carbamide, centrifugal, supernatant is carried out dialysed overnight, obtains crude separation sample.
The sample just separated uses vertical electrophoresis apparatus to carry out repurity: the sample just separated is used 4% concentration glue and
The separation gel of 12% carries out SDS-PAGE electrophoresis, uses pre-dyed protein standard substance as molecular weight reference simultaneously.80v electrophoresis 2h
After, cut, with reference to pre-dyed protein standard substance, the protein gel band that molecular weight is 70kd.After protein gel uses mortar pulverize,
Add electroelution instrument, use 7M carbamide 10A electric current eluting 8h.The rich protein collected in cap is dialysed, lyophilizing.Purification
The SDS-PAGE spectrum of 70kDa Cordyceps glycoprotein is as it is shown in figure 1, the Cordyceps 70kDa glycoprotein of preparation is used for
Immunogen, coating antigen and kit standard solution.
The preparation and purification of embodiment two anti-Cordyceps antibody.
By immunogen 6 weeks female balb/c Mus of immunity respectively of embodiment one preparation, often group 3.During first immunisation injection,
The immunizing antigen 100 μ L of 100 μ g/mL, fully emulsified with equivalent Freund's complete adjuvant respectively, abdominal cavity direct injection.It is spaced two weeks
After, take the antigen of sample, with 100 μ L Freund's incomplete adjuvant emulsifyings, same method is injected.
Cell merge before 1d or drew the same day neck put to death Kunming mouse, be immersed in 70% ethanol, body surface sterilization；Use pin
Fixing Kunming mouse on stencil plate, abdominal cut on superclean bench, provoke peritoneum with pincet, inject 5mL RPMI-1640 complete
Full culture fluid (added 15% hyclone by GIBICO RPMI-1640 basic culture solution and obtain), rubs abdominal cavity gently with hands,
Its internal liquid aseptic straw immigration 75mL HAT complete culture solution (is added by 74.25mL RPMI-1640 complete culture solution
Enter 0.75mL 100 × HAT liquid and obtain) in, mixing with suction pipe, spread 24 orifice plates, every hole adds 0.5mL, is placed in 37 DEG C of CO2 incubators
In.
Mouse orbit blood-letting, collects serum, draws neck to put to death, 70% alcohol-pickled sterilization body surface, and aseptic taking-up spleen is put into
In RPMI-1640 basic culture solution (purchased from GIBICO, article No. is A10491-01), and carefully reject fascia and fat, shred,
It is placed in the stainless steel sift of 100 mesh, aseptic grinding, discharges single splenocyte, draw the liquid containing splenocyte and be placed in 50mL
In sterile centrifugation tube, centrifugal.
Myeloma cell and the above-mentioned splenocyte prepared are added the centrifuge tube of same 50mL with the ratio of number 5:1
In, adding the incomplete culture fluid of RPMI-1640 (purchased from GIBICO, article No. the is 61870-036) 20mL of 37 DEG C of temperature baths, mixing is all
Even, 1500r/min is centrifuged 6min, supernatant discarded, bottom finger tapping down centrifuge tube, makes precipitation mix such as pasty state；Use pipet
Take the PEG 1mL of 37 DEG C of preheatings, instill centrifuge tube, after standing 1min, in 37 DEG C of water-baths, in 2min, drip RPMI-1640 complete
Full culture fluid 10mL, 1000r/min are centrifuged 6min, supernatant discarded, add 75mL HAT culture fluid, mix gently, will mix suspension
Be sub-packed in 24 orifice plates of feeder cells, every hole 0.5mL, in 37 DEG C, the incubator of 5%CO2 saturated humidity is hatched.
After fusion 6～9d, change liquid 1 time by HAT culture fluid half amount, behind 12～14d, use RPMI-instead according to proliferative conditions
1640 complete culture solutions；Until cell attachment to when accounting for plate hole 1/3, count hole count and the total cellular score of Growth of Hybridoma Cell, take
Clear liquid, indirect ELISA selects titer high and indirect competitive ELISA selects the positive hybridoma cell that Drug inhibition is strong.
Using indirect ELISA and indirect competitive ELISA method to carry out positive hybridoma cell screening, display is positive and occurs competing
The hole striving suppression reaction is the hole producing Cordyceps antibody, and can be used for further sub-clone.
Under aseptic condition, the cell in eluting positive hole, it is transferred to spread with feeder cells in advance by cell with elbow straw
In 96 well culture plates of plate, each archioporus clones into 8 holes, at the bottom of cell attachment covers with 1/2～1/3 hole after, take supernatant,
Connect ELISA detection；Take the strong sub-clone that is positive, 2～5 times the most repeatedly, wait antibody positive in the supernatant of hole, cloned 8
When rate is 100%, picking single cell clone, it is detected as full positive and is transferred to 24 porocyte culture plates or 25mL Tissue Culture Flask
Amplification culture, builds strain and with subpackage, frozen.Carry and inject 0.5mL norphytane the last week to Balb/c mouse peritoneal.Take freeze-stored cell
Strain, after recovery, breeds through mass propgation, collects cell, after full culture medium of cannoing be used up washing secondary, more not exclusively cultivates with 10mL
Base suspends, counting；By cell, (every mice 1mL, containing 3.1 × 107Individual cell) intraperitoneal injection of mice abdominal part, after 10～15d, treat
By No. 16 syringe aseptic collection ascites when mouse web portion substantially expands；2000r/min is centrifuged 10min, remove upper-layer fat and
Lowermost fibre albumen and cell, collect middle level, and subpackage-70 DEG C is frozen standby.
Take ascites centrifugal after middle layer segment 3mL, add 0.06mol/L, pH 4.5 sodium-acetate buffer of 2 times of volumes.
Caprylic acid is dropwise slowly added in sample, to final concentration 33 μ g/mL ascites, stirring while adding, continue stirring after adding
30min, at 4 DEG C, 10000r/min is centrifuged 30min, goes precipitation (albumin and other non-IgG albumen).Take supernatant micro-through 0.45 μm
Pore membrane filters, and mixes with 1/10 volume 10 × PBS that (10 × PBS is by 80gNaCl, 2g KCl, 11.5g Na2HPO4、2g
KH2After PO4,0.5845g EDTA dissolves with 950mL distilled water, adjust pH to 7.4 and be settled to 1000mL and obtain), use 1mol/L
NaOH solution adjusts pH value to 7.4.Supernatant is cooled to 4 DEG C, adds ammonium sulfate extremely final concentration of 0.277g/mL.Stirring 30min, at 4 DEG C
10000r/min is centrifuged 30min, abandons supernatant.With a small amount of PBS solution dissolution precipitation, by the PBS mistake of 50～100 times of volumes
At night, change liquid 3 times.Obtain anti-Cordyceps antibody after purification, preserve standby at 4 DEG C.
The preparation of embodiment winter worm summer grass standard substance.
The structure of embodiment four Cordyceps enzyme-linked immunologic detecting kit.
Pre-coated elisa plate:
The coating antigen (see embodiment one) of 0.1mL 1mg/L is added high adsorptive enzyme target, is coated overnight, use confining liquid
Close 2h, the formula of confining liquid be (1% bovine serum albumin, 1% N of casein, 0.5% soybean protein powder, 0.05% tween-
80, it is dissolved in 0.01M pH7.4PBS), wash version vacuum drying after closing.
Anti-Cordyceps enzyme labelled antibody:
Weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water, add the 0.1M NaIO that 0.2ml newly joins4Solution,
Under room temperature, lucifuge stirs 20 minutes.Above-mentioned solution is loaded in bag filter, the sodium-acetate buffer of 1mM PH4.4 is dialysed, 4 DEG C
Overnight.Adding 20 μ l 0.2M PH9.5 carbonate buffer solutions, pH is increased to 9.0～9.5, adds the 10mg worm summer in anti-winter immediately after
Grass monoclonal antibody is in 1ml 0.01M carbonate buffer solution, and room temperature lucifuge is gently mixed 2 hours.Add the 4mg/ that 0.1ml newly joins
ml NaBH4Liquid, mixing, then put 4 DEG C 2 hours.Above-mentioned liquid is loaded in bag filter, 0.15M pH7.4PBS is dialysed, 4 DEG C of mistakes
At night, the 0.01M pH7.4PBS divided with the Hydrazoic acid,sodium salt of bovine serum albumin, 0.05% tween 80 and 15mM containing 1% is dilute
Release 8000 times, subpackage 14mL to each test kit.
Substrate nitrite ion: mixed by developer A and developer B equal-volume and obtained；Weigh 23mg tetramethyl benzidine, add
1mL DMSO dissolves, and then adds 0.1M pH5.5 acetic acid-sodium-acetate buffer 66mL, obtains developer A；Take distilled water 100mL,
Add urea peroxide 10 μ g, obtain developer B.Nitrite ion A and nitrite ion B 1:1 by volume is mixed, subpackage 14mL to each examination
Agent box.
Stop buffer: for 2M sulfuric acid solution, subpackage 7mL to each test kit.
The Cordyceps immunity detection reagent that the present invention provides includes: Cordyceps titer 7 bottles, concentration is respectively 0 μ
g/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、64μg/mL；Anti-Cordyceps enzyme labelled antibody；It is coated the winter
The ELISA Plate of worm summer grass, substrate nitrite ion, stop buffer.
The Cordyceps immunity detection reagent that the present invention provides is preserved under 4 DEG C of environment, can guarantee the quality more than 1 year.
Cordyceps Related product is detected by embodiment five Cordyceps enzyme-linked immunologic detecting kit.
Sample pre-treatments:
Cordyceps and Cordyceps goods: by sample homogenizing, weigh 1g, and the PBS adding 20mL 0.01M pH7.4 surpasses
Sound extracts 2min continuously, and centrifugal, taking supernatant is sample detection solution, if the testing result of sample detection solution exceedes linearly
Range limit, need to take the circumstances into consideration dilution again.
Cordyceps enzyme-linked immunologic detecting kit detects:
1, being taken out from cold storage environment by required reagent, at room temperature balance more than 30min, various reagent must before using
Shake up；
2, adding the Cordyceps titer of series concentration or sample detection solution 50ul in corresponding micropore, titer is equal
Need to do 2 parallel tests, then add 50 μ l enzyme labelled antibody working solutions, room temperature lucifuge is reacted 30 minutes；
3, carefully open cover plate film, discard liquid in micropore, and pat dry micropore remains residual liquid in absorbent paper, toward micro-
Hole fills cleaning mixture, vibrates gently, place 2 minutes, discard liquid in micropore, and pat dry in absorbent paper, repeated washing 4 times
Or machine-wash 5 times；
5, add 100 μ l substrate nitrite ions in corresponding micropore, and at room temperature lucifuge is reacted 15 minutes；
6, add 50 μ l stop buffers in corresponding micropore, use microplate reader to measure OD value under 450nm wavelength.
Experimental data Criterion curve according to Cordyceps titer, result is as shown in Figure 2.The recurrence of standard curve
Equation R2> 0.99, illustrate that OD value and Cordyceps concentration have good linear relationship.Linear regression side according to standard curve
Journey.During for qualitatively judging, sample absorbance value, less than the point of standard curve least concentration, can be judged as that Cordyceps is positive
Sample.No person is negative, and Cordyceps enzyme linked immunosorbent detection standard curve is as shown in Figure 2.
The specificity of embodiment six Cordyceps enzyme-linked immunologic detecting kit.
Use Cordyceps enzyme-linked immunologic detecting kit that the common proteins in food is detected, result such as table 2 institute
Show.As known from Table 1, Cordyceps enzyme-linked immunologic detecting kit and other protein ingredient no cross reactions in food, specificity
Good, testing result will not be impacted.
Table 1 Cordyceps enzyme linked immunological kit and the cross reaction of other protein ingredients in food
The repeatability of embodiment seven Cordyceps enzyme-linked immunologic detecting kit.
Use Cordyceps enzyme-linked immunologic detecting kit to Cordyceps sample (including negative sample and positive)
Carrying out duplicate detection between different micropore and different ELISA Plate respectively, result is as shown in Table 3 and Table 4.As may be known from Table 3 and Table 4, winter worm
Summer grass enzyme-linked immunologic detecting kit carry out difference between the micropore of sample detection < 2%, difference between ELISA Plate < 5%, reproducible.
Table 2 Cordyceps enzyme-linked immunologic detecting kit carries out difference between the micropore of sample detection
Table 3 Cordyceps enzyme-linked immunologic detecting kit carries out difference between the ELISA Plate of sample detection
The matrix effect of embodiment eight Cordyceps enzyme-linked immunologic detecting kit.
Use Cordyceps enzyme-linked immunologic detecting kit to Sal, Portugal in sample (including negative sample and positive)
Some common interference things such as grape sugar and fructose carry out the detection of matrix effect, and result is as shown in table 4.As shown in Table 3, to feminine gender
In the detection of sample and positive, the glucose of mass concentration 1%, the fructose of 1%, the starch of 1%, the sodium chloride pair of 1%
The impact of testing result all without significant difference (p > 0.05), illustrates the Cordyceps enzyme-linked immunologic detecting kit that the present invention provides
Anti-matrix interference is effective, only sample need to be carried out supersound extraction and dilution, can complete sample pre-treatments.
The impact on Cordyceps enzyme-linked immunologic detecting kit testing result of the table 4 common interference thing
A kind of Cordyceps enzyme-linked immunologic detecting kit that the present invention provides and preparation method thereof, anti-Cordyceps feature
Protein antibodies for antigenic determinant heat-resisting, can quickly differentiate the Cordyceps true and false in sample, applied widely, be conducive to
The guarantee of consumer legitimate right；Specificity is good, highly sensitive, and anti-matrix interference is effective, and testing result accuracy is good, repeats
Property is good；The pre-treatment of sample is simple, detects simple to operate, and reagent all provides with the form of working solution, it is convenient to carry out a large amount of
The examination of sample.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of present inventive concept, it is also possible to make some simple deduction or replace, all should be considered as belonging to the present invention's
Protection domain.
Claims (5)
1. the preparation method of a Cordyceps enzyme-linked immunologic detecting kit, it is characterised in that comprise the following steps:
S1, Cordyceps 70kDa glycoprotein extract and separate: by Liquid isoelectric focusing, protein electrophoresis, electroelution instrument protein purification
System prepares Cordyceps 70kDa glycoprotein, in this, as the standard substance in antigen, coating antigen and test kit；
S2, the preparation of monoclonal antibody；
S3, the foundation of enzyme-linked immune detection method and assessment；
S4, the assembling of test kit.
The preparation method of Cordyceps enzyme-linked immunologic detecting kit the most according to claim 1, it is characterised in that step
S2 includes: prepare anti-Cordyceps 70kDa glycoprotein monoclonal antibody by cell-fusion techniques and antibody purification technology.
The preparation method of Cordyceps enzyme-linked immunologic detecting kit the most according to claim 1, it is characterised in that step
S3 includes: sets up Cordyceps enzyme-linked immune detection method and comments its specificity, anti-matrix interference ability and repeatability
Estimate.
The preparation method of Cordyceps enzyme-linked immunologic detecting kit the most according to claim 1, it is characterised in that step
S4 includes: by the ELISA Plate of pre-coated Cordyceps 70kDa glycoprotein, Cordyceps 70kDa glycoprotein standard solution, Radix Cochleariae officinalis mistake
Oxide enzyme labelling Cordyceps 70kDa glycoprotein monoclonal antibody, nitrite ion and stop buffer are assembled into test kit.
5. a Cordyceps enzyme-linked immunologic detecting kit, it is characterised in that: by as described in any one of Claims 1-4
The preparation method of Cordyceps enzyme-linked immunologic detecting kit be prepared from.
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