CN105866409A - Candida mannan antigen immunoassay kit and preparation method and application thereof - Google Patents
Candida mannan antigen immunoassay kit and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a candida mannan antigen immunoassay kit, comprising a Mn-coated ELISA plate and an anti-Mn polyclonal enzyme-labeled antibody. According to the kit of the invention, the ELISA plate is coated with Mn, a sample or standard antigen to be detected and a coating antigen are subjected to competitive binding with a limited antibody binding site and then to rendering reaction via horse radish peroxidase and a substrate, a standard curve is drawn according to an Mn standard article, and concentration value of the antigen to be detected is calculated according to the standard curve. The kit of the invention has good sensitivity and specificity, results show high accordance rate with a reference reagent, more accurate and more reliable detection results can be provided, the kit is simple and easy to operate, detecting is fast and sensitive, an ELISA apparatus used herein is simple and popular and is low in cost, and the kit of the invention is an effective tool for quantitative detection of Mn.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of Candida mannan
(Candida mannan, Mn) antigen immune detection kit and preparation method and application.
Background technology
Studies of invasive fungal infections (invasive fungal disease, IFD) or aggressive are true
Bacterium infects (invasive fungal infection, IFI) and refers to that pathogenic fungus invades skin
The fungal infectious disease that undertissue, mucosa, muscle and internal organs etc. are caused.Candidiasis
Genus is modal fungus in opportunistic fungus or opportunistic fungus, and it is mainly invaded mucosa, enters
One step can be sent out in blood, causes disseminated infections；It is in the aggressive caused by fungal infection
Mycosis (invasive fungal sisease, IFD) accounts for first place.Candida holds very much
Easily breed in respiratory system, cause fungal pneumonia.And the clinical manifestation of fungal pneumonia and
The clinical manifestation of bacterial pneumonia is essentially identical, and easy mistaken diagnosis is bacterial pneumonia so that fungus sense
Dye increases the weight of further, even sends out whole body.
The attribution case fatality rate of Invasive candidiasis may be up to 47% according to statistics, attribution case fatality rate
Adult patient is 15%-25%, is 10%-15% in neonate and child.Aggressive is read
ICU can be made to move in the time for pearl bacterium disease and the hospital stays increases 12.7d and 15.5d respectively.Make
Become main reason is that of this high mortality in early days candidiasis can not be examined in the course of disease
Survey diagnosis, so that patient can not get treating timely and effectively and dead, therefore select to detect in early days
Diagnostic method has great importance.
Mannan (Mannan) is the major antigen composition of Candida cell wall.For reading
The diagnosis that pearl bacterium infects, in addition to traditional cultural method, measures Mn in serum, by
Gradually become simple, method, the inspection carried out at present efficiently of Invasive candidiasis early diagnosis
Survey method mainly has following 5 big classes:
(1) histopathological examination, this method is invasive inspection, not easy to drawing materials；
(2) hemoculture method, this method sensitivity is poor, the longest, easily pollutes, some fungus
Sample collection is difficult, and positive rate is the highest；
(3) direct microscopy method, this method reliability is low, and limitation is big, is suitable only for detecting shallow
Portion's mycosis；
(4) imaging examination, this method poor specificity, sexy with involvement of blood vessel on iconography
Contaminate similar, be not easily distinguishable.
(5) immunological detection: specificity realizes the detection to candidiasis for the antibody of Mn,
Detection blood sensitivity reaches more than 80%.Immunological detection method sensitivity and specificity are the most relatively
Front four kinds of methods have obvious advantage, are increasingly becoming the common method of candidiasis detection.In state
In the diagnostic criteria of inside and outside IFI, all think that the serum candida antigens positive can be as microbiology
Diagnostic criteria, the Mn level in serum that therefore measures contribute to oidiomycotic early diagnosis,
Early treatment.
Immunoassay technology is to utilize the specific binding reaction of energy between antigen-antibody, is marked by detection
Note label on reactant, the method that antigen or antibody are realized qualitative or quantitative detection.
According to the difference of label, it is divided into enzyme immunoassay technology (Enzyme-Linked
Immunosorbant Assay, ELISA), Immunofluorescence test technology, chemiluminescence immunoassay
Analytical technology, immune microsphere technology, immune colloidal gold technique etc..Wherein elisa technique has
Simple to operate, highly sensitive, detection time short feature, in Clinical detection and scientific research
It is widely used.
In the Clinical detection of candidiasis, on current international market, immune detection product is few in number,
The existing ELISA method detection kit product having Bio-Rad company of the U.S., this product uses
Double-antibody method, its method is first to be coated in ELISA Plate by the specific monoclonal antibody of candidiasis,
Add sample to be checked and the same monoclonal antibody of horseradish peroxidase (HRP) labelling,
37 DEG C are reacted 1.5 hours, and the antigen in sample to be checked can be combined also with specific monoclonal antibody
Form sandwich structure, add tetramethyl benzidine (TMB) colour developing, the depth of color and treating
The concentration of inspection antigen is proportionate, thus realizes the detection of Mn.
Summary of the invention
In the full text of the present invention, the following term represented respectively of writing a Chinese character in simplified form is:
Mn-Candida mannan；
EDTA-disodiumedetate；
PBS-phosphate buffered solution；
SDS-dodecyl sodium sulfate；
CBS-carbonate buffer solution；
Tirs-trishydroxymethylaminomethane buffer solution；
BSA-bovine serum albumin；
TMB-tetramethyl benzidine；
HRP-horseradish peroxidase；
OD-absorbance under setted wavelength.
For the composition of EDTA, PBS, CBS, Tris, TMB, concentration, pH etc.,
Use the common type in immunobiology.
It is an object of the invention to provide a kind of Candida mannan Antigen extraction method.
It is an object of the invention to provide a kind of Candida mannan antigen immune detection kit.
It is a further object to provide above-mentioned bent Candida mannan antigen immune inspection
The preparation method of test agent box.
The present invention a further object is offer above-mentioned Candida mannan antigen immune detection
The application of test kit.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of Candida mannan Antigen extraction method:
1) Candida albicans is cultivated and is used general Candida albicans condition of culture, specifically
Saying that culture medium uses YM broth bouillon, composition is that every liter of culture medium yeast Han 3g carries
Take thing, 5g peptone, 3g maltose extract, 10g D-Glucose.Cultivate temperature
Degree is 30 DEG C, and concussion speed is 200rpm, incubation time about 28h.
2) above-mentioned culture fluid is centrifuged, precipitation i.e. albicans cell 0.9wt% chlorine
Changing sodium to wash twice, 56 DEG C of heating 2h kill cell, obtain white with acetone drying
Candidiasis mycopowder.
3) gained Candida albicans mycopowder is at pH=7.0, and concentration is the citric acid of 0.02M
Salt buffer mesohigh sterilizing 90min, centrifugal, retain supernatant, precipitation glue
Sterilizing under thing the same terms, centrifugal, merge supernatant.
4) above-mentioned supernatant joins in methanol, precipitation occurs；It is centrifugally separating to obtain precipitation,
Precipitation is dissolved in deionized water, dialyses with deionized water, lyophilizing, obtains rough manna and gathers
Sugar.
5) rough mannan is dissolved in deionized water, adds cetyl trimethyl bromination
The aqueous solution of ammonium, mixed liquor static a period of time appearance precipitation.
6) centrifugation precipitation, precipitate with deionized water is washed, and merges cleaning mixture and 5)
Step supernatant, amalgamation liquid adds the boric acid solution of 1wt%.
7) above-mentioned mixed liquor adds 2M sodium hydroxide while stirring, regulates pH to 8.8,
Generate cetyl trimethylammonium bromide-mannan complex compound sediment, collect precipitation,
With the 0.5% sodium acetate washing of pH=8.8, then use 2% acetic acid, gained solution
Precipitate in ethanol.
8) centrifugal, abandon supernatant, precipitate and wash with the ethanol acetate solution of 2%, be dissolved in water
Neutralize with 2M sodium hydroxide afterwards, distilled water is dialysed, lyophilizing, obtain Candida albicans
Bacterium mannan product.
In the present invention, albicans cell is after citrate processes, and supernatant exists
Methanol precipitates and i.e. can get mannan crude product, add cetyl trimethyl bromine afterwards
Changing ammonium, its purpose is as follows: 1) under neutrallty condition, goes out protein and nucleic acid；2) alkali
Under the conditions of property, and mannan combines and forms complex compound sediment, purified polysaccharide.Therefore,
In the present invention, use cetyl trimethylammonium bromide to prepare mannan, eliminate
The process of gel chromatography, not only makes preparation technology simplify, and can prepare high-purity simultaneously
The mannan of degree.
A kind of Candida mannan antigen immune detection kit, including Mn coated enzyme mark
Plate and anti-Mn polyclone enzyme labeling antibody.
Further, above-mentioned Candida mannan antigen immune detection kit also includes Mn
Standard substance, sample treatment solution, concentration washing liquid, Sample dilution, substrate solution and stop buffer.
The operation principle of the Candida mannan antigen immune detection kit of the present invention is as follows:
1) Mn is coated on solid phase carrier, makes solid phase antigen；
2) will add with biotin labeled anti-Mn polyclone enzyme labeling antibody after sample process to be checked
1), in solid phase, the antibody combining site that antigen to be checked is limited with envelope antigen competition binding is made；
3) through isothermal reaction and thoroughly wash, add HRP labelling streptavidin；
4) through isothermal reaction and thoroughly wash, add the reaction substrate TMB colour developing of enzyme,
In the depth of color and sample to be checked, Mn concentration is negative correlation；
5) under certain wavelength, measure absorbance by microplate reader, realize antagonism by standard curve
The detection of original content.
The relevant technical information of Mn of the present invention and anti-Mn polyclone enzyme labeling antibody is as follows:
Described anti-Mn polyclonal antibody, can be specific binding with Mn, shows with SDS-PAGE
For homogeneous antibody products, being coated with Mn, indirect ELISA detection shows that its titer is not less than
1:1×105, described antibody is obtained by following method: move as immunogen immune with Mn
The serum that thing obtains carries out isolated and purified, obtains antibody, and described purification process is saturated ammonium sulfate
Salt precipitation method and/or affinity chromatography, wherein, immunization method can be subcutaneous injection, spleen
Interior injection, intravenous injection or lumbar injection, immunizing dose is 0.3-2mL/, moving of immunity
Thing is rat, mice, Cavia porcellus, rabbit, chicken, sheep, horse, pig or donkey, preferably rabbit.
The preparation method of above-mentioned anti-Mn polyclonal antibody, concretely comprises the following steps:
(1) Candida albicans mannan antigen is extracted；
(2) by Candida albicans mannan antigen immune animal.Collect Mn antigen immune
The carotid artery blood of animal, separates antiserum；
(3) preliminary purification is carried out by saturated ammonium sulfate salt precipitation method and/or affinity chromatography:
Described anti-Mn polyclonal antibody, is that the anti-Mn polyclonal antibody of above-mentioned purification is biological
Element is marked.
In the present invention, described concentration washing liquid, Sample dilution, sample treatment solution, substrate
Solution and stop buffer are all immunobiology experiment field common agents, its constituent and proportioning
As follows:
Concentration washing liquid: 96.0 parts of sodium chloride of meter by weight, 2.40 parts of potassium chloride, 12
Hypophosphite monohydrate disodium hydrogen 42.96 parts, potassium dihydrogen phosphate 2.88 parts, tween 20 0.05 part,
Ultra-pure water 1000 parts；
Enzyme marker: HRP labelling streptavidin；
Sample dilution: artificial serum；
Sample treatment solution: can take following albuminous degeneration solution (pH2-pH10):
0.05-0.2mol/L EDTA solution；0.07-0.2mol/L glycine-HCL (glycine-salt
Acid) solution；0.05-15mg/mL pronase；0.01-0.1mol/L SDS (dodecane
Base sodium sulfonate) solution or 1mol/L-8mol/L carbamide；
Substrate solution: TMB；
Stop buffer: 2mol/L sulfuric acid solution.
Above-mentioned anti-Mn polyclonal antibody and Mn standard substance a, Mn standard substance b, Mn standard
Product c, Mn standard substance d, Mn standard substance e, enzyme marker, sample treatment solution, concentration washing liquid,
Sample dilution, substrate solution and stop buffer are respectively put in each reagent bottle, above-mentioned each reagent
Bottle is fixed by sponge bracket, and is together placed in test kit with the coated ELISA Plate of Mn and shrouding film
In box body.
In the present invention, between the plurality of Mn standard substance, there is concentration difference thus drawn
Standard curve, it is preferred that above-mentioned Candida mannan antigen immune detection kit, Mn
The concentration of standard substance a be the concentration of 10ng/ml, Mn standard substance b be 5ng/ml, Mn standard
The concentration of product c be the concentration of 2.5ng/ml, Mn standard substance d be 1ng/ml, Mn standard substance e
Concentration be 0.5ng/ml.
Accordingly, the invention discloses above-mentioned Candida mannan antigen immune detection kit
Preparation method, specifically comprise the following steps that
(1) ELISA Plate is prepared
A () Mn is coated the preparation of liquid: use following buffer solution that Mn is diluted to 25ng/100
μ L-2 μ g/100 μ L:0.01M-0.2M PBS, its pH7.0-pH8.0；0.05-0.2M
CBS, its pH9.0-pH9.6；0.05mol/L Tris buffer, its pH10.0-pH10.6；
The preparation of (b) confining liquid: under the defatted milk powder of 3%-5% or 1%-4%BSA are added
State in buffer solution, be configured to confining liquid: buffer solution, 0.01M-0.2M PBS, its
pH7.0-pH8.0；
(c) coated elisa plate:
1. the Mn of preparation being coated liquid add in ELISA Plate hole, every hole is separately added into 60 μ L-200
μ L is coated liquid；
2. ELISA Plate is coated 8-16h under being placed in 2-8 DEG C of environment；
3. being added in ELISA Plate hole by the confining liquid of preparation, every hole is separately added into 60 μ L-200 μ
L confining liquid, is placed in 37 DEG C of incubators, 30-90 minute；
4. take out after ELISA Plate from incubator and discard confining liquid, 37 DEG C of constant temperature 30-90 minute, i.e.
?；
(2) preparation (foundation of quantitation curves) of standard substance
The preparation of standard substance is that Mn is formulated with artificial serum, and diluted concentration is respectively
10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0.5ng/ml；
(3) preparation of anti-Mn polyclone enzyme labeling antibody solution: by biotin labeled anti-Mn
Polyclonal antibody conjugate stabilizer forms with the dilution proportion of 1:2000-1:20000；
(4) preparation of HRP labelling streptavidin solution: HRP labelling streptavidin is even
Connection thing stabilizer forms with the dilution proportion of 1:2000-1:20000；
(5) preparation of sample treatment solution: be configured to following pH 2-pH10 with ultra-pure water respectively
Sample treatment solution: 0.05-0.2mol/L EDTA solution；0.07-0.2mol/L glycine
-HCL (glycine-HCI) solution；0.05-15mg/mL pronase；0.01-0.1mol/L
SDS solution or 1mol/L-8mol/L carbamide；
(6) preparation of washing liquid (20 × 0.01M PBS) is concentrated: weigh chlorination by weight
96.0 parts of sodium, 2.40 parts of potassium chloride, disodium hydrogen phosphate dodecahydrate 42.96 parts, di(2-ethylhexyl)phosphate
2.88 parts of hydrogen potassium, tween 20 0.05 part, ultra-pure water 1000 parts, mix homogeneously；
(7) Sample dilution is: artificial serum；
(8) substrate solution is: TMB；
(9) preparation of stop buffer: concentrated sulphuric acid is diluted with ultra-pure water 1:8, is configured to 2mol/L
The stop buffer of sulfuric acid solution.
Further, the invention also discloses the detection examination of above-mentioned Candida mannan antigen immune
The application in terms of detection Mn concentration of the agent box.
Preferably, above-mentioned Candida mannan antigen immune detection kit detection Mn concentration
Application use competitive ELISA method (one-step method), including the coated ELISA Plate of Mn, Mn
The rabbit source polyclone biotin labelled antibodies of immunity, Mn standard substance a, Mn standard substance b, Mn
Standard substance c, Mn standard substance d, Mn standard substance e, enzyme marker, concentration washing liquid, sample is dilute
Release liquid, sample treatment solution, substrate solution and stop buffer, specifically comprise the following steps that
A) take sample to be checked and sample treatment solution mix with the volume ratio of 1:1-5:1 and boil
It is centrifuged after 1-10min and obtains thing to be detected；
B) thing to be detected of step a) is resisted with biotin labeled anti-Mn polyclone enzyme mark
Body equal-volume mixes and adds in the coated ELISA Plate of Mn and hatches simultaneously
20-60min, washes plate after hatching；
C) in the ELISA Plate of step b), add HRP labelling streptavidin and hatch 20-60min,
Plate is washed after hatching；
D), after adding TMB colour developing 15min in the ELISA Plate of step c), add and terminate
Detect after liquid, in microplate reader, read the absorption photometric value of 450 nanometers.
Compared with existing procucts, the technology difference of the present invention is: 1, method is different: Bio-Rad
Candidiasis bacterium test kit uses double-antibody method, and the present invention uses competition law, is first coated by Mn
In ELISA Plate, more limited with envelope antigen competition binding to sample to be checked or standard antigen is resisted
Body binding site, the shade of measurement result and the concentration of antigen to be checked are negative correlation.2、
Antibody is different: the candidiasis monoclonal antibody that Bio-Rad candidiasis test kit uses, anti-used by the present invention
Body is anti-Mn polyclone enzyme labeling antibody, can draw standard curve according to Mn standard substance, according to mark
Directrix curve calculates the concentration value of antigen to be checked, and this detection kit has preferable sensitivity and spy
The opposite sex, result is high with reference reagent coincidence rate, is provided that assay more accurately and reliably, institute
Stating test kit easy to operation, detect rapid sensitive, microplate reader used is simple, universal, valency
Lattice are cheap, and this detection kit is that Mn detection by quantitative provides a kind of effective tool.
Accompanying drawing explanation
Fig. 1 shows the result of the SDS-PAGE detection of polyclonal antibody.
Fig. 2 shows the result of the titration of polyclonal antibody.
Fig. 3 shows Mn kit standard curve chart.
Detailed description of the invention
The preparation of embodiment 1 anti-Mn polyclonal antibody
1. immune animal
Candida albicans mannan antigen is used the back of the body to 3 Adult female new zealand white rabbits
Portion's multiple spot hypodermic injection carries out immunity, and rabbit ear edge venous blood is removed in immunity for first 1 week, separates serum
As negative control, immunizing dose is 1.0mL/ time, every time filling 1.0mL Freund during immunity
Adjuvant, as immunological adjuvant, is strengthened for the 7th, 14,21 and 28 days after initial immunity again
Immunity is once.
2. the acquisition of polyclonal antibody
1) titration: after 3 immunity, takes blood, blood sampling volume from rabbit auricular vein
For 1mL/ only, separate serum, with the antibody titer in indirect elisa method detection serum.Should
Polyclonal antibody titer is the highest, more than 1:1 × 105。
2) antiserum is separated: when rabbit titer is higher than 1:1 × 105Time, with carotid artery blood-letting
Method is taken a blood sample in a large number, at room temperature places about 2h after taking blood, and then 4 DEG C stand overnight and make it coagulate
Gu, 2500rpm is centrifuged 15min, takes supernatant.
3) preliminary purification is carried out with saturated ammonium sulfate salting out method
1. saturated ammonium sulfate primary sedimentation
Take the above-mentioned serum of 17ml to put in beaker, and add isopyknic normal saline (17ml),
Agitator stirs, is slowly added dropwise the saturated ammonium sulfate (34ml) of cumulative volume 50%, stir 3-5
Minute, preserve overnight in forwarding 4 DEG C of refrigerators to.
2. saturated ammonium sulfate secondary precipitation and dialysis
(1) the same serum albumin of primary sedimentation is divided in 2 50ml centrifuge tubes,
5000rpm is centrifuged 15min；
(2) remove supernatant, in centrifuge tube, put into the physiology of cumulative volume 60% (10.27ml)
Saline, is allowed to be completely dissolved, blows and beats in course of dissolution, is slowly dripping cumulative volume 40%
Adding saturated ammonium sulfate (6.8ml), constantly shake, preserve in forwarding 4 DEG C of refrigerators to, precipitation is at least
2.5 hour.
(3) the serum solution machine by centrifugation after 2 precipitations that takes is centrifuged, and 5000rpm is centrifuged 15min,
Remove supernatant.Add 2-3ml high pressure PBS according to precipitation capacity, carry out piping and druming and be allowed to the most molten
Solve, take dialyzer, the serum dissolved is put in dialyzer and dialyses, forward 4 DEG C of ice to
Preserve in case.Change liquid once every 2-3h, change 3-4 time altogether.
3. it is purified with affinity chromatography
(1) Protein A Sepharose dry powder 100mg is weighed, through PBS 4 DEG C overnight
The most swelling, balance with PBS after dress post.
(2) the polyclonal antibody crude extract after dialysis is crossed at 4 DEG C repeatedly post 20 times, mistake
Post liquid retains.
(3) with 0.01M PB 0.14M NaCl pH7.4 abundant drip washing pillar, to light splitting light
Degree mensuration OD280 approximates 0 or OD280 value the most no longer to be reduced.
(4) changing 0.1M glycine-HCl pH2.8 eluting, 500 μ l for the first time, eluting produces
Thing neutralizes with the 1.0M Tris-HCl pH9.0 of about 20 μ l immediately.Eluent successively decreases successively,
Collect eluted product.Obtain Candida albicans polyclonal antibody.
The detection of embodiment 2 anti-Mn polyclonal antibody
1.SDS-PAGE electrophoresis detection
The antibody preparing embodiment 1 carries out SDS-PAGE electrophoresis, carries out the gel obtained
Coomassie brilliant blue staining.Experimental result is shown in Fig. 1.Be can be seen that by figure, at 25KD and 50KD
There is clear significantly band molecular weight area, illustrates that antibody purity is the highest.
2. titration
It is measured by indirect elisa method antagonist titer.Coating antigen used is Candida albicans
Mannan antigen, ELIAS secondary antibody used is the goat anti-rabbit igg of horseradish peroxidase-labeled.
Testing result is shown in Fig. 2.Can be seen that from result, this polyclonal antibody titer is the highest, more than 1:1
×105。
The preparation of embodiment 3 Candida mannan antigen immune detection kit
One, the preparation of ELISA Plate
1, the preparation of liquid it is coated:
Described Mn is coated liquid, uses 0.01M PBS solution that Mn is diluted to 25ng/100
μL pH7.0-pH 7.4。
2, the preparation of confining liquid:
1) confining liquid described in, uses 0.01M PBS solution, its pH7.0-pH7.4；
2) defatted milk powder of 3% is added in above-mentioned solution, be configured to confining liquid.
3, the method for coating of ELISA Plate:
1) being added in ELISA Plate hole by the liquid that is coated of preparation, every hole is separately added into 60 μ L and is coated
Liquid；
2) above-mentioned ELISA Plate is coated 8h under being placed in 2-8 DEG C of environment；
3) adding in ELISA Plate hole by the confining liquid of preparation, every hole is separately added into 60 μ L and closes
Liquid, is placed in 37 DEG C of incubators, 30 minutes；
4) take out after ELISA Plate from incubator and discard confining liquid, 37 DEG C of constant temperature 30 minutes.
Two, the preparation (foundation of quantitation curves) of standard substance
The preparation of standard substance is that Mn is formulated with artificial serum, and diluted concentration is respectively
10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0.5ng/ml.
Three, the preparation of the polyclone biotin labelled antibodies solution of anti-Mn
The preparation of the polyclone enzyme labeling antibody solution of anti-Mn is by many for biotin labeled anti-Mn
Clone's enzyme labelled antibody conjugate stabilizer forms with the dilution proportion of 1:2000.
Four, the preparation of enzyme marker
The preparation of HRP labelling streptavidin solution is by steady for HRP labelling streptavidin conjugate
Determine agent to form with the dilution proportion of 1:2000.
Five, the preparation of sample treatment solution
Can be selected for following treatment fluid: dissolve disodiumedetate with ultra-pure water, be configured to
0.05mol/L EDTA solution, pH 4.0-pH4.8.
Six, washing liquid (20 × 0.01M PBS) is concentrated
Concentration washing liquid: 96.0 parts of sodium chloride, 2.40 parts of potassium chloride, 12 hypophosphite monohydrate hydrogen two
42.96 parts of sodium, potassium dihydrogen phosphate 2.88 parts, tween 20 0.05 part, ultra-pure water 1000
Part.
Seven, Sample dilution
Artificial serum.
Eight, substrate solution
TMB。
Nine, stop buffer
Concentrated sulphuric acid is diluted with ultra-pure water 1:8, is configured to 2mol/L sulfuric acid solution as termination
Liquid.
The preparation of embodiment 4 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.1M PBS solution that Mn is diluted to 25ng/100 μ
L, pH7.6-pH8.0, remaining step is with embodiment 3.
The preparation of embodiment 5 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.2M PBS solution that Mn is diluted to 25ng/100 μ
L, pH7.6-pH8.0, remaining step is with embodiment 3.
The preparation of embodiment 6 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.05M CBS solution that Mn is diluted to 25ng/100
μ L, pH9.0-pH9.6, remaining step is with embodiment 3.
The preparation of embodiment 7 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.1M CBS solution that Mn is diluted to 25ng/100 μ
L, pH9.0-pH9.6, remaining step is with embodiment 3.
The preparation of embodiment 8 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.2M CBS solution that Mn is diluted to 25ng/100 μ
L, pH9.0-pH9.6, remaining step is with embodiment 3.
The preparation of embodiment 9 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.05M Tris buffer solution to be diluted to by Mn
25ng/100 μ L, pH10.0-pH10.6, remaining step is with embodiment 3.
The preparation of embodiment 10 Candida mannan antigen immune detection kit
Described Mn is coated liquid, uses 0.01M PBS solution that Mn is diluted to 500ng/100
μ L, its pH7.2-pH7.4.
Described confining liquid, uses 0.1M PBS phosphate buffered solution, its pH7.6-pH8.0,
Defatted milk powder by 5% adds in above-mentioned solution, is configured to confining liquid,
Remaining step is with embodiment 3.
The preparation of embodiment 11 Candida mannan antigen immune detection kit
Described confining liquid, uses 0.2M PBS phosphate buffered solution, its pH7.6-pH8.0,
Defatted milk powder by 5% adds in above-mentioned solution, is configured to confining liquid,
Remaining step is with embodiment 10.
The preparation of embodiment 12 Candida mannan antigen immune detection kit
Described confining liquid, uses 0.2M PBS phosphate buffered solution, its pH7.6-pH8.0,
BSA by 1% adds in above-mentioned solution, is configured to confining liquid, and remaining step is with embodiment 10.
The preparation of embodiment 13 Candida mannan antigen immune detection kit
Described confining liquid, employing 0.2M PBS phosphate buffered solution, its pH7.6-pH8.0,
BSA by 2% adds in above-mentioned solution, is configured to confining liquid, and remaining step is with embodiment 10.
The preparation of embodiment 14 Candida mannan antigen immune detection kit
Described confining liquid, uses 0.2M PBS PBS solution, and its pH7.6-pH8.0, by 4%
BSA add in above-mentioned solution, be configured to confining liquid, remaining step is with embodiment 10.
The preparation of embodiment 15 Candida mannan antigen immune detection kit
Described sample treatment solution can be selected for: dissolves disodiumedetate with ultra-pure water, joins
Make 0.2mol/L EDTA solution, pH 4.0-pH4.8.Remaining step is with embodiment 10.
The preparation of embodiment 16 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolve dodecyl sodium sulfate with ultra-pure water, preparation
Becoming 0.01mol/L SDS solution, Ph8.5-pH10, remaining step is with embodiment 15.
The preparation of embodiment 17 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolve dodecyl sodium sulfate with ultra-pure water, preparation
Becoming 0.05mol/L SDS solution, Ph8.5-pH10, remaining step is with embodiment 15.
The preparation of embodiment 18 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolve dodecyl sodium sulfate with ultra-pure water, preparation
Becoming 0.1mol/L SDS solution, Ph8.5-pH10, remaining step is with embodiment 15.
The preparation of embodiment 19 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves glycine with ultra-pure water, is configured to
0.07mol/L glycine, then with dense HCL solution regulation pH to pH2.2-pH 2.8 remaining
Step is with embodiment 15.
The preparation of embodiment 20 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves glycine with ultra-pure water, is configured to
0.13mol/L glycine, then with dense HCL solution regulation pH to pH2.2-pH 2.8 remaining
Step is with embodiment 15.
The preparation of embodiment 21 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves glycine with ultra-pure water, is configured to
0.2mol/L glycine, then with dense HCL solution regulation pH to pH2.2-pH 2.8 remaining
Step is with embodiment 15.
The preparation of embodiment 22 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves pronase with ultra-pure water, is configured to
0.05mg/mL pronase solution, pH8.0-pH 9.0, remaining step is with embodiment 15.
The preparation of embodiment 23 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves pronase with ultra-pure water, is configured to 5
Mg/mL pronase solution, pH8.0-pH 9.0, remaining step is with embodiment 15.
The preparation of embodiment 24 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: dissolves pronase with ultra-pure water, is configured to
15mg/mL pronase solution, pH8.0-pH 9.0, remaining step is with embodiment 15.
The preparation of embodiment 25 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: uses ultra-pure water dissolved urea, is configured to 1mol/L
Carbamide, pH7.2-pH8.0, remaining step is with embodiment 15.
The preparation of embodiment 26 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: uses ultra-pure water dissolved urea, is configured to 4mol/L
Carbamide, pH7.2-pH8.0, remaining step is with embodiment 15.
The preparation of embodiment 27 Candida mannan antigen immune detection kit
The compound method of sample treatment solution is: uses ultra-pure water dissolved urea, is configured to 8mol/L
Carbamide, pH7.2-pH8.0, remaining step is with embodiment 15.
Embodiment 28Mn immunity detection reagent detecting step
One, the process of sample
1) boiling water bath 5min after sample to be checked being mixed with the volume ratio of 3:1 with sample treatment solution.
2) the mixed liquor 5,000g after water-bath is centrifuged 5min.
3) centrifuged supernatant is used for detecting.
Two, detecting step
1) 96 hole ELISA Plate of the most pre-coated antigen are taken out；
2) preparation work cleaning mixture: concentrate washing liquid dilution 20 × (1 part concentrate washing liquid (20 ×
0.01M PBS) add sterile deionized water or the ultra-pure water of 19 parts)；
3) sample-adding: bidding directrix curve group, testing sample group respectively, wherein
Standard curve group: each standard curve point (0.5ng/ml-10ng/ml)
Sample to be tested group: the sample to be tested after process
Two groups are added in ELISA Plate hole with anti-Mn biotin labelled antibodies equal-volume respectively,
40min is hatched at 37 DEG C；
5) washing: getting rid of reactant liquor, every hole adds the washing liquid no less than 300 μ L every time, quiet
Pat dry after putting 40s, repeat above-mentioned washing operation, altogether washing 3 times；
6) sample-adding: enzyme labelling streptavidin is added in ELISA Plate hole, hatches at 37 DEG C
40min；
7) washing: getting rid of reactant liquor, every hole adds the washing liquid no less than 300 μ L every time, quiet
Pat dry after putting 40s, repeat above-mentioned washing operation, altogether washing 3 times；
8) colour developing: after washing terminates, every hole adds substrate solution 80 μ L, hatches at 37 DEG C
15min, lucifuge；
9) terminate: in every hole, add 50 μ L stop buffers, after mixing, read at OD450nm
Number；
10) result judges: input titer and the absorbance of testing sample the most respectively
Measured value, the semilog standard curve drawn according to software for calculation and equation, can automatically calculate
Go out the concentration value of Mn in each testing sample.
The clinical practice of embodiment 34 test kit (applies test kit and the reality of embodiment 10
Execute the detecting step of example 28)
The determination of Mn immunity detection reagent reference value
Clinical definite is monilial infection positive sample 60 example, simultaneously detection normal person's sample 300
Example, measures OD450 value, calculates antigen concentration value (table according to standard curve (table 1, Fig. 3)
2) reference value criterion (table 3), is finally determined.
Table 1 examination criteria curve
The determination ELISA clinic inspection of table 2Mn immunity detection reagent reference value
Survey result
Note: * represents and compares P < 0.01 with normal person；
Table 3
If the testing result of sample falls in suspicious interval, then need to carry out second time and detect.
Result according to standard curve calculates the concentration of detection antigen, normal by detecting 300 examples
People's sample, the concentration value of the antigen taking 95% confidence interval is the Cut-off upper limit:(average
Value)+2s (standard deviation)=0.5+2*0.25=1.0, by detecting 60 example positive patients,
The concentration value of the antigen taking 95% confidence interval is Cut-off lower limit:(meansigma methods)-2s
(standard deviation)=2.5-2*0.9=0.7, the concentration value of antigen is at 0.7ng/ml-1.0ng/ml
Between be patient suspected.
The methodological study of embodiment 35 test kit (apply embodiment 22 test kit and
The detecting step of embodiment 32)
1, sensitivity experiments
Collect clinical definite sample 20 example to test.
Diagnostic sensitivity=positive sample detection total number of cases × 100% of number of cases/positive sample, by testing
Result illustrates that the sensitivity of this experiment is more than 85%.
Table 4 sensitivity Detection experimental result
2, specificity experiments
Detect 20 example Healthy People samples.
Specificity=negative sample detection total number of cases × 100% of number of cases/negative sample, by experimental result
Illustrate that the sensitivity of this experiment is more than 90%.
Table 5 specific detection experimental result
3, response rate experiment
Normal human blood is selected to detect after adding Candida mannan antigen 2 μ g/L, 3 μ g/L,
Calculate the ratio of actual value and expected value, be recycled rate.The response rate is between 80-120%
Think qualified.Illustrated that the response rate of this experiment, between 80%-120%, is returned by experimental result
Yield is good.
Table 6 response rate result is tested
4, repeated experiment
1) betweenrun precision
Criterion of acceptability: same specimen tested once a day, on continuous 10 working days, calculates it
Average M, standard deviation SD and coefficient of variation CV, coefficient of variation CV≤25% is qualified.Conclusion:
This product betweenrun precision (i.e. coefficient of variation CV) is 24%, less than 25%, and conformance with standard,
Prove that this product betweenrun precision is good.
Table 7 betweenrun precision result is tested
2) withinrun precision
Criterion of acceptability: by same specimen 10 groups of data of parallel assay in same batch is tested.
Calculating its average M, standard deviation SD and coefficient of variation CV, coefficient of variation CV≤15% is qualified.
This product withinrun precision (i.e. coefficient of variation CV) is 14%, less than 15%, and conformance with standard,
It is qualified to verify.
Table 8 withinrun precision result is tested
5, stability experiment
The test kit assembled is placed in 37 DEG C of environment, does standard curve every day and detected
Know the antigenic solution of concentration, continuous detecting 5 days, detected value rate of change (i.e. coefficient of variation CV)
Less than 20%, it was demonstrated that stabilization of kit.Its result shows coefficient of variation CV≤20% of 5 days,
Illustrate that the present invention has good stability.
Table 9 stability test result
Above-mentioned detailed description of the invention is to Candida mannan antigen immune detection kit of the present invention
And preparation method and application the detailed description that carries out, be illustrative rather than determinate,
Can according to restriction scope list several embodiments, therefore without departing from the overall structure of the present invention
Changing and modifications under Siing, within should belonging to protection scope of the present invention.
Claims (8)
1. a Candida mannan antigen immune detection kit, it is characterised in that: bag
Include the coated ELISA Plate of Mn and anti-Mn polyclone biotin labelled antibodies.
Candida mannan antigen immune detection kit the most according to claim 1,
It is characterized in that: also include Mn standard substance, sample treatment solution, enzyme marker, concentration washing liquid,
Sample dilution, substrate solution and stop buffer.
Candida mannan antigen immune detection kit the most according to claim 2,
It is characterized in that: each component composition and proportioning are as follows:
Concentration washing liquid: 96.0 parts of sodium chloride of meter by weight, 2.40 parts of potassium chloride, 12
Hypophosphite monohydrate disodium hydrogen 42.96 parts, potassium dihydrogen phosphate 2.88 parts, tween 20 0.05 part,
Ultra-pure water 1000 parts；
Sample dilution: artificial serum；
Sample treatment solution: can take following albuminous degeneration solution, its pH2-pH10,
0.05-0.2mol/L EDTA solution；0.07-0.2mol/L glycine-HCL solution；
0.05-15mg/mL pronase；0.01-0.1mol/L SDS solution or
1mol/L-8mol/L carbamide；
Substrate solution: TMB solution；
Stop buffer: 2mol/L sulfuric acid solution.
Candida mannan antigen immune detection kit the most according to claim 2,
It is characterized in that: between multiple Mn standard substance, there is concentration difference.
5. the Candida mannan antigen immune detectable that one of claim 1-4 is described
The preparation method of box, it is characterised in that: comprise the following steps that,
(1) ELISA Plate is prepared
A () Mn is coated the preparation of liquid: use following buffer solution that Mn is diluted to 25ng/100
μ L-2 μ g/100 μ L:0.01M-0.2M PBS, its pH7.0-pH8.0；0.05-0.2M
CBS, its pH9.0-pH9.6；0.05mol/L Tris buffer, its pH10.0-pH10.6；
The preparation of (b) confining liquid: defatted milk powder or the 1%-4%BSA of 3%-5% are added slow
Dissolved liquid is configured to confining liquid；Wherein buffer solution is 0.01M-0.2M PBS, its
pH7.0-pH8.0；
(c) coated elisa plate:
1. the Mn of preparation being coated liquid add in ELISA Plate hole, every hole is separately added into 60 μ L-200
μ L is coated liquid；
2. ELISA Plate is coated 8-16h under being placed in 2-8 DEG C of environment；
3. being added in ELISA Plate hole by the confining liquid of preparation, every hole is separately added into 60 μ L-200 μ
L confining liquid, is placed in 37 DEG C of incubators, 30-90 minute；
4. take out after ELISA Plate from incubator and discard confining liquid, 37 DEG C of constant temperature 30-90 minute, to obtain final product；
(2) preparation of standard substance
Standard substance use Mn formulated with artificial serum, and diluted concentration is respectively
500ng/ml-0.1ng/ml；
(3) preparation of anti-Mn polyclone biotin labelled antibodies solution: by biotin labeled
Anti-Mn polyclone enzyme labeling antibody conjugate stabilizer is with the dilution proportion of 1:2000-1:20000
Form；
(4) preparation of HRP labelling streptavidin solution: HRP labelling streptavidin is even
Connection thing stabilizer forms with the dilution proportion of 1:2000-1:20000；
(5) preparation of sample treatment solution: be configured to following pH2-pH10's with ultra-pure water respectively
Sample treatment solution: 0.05-0.2mol/L EDTA solution；0.07-0.2mol/L glycine-HCL
Solution；0.05-15mg/mL pronase；0.01-0.1mol/L SDS solution or
1mol/L-8mol/L carbamide；
(6) preparation of washing liquid (20 × 0.01M PBS) is concentrated: weigh chlorination by weight
96.0 parts of sodium, 2.40 parts of potassium chloride, disodium hydrogen phosphate dodecahydrate 42.96 parts, di(2-ethylhexyl)phosphate
2.88 parts of hydrogen potassium, tween 20 0.05 part, ultra-pure water 1000 parts, mix homogeneously；
(7) Sample dilution is: artificial serum；
(8) substrate solution is: TMB solution；
(9) preparation of stop buffer: concentrated sulphuric acid is diluted with ultra-pure water 1:8, is configured to 2mol/L
Sulfuric acid solution is as stop buffer.
Candida mannan antigen immune detection kit the most according to claim 5
Preparation method, it is characterised in that: the preparation of described anti-Mn polyclonal antibody includes following step
Rapid:
(1) Candida albicans mannan antigen is extracted；
(2) by Candida albicans mannan antigen immune animal, Mn antigen immune is collected
The carotid artery blood of animal, separates antiserum；
(3) it is purified by saturated ammonium sulfate salt precipitation method and affinity chromatography.
7. the Candida mannan antigen immune detectable that one of claim 1-4 is described
Box application in terms of detection Mn concentration.
Candida mannan antigen immune detection kit the most according to claim 7
The application of detection Mn concentration, it is characterised in that use competitive ELISA method, be coated including Mn
ELISA Plate, Mn standard substance, concentrate washing liquid, Sample dilution, sample treatment solution, substrate
Solution and stop buffer, specifically comprise the following steps that
A) take sample to be checked and sample treatment solution mix with the volume ratio of 1:1-5:1 and boil
It is centrifuged after 1-10min and obtains thing to be detected；
B) by many grams of the anti-Mn of the thing to be detected of step a) Yu horseradish peroxidase-labeled
Grand enzyme labelled antibody equal-volume mixes and adds in the coated ELISA Plate of Mn and incubates simultaneously
Educate 20-60min, after hatching, wash plate；
C), after adding TMB colour developing 15min in the ELISA Plate of step b), add and terminate
Detect after liquid, in microplate reader, read the absorption photometric value of 450 nanometers.
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| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
| CN114933652A (en) * | 2022-06-24 | 2022-08-23 | 丹娜(天津)生物科技股份有限公司 | Anti-candida mannan monoclonal antibody and application thereof |
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| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
| CN110954691B (en) * | 2019-12-17 | 2021-02-26 | 丹娜(天津)生物科技股份有限公司 | Sample pretreatment reagent and application thereof |
| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
| CN114933652A (en) * | 2022-06-24 | 2022-08-23 | 丹娜(天津)生物科技股份有限公司 | Anti-candida mannan monoclonal antibody and application thereof |
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Application publication date: 20160817 |