CN1645145A - Method and reagent box for inspecting mycelian protein antibody of white candida - Google Patents
Method and reagent box for inspecting mycelian protein antibody of white candida Download PDFInfo
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Abstract
The invention discloses a method and a reagent box for inspecting a candida albicans hypase protein antibody and diagnosing attack infection of candida albicans. The method performs filtering by using a monoclonal antibody of anti-candida albicans hypase protein P47 in a bacteriophage surface display peptide library, prepares simulated antigen or candida albicans hypase protein P47 natural antigen by expanding and purifying, then uses the antigen peridiuming a solid support, adds a biologic sample to be inspected to incubate and wash, adds a marked second antibody for human antibodies to incubate and wash, detects existing of an immune combined composition in an incubated mixture. The reagent box includes an ELISA reagent box and a colloid gold reagent box. The candida albicans hypase protein antibody can be inspected by usign the method simply and in high speed and efficiency; the reagent box has a low cost, convenient usage and high specificity.
Description
Technical field
The present invention relates to candida albicans aggressive INFECTION IN DETECTION method, particularly relate to a kind of method and kit that detects mycelian protein antibody of white candida.
Background technology
Candida albicans (C.albican), claim the candida albicans bacterium again, often being present in human oral cavity, throat, enteron aisle, vagina mucosa etc. locates, it is important conditioned pathogen, (as organ transplant, diabetes, malignant tumour, blood disease, severe malnutrition etc.) obviously descend in immunologic function, or when using antibiotic to cause flora imbalance in a large number, this bacterium can cause serious invasive systemic infection.The laboratory diagnosis difficulty that the candida albicans aggressive infects is bigger, because this bacterium is a conditioned pathogen, perches in human body multi-section position, often causes that shallow table infects, and clinical separation is difficult for judging whether to cause a disease when positive; In addition, many deeps sample can't be gathered, and can not separate with cultivation, and these have all reduced diagnosis.Though think at present isolate candida albicans in the single blood culture can clear and definite aggressive diagnosis of infection, the susceptibility of blood culture method is low.Explore serological diagnostic method and at home and abroad carried out many years, the antibody of the anti-full bacterium of people detection is arranged, because this antibody is prevalent in people's the serum, diagnostic value is undesirable.Along with going deep into of research, it is found that the fibrin adhesion receptors that exist in a large number on the mycelia wall of candida albicans are that candida albicans can be invaded human endothelial cells and basement membrane causes the important substance that aggressive infects, thereby think the detection mycelian protein antibody of white candida, bigger to aggressive diagnosis of infection meaning.Specificity and susceptibility that external report detects the method diagnosis infection by Candida albicans of antibody can reach 93% and 80% respectively, and 73% positive findings can be early than blood culture 2 days, and some case can obtain diagnostic result in 15 days early than blood culture.These information indicatings infect to aggressive that the antibody of diagnostic value is arranged is the antibody of mycelian protein P47, detect mycelian protein antibody of white candida and help to implement as early as possible antifungal therapy.
Detecting mycelian protein antibody of white candida needs P47 albumen (SDS-PAGE electrophoresis showed relative molecular weight 47000, down together) as detectable, but the extraction of this albumen and purge process complexity, preparation is difficulty relatively, therefore needs the improvement preparation method.In novel diagnostic reagent research, phage display technique is a kind of quick, effective, easy method.The principle of display technique of bacteriophage is that the random oligonucleotide sequence of chemosynthesis is inserted phage genome, give expression to the small peptide that each seed amino acid random groups is closed at phage surface, by with the reaction of specific target, the bacteriophage of showing specific polypeptide is screened from phage peptide library, make the bacteriophage amplification of selecting by Escherichia coli, carry out sequencing then, obtain amino acid sequence corresponding, 26S Proteasome Structure and Function information, realize high flux screening, become a kind of effective molecular screening method.
Summary of the invention
The purpose of this invention is to provide the method that detects mycelian protein antibody of white candida fast, simply, efficiently.
Another object of the present invention provides fast, the ELISA kit of efficient detection mycelian protein antibody of white candida and immune colloid gold reagent box.
Technical scheme of the present invention is a kind of new technical scheme that has the function design of simulation native antigen epi-position according to the phage display peptide.The present invention carries out affine naughty sieve with monoclonal antibody specific to phage random displayed polypeptide storehouse, prepares candida albicans hyphal albumen analogue antigen.Experimental results show that analogue antigen and P47 have identical reactivity, have stronger affinity, react good with the infected person anteserum with anti-mycelian protein antibody of white candida.
The objective of the invention is to realize by following measures:
A kind of method that detects mycelian protein antibody of white candida, it is as follows specifically to detect step:
A. prepare analogue antigen or native antigen: analogue antigen is to screen acquisition with the monoclonal antibody of anti-candida albicans hyphal albumen P47 from phage random displayed polypeptide storehouse; Native antigen is candida albicans hyphal albumen P47;
B. use above-mentioned analogue antigen or native antigen bag by solid support;
C. biological sample to be measured is added on the solid support of bag quilt, under the condition that is fit to the antigen-antibody combination, hatch, clean and remove any unconjugated composition;
D. add second antibody, under the condition that is fit to the antigen-antibody combination, hatch, clean and remove any unconjugated composition at people's antibody through mark;
E. detect immune existence in the mixtures incubated in conjunction with compound.
The method of described detection mycelian protein antibody of white candida, wherein analogue antigen is the analogue antigen that bacteriophage that the monoclonal antibody with candida albicans hyphal albumen P47 filters out from phage display peptide storehouse obtains through amplification, purifying.
The method of described detection mycelian protein antibody of white candida, wherein analogue antigen be illustrated in the 12 following peptides of the amino acid sequence of phage surface any or several:
1)GlyGlnGluAlaAlaIleProThrSerAspMetPhe(SEQ?ID?No.1)
2)TyrPrpProLeuMetSerAlaLeuLysLysLeuPro(SEQ?ID?No.2)
3)HisTrpGluTyrThrThrSerProHisProArgLeu(SEQ?ID?No.3)。
The method of described detection mycelian protein antibody of white candida, wherein native antigen is meant through the sonicated candida albicans hyphal, again through the candida albicans hyphal albumen P47 of Con A-Sepharose column chromatography purification.
Described a kind of method that detects mycelian protein antibody of white candida, the condition that wherein is fit to the antigen-antibody combination is meant the temperature and time of hatching the antigen-antibody potpourri, and wherein temperature is 4 ℃～50 ℃, and the time is 2 minutes to 24 hours; Preferred temperature is a room temperature to 43 ℃, and the time is 2 minutes to 2 hours.
Described a kind of method that detects mycelian protein antibody of white candida, wherein detection method can adopt ELISA method or immune colloid gold percolation; The temperature of hatching the antigen-antibody potpourri when adopting the ELISA method is 37 ℃～43 ℃, and the time is 30 minutes to 1 hour; The temperature of hatching the antigen-antibody potpourri when adopting the immune colloid gold percolation is a room temperature, and the time is several minutes.
The method of described detection mycelian protein antibody of white candida, wherein biological sample is meant blood, serum or the blood plasma from the experimenter.
The method of described detection mycelian protein antibody of white candida, wherein second antibody is meant anti-human IgG antibody or the anti-human IgM antibody with horseradish peroxidase or alkali phosphatase enzyme mark, or the anti-human IgG of colloid gold label or anti-human IgM antibody.
A kind of ELISA kit that detects mycelian protein antibody of white candida, its kit consists of:
1) envelope antigen: the analogue antigen or the native antigen that obtain by method for preparing;
2) solid support: can adopt microwell plate, be used for bag by above-mentioned analogue antigen or native antigen;
3) enzyme labelled antibody: with the anti-human IgG of candida albicans hyphal albumen P47 or the anti-people IgM of horseradish peroxidase or alkali phosphatase enzyme mark;
4) positive control serum or negative control sera;
5) enzyme labelled antibody dilution: PBS
6) substrate solution: A liquid: 0.06% urea peroxide, 0.01M pH4.5 citrate buffer solution;
B liquid: 0.06%TMB, 0.01M pH4.5 citrate buffer solution;
7) cleansing solution: PBS contains 0.01% Tween-20;
8) stop buffer: 1M sulfuric acid.
A kind of immune colloid gold reagent box that detects mycelian protein antibody of white candida, its kit consists of:
1) envelope antigen: the analogue antigen or the native antigen that obtain by method for preparing;
2) reaction unit: the one, small flat box divides box body and lid, and circular hole is arranged on the lid; The 2nd, the suction filler is contained in the box body, and the 3rd, place on the absorbent material, the nitrocellulose filter under the lid; Nitrocellulose filter is used for fixing above-mentioned analogue antigen or native antigen and as the human IgG or the IgM of Quality Control;
3) colloid gold label antibody: with the anti-human IgG or the anti-people IgM of colloid gold label.
Beneficial effect of the present invention:
1, can obtain the candida albicans hyphal albumen analogue antigen of the high titre of purifying: with immobilised anti-candida albicans hyphal protein monoclonal antibody phage display peptide library is carried out many wheels and wash in a pan sieve, can make the bacteriophage that combines with antibody specificity be able to enrichment greatly.Select and the strongest phage clone of candida albicans hyphal protein specific antibody reaction, breed in a large number in Escherichia coli ER2738 strain, the culture supernatant precipitates 2 times through PEG8000, obtains the phage particle of purifying, learns that through titration phage titre is 10
12/ ml is used for bag by 10000 times of time dilutions, and the bacteriophage number of bag quilt is about 10
7/ hole.
2. special, efficient, the good reproducibility of detection method: the present invention is on the basis for preparing P47 protein monoclonal antibody (seeing embodiment 1), wash in a pan the analogue antigen that sieves candida albicans hyphal albumen from the phage display random peptide library, be used to detect candida albicans antibody in the patient body.
10 parts through cultivate conclusive evidence for the candida albicans aggressive infect, with the patients serum of P47 albumen as the Detection of antigen antibody positive, all positive with the analogue antigen detection, susceptibility is 100%, and positive degree with conform to P47 Detection of antigen result; The patient specimen that 1 routine candida albicans phlegm is cultivated and blood culture is positive simultaneously detects the many parts of samples of leaving and taking in 3 months with this method, and antibody is all positive; And other 17 examples only phlegm cultivate the blood specimen of positive patient, it is all negative to detect antibody repeatedly.Take among the patient of immunodepressant, many parts of blood samples of 16 routine patients have been made replication, wherein 3 routine patients are sampled 3 times, 5 example samplings 2 times, 2 example samplings 4 times, 1 example sampling 5 times, all obtain negative findings, other 1 routine patient samples 5 times, and 4 times the result is positive, and 1 time negative, 3 example samplings 2 times, 1 example sampling 4 times, the result is all positive, and the specificity of illustration method and repeatability are good.
3. clinical application effect is good: make ELISA with analogue antigen, detected 100 volunteer doner of blood's serum candida albicans hyphal egg P47 antibody, 2 positives, positive rate 2%; Patient's 43 examples that 134 examples are taken immune suppressive cyclosporin A detect candida albicans albumen P47 antibody, positive rate 33%, and its concrete outcome sees Table 1.
Table 1 the present invention takes immune suppressive cyclosporin A patient testing result to 134 examples
The negative routine number positive rate (%) of the positive routine number of patient's classification
Renal transplantation recipients 34 61 36
All kinds of ephritis 8 19 30
Other are 1 11 8 years old
Add up to 43 134 33
Annotate: all kinds of ephritis comprise chronic nephritis, chronic kidney hypofunction, nephrotic syndrome; Other groups comprise Huppert's disease, diseases such as systemic loupus erythematosus.
Through x
2Testing identity, 3 groups of patients that take immunodepressant detect positive rate no significant difference, P=4.62; Healthy population (2% detects the positive) is organized difference with patient conspicuousness.
The ELISA method that the present invention sets up with analogue antigen, its susceptibility, special and direct use P47 proteantigen testing result conform to; 10 examples are diagnosed as the patients serum that the candida albicans aggressive infects, and use this law and detect all positive; 17 examples only phlegm are cultivated the positive and the blood culture negative patient uses none example of this method and detects antibody, the invasive systemic infection that this explanation phlegm is cultivated positive patient and do not meant that candida albicans is used the deep aggressive that this method can the auxiliary diagnosis candida albicans and is infected.Kit provided by the invention is easy to use, specificity is high, cost is low.
Embodiment
The present invention is further illustrated by the following examples.
1. sample is originated: normal control group serum is taken from 100 normal blood donors.Candida albicans is cultivated positive group and comprises that clinical definite is for the Candida albicans aggressive infects, repeatedly candida albicans is turned out in the multi-section position, 10 parts of the patients serums of anti-P47 antibody positive; Blood culture and phlegm are cultivated 1 part of the patients serum of the candida albicans that grows simultaneously, and only phlegm is cultivated 17 parts of positive patient serum specimens.Take immunodepressant patient group and be patient's 134 examples such as the kidney transplant of taking immune suppressive cyclosporin A and ephritis totally 166 parts of blood specimens.
2. main agents:
12 peptide phage display libraries and corresponding host bacterium E.coli ER2738 are BioLabs company product at random.
Coating buffer: 0.1mol/L NaHCO
3, pH9.6.
Confining liquid: 5.0g/L bovine serum albumin(BSA), 0.2g/L NaN
3, be dissolved in the coating buffer.
Free liquid: 0.2mol/L glycocoll-HCl, pH2.2,1.0g/L bovine serum albumin(BSA).
Neutralizer: 1mol/L Tris-HCl, pH9.1.
Stop buffer: 1M sulfuric acid.
TBS：50mmol/L?pH7.5?Tris-HCl，150mmol/L?NaCl。
Add 0.1%Tween 20 among the TBS-T:TBS.
PBS:KH
2PO
40.2g, Na
2HCO
4.12H
2O 2.9g, NaCl 8.0g, KCl 0.2g distills water-soluble to 1000ml.
Add 0.05%Tween 20 among the PBS-T:PBS.
Substrate solution: A liquid: 0.06% urea peroxide, 0.01M pH4.5 citrate buffer solution;
B liquid: 0.06%TMB, 0.01M pH4.5 citrate buffer solution;
3. statistical procedures adopts x
2Check.
Embodiment 1
1.1 the preparation of natural candida albicans hyphal albumen P47
Candida albicans C1 strain (introducing from institute of internal medicine of the Chinese Academy of Sciences) is inoculated in the RPMI1640 nutrient solution that contains 15% calf serum, 37 ℃ of concussions were cultivated 7 hours, the thalline that mirror is observed more than 95% down transfers the mycelia phase to, 3000 * g collected thalline in centrifugal 30 minutes, after physiological saline is washed 3 times, prepare bacteria suspension with the distilled water that contains the 1mmol/L phenylmethylsulfonyl fluoride, ultrasonic broken wall is pulverized under the condition of ice bath, 3000 * g removed cell fragment in centrifugal 30 minutes, supernatant is saltoutd with 70% saturated ammonium sulfate, with the PBS desalination of dialysing.Cross Con A-Sepharose 4B affinity column and remove mannose and Mannoproteins, collect the albumen of relative molecular weight 47000, obtain P47 albumen, i.e. native antigen.
1.2 the preparation of candida albicans hyphal albumen P47 analogue antigen
1.2.1 preparation candida albicans hyphal albumen P47 monoclonal antibody: with P47 protein immunization Balb/c mouse 0.2mg/ time, each interval 7 days, treat that the two-way immunodiffusion of serum is tired and reach 1: 32, prepare monoclonal antibody with hybridoma technology.Purification of Monoclonal Antibodies adopts polyglycol (PEG) precipitation method.Get hybridoma ascites 5ml, with VBS (4mmol/L barbital, 0.15mol/L NaCl, 0.8mmol/L Mg
2+, 0.3mmol/L Ca
2+) dilute one times, add SiO
2Powder 260mg shakes frequently, and is centrifugal behind the 30min, gets supernatant, to remove the fat in the ascites.Use the polyglycol (PEG6000) of final concentration 6.25% and 5% respectively to precipitate once again, the centrifugal sediment of last contains the VBS dissolving of 0.5mol/L NaCl with 2ml.Antibody behind the purifying is used for bag by microwell plate.
1.2.2 affine naughty sieve: carry out with reference to BioLabs company phage display peptide library service manual.The equal sterile working of each step in the screening process.Coating buffer with pH9.6 is 120 μ g/ml with the dilution of P47 monoclonal antibody, adds the lath micropore, 150 μ l/ holes, and 4 ℃ are spent the night.Get rid of coating buffer, at the most raffinate of aseptic thieving paper arsis, confining liquid is filled it up with in each hole, and 4 ℃ of 1h discard confining liquid, wash 6 times with TBS-T.From original peptide storehouse, get 10 μ l and mix the micropore that the back adds monoclonal antibody bag quilt with 1ml TBS-T, 100 μ l/ holes, totally 10 holes, room temperature jog 30min.Wash plate 10 times with TBS-T.Add and free liquid 100 μ l/ holes, room temperature jog 8min, each hole eluent of sucking-off is merged into a pipe, adds 40 μ l neutralizers, and pH is transferred to 7.2.Get 1 μ l eluate titration plaque number, the early stage host bacterium E.coli ER2738 of all the other eluate inoculation logarithmic growths, 4.5h is cultivated in 37 ℃ of violent joltings, carries out the bacteriophage amplification.Amplification cultivation thing centrifuging and taking supernatant spends the night with 8,000 4 ℃ of precipitations of PEG.Next day is centrifugal, suspends with 1ml TBS and precipitates, and carries out second and takes turns naughty sieve.So carry out the three-wheel screening.Select with the high dozens of phage clone of affinity of antibody and carry out determined dna sequence, the amino acid sequence that obtains three advantages clones is:
1)GlyGlnGluAlaAlaIleProThrSerAspMetPhe(SEQ?ID?No.1)
2)TyrPrpProLeuMetSerAlaLeuLysLysLeuPro(SEQ?ID?No.2)
3)HisTrpGluTyrThrThrSerProHisProArgLeu(SEQ?ID?No.3)。
1.2.3 the amplification of analogue antigen and purifying: carry out with reference to BioLabs company phage display peptide library service manual.Choose the single bacterium colony of host bacterium E.coli ER2738 to liquid LB nutrient culture media, exponential phase is cultivated in 37 ℃ of concussions, and the bacteriophage that inoculation filters out continues concussion and cultivates 4.5h.Culture is transferred to centrifuge tube, and 4 ℃ of centrifugal 25min of following 12000r/min get supernatant, and the 4 ℃ of precipitations of PEG/NaCl (20%PEG8000,2.5mol/L NaCl) that add 1/6 volume are spent the night.4 ℃ once more, the centrifugal 25min of 12000rpm abandons supernatant, and precipitation is dissolved with TBS, 4 ℃, the centrifugal 10min of 12000rpm, supernatant precipitate 1h at least in the PEG/NaCl ice bath with 1/6 volume again, the centrifugal supernatant that goes, precipitation promptly obtains required analogue antigen with the TBS dissolving.
1.3 ELISA method: analogue antigen (or native antigen) is used pH9.6,0.1mol/L NaHCO
3Coating buffer dilution (1 μ g/ml), bag is by the polystyrene reactant plate, and 4 ℃ are spent the night.Next day, every hole added confining liquid 100 μ l, 37 ℃ of 1h.Wash 5 times each 3min with TBS-T.Respectively experimenter's serum (or blood or blood plasma) is done dilution in 1: 100 with the PBS solution that contains 10% calf serum, add micro reaction plate, each experiment is all established blank, negative control and positive control, 37 ℃ of incubation 45min; PBS-T washes 5 times.Add the anti-human IgG antibody of horseradish peroxidase-labeled or the anti-human IgM antibody of horseradish peroxidase-labeled (the abbreviation enzyme is marked anti-human IgG antibody, enzyme is marked anti-human IgM antibody, down together), 37 ℃ of 45min; PBS-T washes 3 times.Add substrate solution, 37 ℃ of 10min add stop buffer, survey the 450nm absorbance, and it is the normal value upper limit that result's judgement adds 3SD with the mean value of normal control group absorbance, surpasses this upper limit person and is judged to the positive.
Embodiment 2
The ELISA kit that detects mycelian protein antibody of white candida consists of:
1) envelope antigen: amino acid sequence is any in the analogue antigen of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or three kinds of mixed in equal amounts;
2) solid support: the polystyrene board micro reaction plate is used for bag by above-mentioned analogue antigen;
3) enzyme labelled antibody: mark anti-human IgG or enzyme is marked anti-people IgM with enzyme;
4) negative control sera;
5) enzyme labelled antibody dilution: PBS;
6) substrate solution: A liquid: 0.06% urea peroxide, 0.01M pH4.5 citrate buffer solution;
B liquid: 0.06%TMB, 0.01M pH4.5 citrate buffer solution;
7) cleansing solution: PBS contains 0.01% Tween-20;
8) stop buffer: 1M sulfuric acid.
Embodiment 3
The ELISA kit that detects mycelian protein antibody of white candida consists of:
1) envelope antigen: candida albicans hyphal albumen P47 antigen;
2) solid support: the polystyrene board micro reaction plate is used for bag by above-mentioned mycelian protein P47 antigen;
3) enzyme labelled antibody: mark anti-human IgG or enzyme is marked anti-people IgM with enzyme;
4) negative control sera;
5) enzyme labelled antibody dilution, PBS;
6) substrate solution: A liquid: 0.06% urea peroxide, 0.01M pH4.5 citrate buffer solution;
B liquid: 0.06%TMB, 0.01M pH4.5 citrate buffer solution;
7) cleansing solution: PBS contains 0.01% Tween-20;
8) stop buffer: 1M sulfuric acid.
Embodiment 4
The colloidal gold kit that detects mycelian protein antibody of white candida consists of:
1) envelope antigen: the analogue antigen or the native antigen that obtain by embodiment 1;
2) reaction unit: above-mentioned analogue antigen or native antigen are put on nitrocellulose filter wherein;
3) colloid gold label antibody: with the anti-human IgG or the anti-people IgM of colloid gold label;
Reaction unit comprises three parts: the one, and small flat box divides box body and lid, and circular hole is arranged on the lid; The 2nd, the suction filler is contained in the box body, and the 3rd, place on the absorbent material, the nitrocellulose filter under the lid; Nitrocellulose filter is used for fixing above-mentioned analogue antigen or native antigen and as the human IgG or the IgM of Quality Control;
The preparation of solid phase point sample film: the disk that nitrocellulose filter is cut into diameter 1cm, add 1 μ l analogue antigen or native antigen (1 μ g/ml) in disk central authorities, 1 μ l (0.2mg/ml) human IgG (being used to measure the anti-mycelian protein antibody of white candida of IgG type) or people IgM (being used to measure the anti-mycelian protein antibody of white candida of IgM type) are put as Quality Control point, drying at room temperature in the top of antigen point.Solid phase point sample film and other materials are assembled into reaction unit.
Assay method: 1, add 100 μ l in the circular hole of reaction unit and contain the 10g/L bovine serum albumin(BSA), the PBS of 0.05%Tween 20 waits to infiltrate; 2, add test serum 50ul, wait to infiltrate; 3, add PBS100 μ l, wait to infiltrate; 4, add 100 anti-human IgGs of μ l colloid gold label or anti-human IgM antibody (corresponding), wait to infiltrate with Quality Control point; 5, add 100 μ l PBS, the unconjugated colloid gold label antibody of flush away.
Observations: positive in 3 minutes if punctation occurs at the antigen point place of film central authorities, otherwise negative; No matter the Quality Control point of film top is that feminine gender or positive findings Quality Control point all should show redness for the test effective marker.
Sequence table
＜110〉Nanjing General Hospital, PLA Nanjing Region
＜120〉method and the kit of detection mycelian protein antibody of white candida
<160>3
<210>1
<211>12
<212>PRT
＜213〉artificial sequence
<400>1
Gly?Gln?Glu?Ala?Ala?Ile?Pro?Thr?Ser?Asp?Met?Phe
1 5 10 12
<210>2
<211>12
<212>PRT
＜213〉artificial sequence
<400>2
Tyr?Prp?Pro?Leu?Met?Ser?Ala?Leu?Lys?Lys?Leu?Pro
1 5 10 12
<210>3
<211>12
<212>PRT
＜213〉artificial sequence
<400>3
His?Trp?Glu?Tyr?Thr?Thr?Ser?Pro?His?Pro?Arg?Leu
1 5 10 12
Claims (10)
1, a kind of detection mycelian protein antibody of white candida method, it is as follows to it is characterized in that specifically detecting step:
A. prepare analogue antigen or native antigen: analogue antigen is to screen acquisition with the monoclonal antibody of anti-candida albicans hyphal albumen P47 from phage display peptide storehouse; Native antigen is candida albicans hyphal albumen P47;
B. use above-mentioned analogue antigen or native antigen bag by solid support;
C. biological sample to be measured is added on the solid support of bag quilt, under the condition that is fit to the antigen-antibody combination, hatch, clean and remove any unconjugated composition;
D. add second antibody, under the condition that is fit to the antigen-antibody combination, hatch, clean and remove any unconjugated composition at people's antibody through mark;
E. detect immune existence in the mixtures incubated in conjunction with compound.
2, a kind of method that detects mycelian protein antibody of white candida according to claim 1 is characterized in that described analogue antigen is the analogue antigen that bacteriophage that the monoclonal antibody with candida albicans hyphal albumen P47 filters out obtains through amplification, purifying from phage display peptide storehouse.
3, a kind of method that detects mycelian protein antibody of white candida according to claim 1 and 2, it is characterized in that described analogue antigen be illustrated in the 12 following peptides of the amino acid sequence of phage surface any or several:
1)GlyGlnGluAlaAlaIleProThrSerAspMetPhe(SEQ?ID?No.1)
2)TyrPrpProLeuMetSerAlaLeuLysLysLeuPro(SEQ?ID?No.2)
3)HisTrpGluTyrThrThrSerProHisProArgLeu(SEQ?ID?No.3)。
4, a kind of method that detects mycelian protein antibody of white candida according to claim 1 is characterized in that described native antigen is meant through the sonicated candida albicans hyphal, again through the candida albicans hyphal albumen P47 of ConA-Sepharose column chromatography purification.
5, a kind of method that detects mycelian protein antibody of white candida according to claim 1, the condition that it is characterized in that described suitable antigen-antibody combination is meant the temperature and time of hatching the antigen-antibody potpourri, wherein temperature is 4 ℃～50 ℃, and the time is 2 minutes to 24 hours; Preferred temperature is a room temperature to 43 ℃, and the time is 2 minutes to 2 hours.
6, a kind of method that detects mycelian protein antibody of white candida according to claim 1 is characterized in that detection method can adopt ELISA method or immune colloid gold percolation; The temperature of hatching the antigen-antibody potpourri when wherein adopting the ELISA method is 37 ℃～43 ℃, and the time is 30 minutes to 1 hour; The temperature of hatching the antigen-antibody potpourri when adopting the immune colloid gold percolation is a room temperature, and the time is several minutes.
7, a kind of method that detects mycelian protein antibody of white candida according to claim 1 is characterized in that described biological sample is meant blood, serum or the blood plasma from the experimenter.
8, a kind of method that detects mycelian protein antibody of white candida according to claim 1 is characterized in that described second antibody is meant anti-human IgG antibody or the anti-human IgM antibody with horseradish peroxidase or alkaline phosphatase or colloid gold label.
9, a kind of ELISA kit that detects mycelian protein antibody of white candida is characterized in that its kit consists of:
1) envelope antigen: the analogue antigen or the native antigen that obtain by claim 1;
2) solid support: can adopt microwell plate, be used for bag by above-mentioned analogue antigen or native antigen;
3) enzyme labelled antibody: with the anti-human IgG or the anti-people IgM of horseradish peroxidase-labeled;
4) positive control serum or negative control sera;
5) enzyme labelled antibody dilution: PBS;
6) substrate solution: A liquid: 0.06% urea peroxide, 0.01M pH4.5 citrate buffer solution;
B liquid: 0.06%TMB, 0.01M pH4.5 citrate buffer solution;
7) cleansing solution: PBS contains 0.01% Tween-20;
8) stop buffer: 1M sulfuric acid.
10, a kind of colloidal gold kit that detects mycelian protein antibody of white candida is characterized in that, its kit consists of:
1) envelope antigen: the analogue antigen or the native antigen that obtain by claim 1;
2) reaction unit: the one, small flat box divides box body and lid, and circular hole is arranged on the lid; The 2nd, the suction filler is contained in the box body, and the 3rd, place on the absorbent material, the nitrocellulose filter under the lid; Nitrocellulose filter is used for fixing above-mentioned analogue antigen or native antigen and as the human IgG or the people IgM of Quality Control;
3) colloid gold label antibody: with the anti-human IgG or the anti-people IgM of colloid gold label.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100066334A CN1294418C (en) | 2004-08-09 | 2005-01-07 | Method and reagent box for inspecting mycelian protein antibody of white candida |
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|---|---|---|---|
| CN200410041634.8 | 2004-08-09 | ||
| CN200410041634 | 2004-08-09 | ||
| CNB2005100066334A CN1294418C (en) | 2004-08-09 | 2005-01-07 | Method and reagent box for inspecting mycelian protein antibody of white candida |
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| CN1294418C CN1294418C (en) | 2007-01-10 |
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| CN (1) | CN1294418C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365639A (en) * | 2017-03-31 | 2017-11-21 | 上海艾瑞德生物科技有限公司 | A kind of collaurum platform cleaning fluid |
| CN108761075A (en) * | 2018-05-24 | 2018-11-06 | 河北伊莱莎生物技术有限公司 | A kind of deoxynivalenol quantifies rapid detection card and its detection method |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
| CN110229221A (en) * | 2019-06-27 | 2019-09-13 | 上海交通大学医学院附属仁济医院 | It is a kind of for detecting the antigen and application thereof of aggressive candidiasis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2274984A1 (en) * | 1998-07-10 | 2000-01-10 | Universite Laval | Candida albicans gene (csa1) encoding a mycelial surface antigen, and uses thereof |
| CN1277358A (en) * | 1999-06-11 | 2000-12-20 | 王丽 | Biological test reagent for monilia albicans infection and its preparation |
| EP1315840A4 (en) * | 2000-09-08 | 2005-11-02 | Univ Texas | Biopanning and rapid analysis of selective interactive ligands (brasil) |
| CN1436857A (en) * | 2002-02-06 | 2003-08-20 | 昆明寰基生物芯片开发有限公司 | Specificity determination and application of one section of candida albicans DNA sequence |
-
2005
- 2005-01-07 CN CNB2005100066334A patent/CN1294418C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365639A (en) * | 2017-03-31 | 2017-11-21 | 上海艾瑞德生物科技有限公司 | A kind of collaurum platform cleaning fluid |
| CN107365639B (en) * | 2017-03-31 | 2020-04-24 | 上海艾瑞德生物科技有限公司 | Colloidal gold platform cleaning solution |
| CN108761075A (en) * | 2018-05-24 | 2018-11-06 | 河北伊莱莎生物技术有限公司 | A kind of deoxynivalenol quantifies rapid detection card and its detection method |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
| CN110229221A (en) * | 2019-06-27 | 2019-09-13 | 上海交通大学医学院附属仁济医院 | It is a kind of for detecting the antigen and application thereof of aggressive candidiasis |
| CN110229221B (en) * | 2019-06-27 | 2022-04-12 | 上海交通大学医学院附属仁济医院 | Antigen for detecting invasive candidiasis and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1294418C (en) | 2007-01-10 |
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