CN103336126B - A kind of agglutinin test chip for saliva sample and disposal route thereof - Google Patents
A kind of agglutinin test chip for saliva sample and disposal route thereof Download PDFInfo
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Abstract
The invention provides a kind of agglutinin test chip for saliva sample, cost of manufacture is lower, test with strong points, the Inherent advantage of binding lectin chip, can detect the information (the N-glycosylation of saliva holoprotein and O-glycosylation sugar chain structure information) of the intermediate result obtained as diabetes B assessment fast, simply, efficiently.Should for the agglutinin test chip of saliva sample, adopt epoxidation sheet base, its special character is: on epoxidation sheet base, only point sample is fixed with at least one agglutinin in PTL-I, LCA and SBA.The present invention is also optimized the preparation of above-mentioned agglutinin test chip and disposal route, thus set up for generating the optimal reaction system detecting holoprotein N-glycosylation and O-glycosylation information in saliva sample, provide convenience for detecting diabetes B patient SGP sugar chain structure fast and efficiently.
Description
Technical field
The present invention relates to a kind of agglutinin test chip and disposal route thereof, specially for saliva sample, obtain the information of the intermediate result as diabetes B assessment.
Background technology
Agglutinin (Lectin) is non-immune origin, does not have a class carbohydrate-binding protein of enzymatic activity, exclusively can identify specific sugar chain sequence in the monose of a certain special construction or glycan and combine with it.It is present in plant, animal and microorganism, finds more than 300 kind of agglutinin from occurring in nature so far.According to the type of combined sugar chain, can the agglutinin divide into several classes type of occurring in nature, can with certain glycan molecule specific binding, be just this sugared agglutinin, generally like this agglutinin can be divided into six large classes: D-MANNOSE or D-Glucose agglutinin (as concanavalin A); 2-Acetamido-2-deoxy-D-glucose agglutinin (agglutinin as Fructus Hordei Germinatus); N-acetylamino galactosamine agglutinin (agglutinin as soybean); D-galactose agglutinin (agglutinin as castor-oil plant); L-fucose agglutinin (agglutinins as chaste tree beans); N-acetyl-neuraminate (sialic acid) agglutinin (as wheat germ agglutinin).Aggegation have various application: agglutinin can be used for the diagnosis of the diseases such as malignant tumour; Aggegation is utilized usually to design antibacterial, antiviral drugs; Agglutinin is utilized to design " biological missile "; Agglutinin is utilized to carry out identification and the analysis of sugar chain structure.The investigative technique of glycoprotein mainly contains: glycoprotein technology in gel, tree species for bio-energy source, technology that agglutinin is affine.
Learn research field along with chip technology being introduced sugar group, agglutinin is more and more noticeable as the important tool of decoding sugar chain structure.Lectin chip is according to the mutual recognition reaction of the specificity between agglutinin and sugar chain, the agglutinin of various separate sources is fixed on epoxidation, aldehyde radical or the sheet base modified through other modes, again with mark after the sample incubation to be detected such as glycoprotein, cell and thalline react, after a series of subsequent treatment, the sugar chain structure in sample to be detected and its agglutinin identified could be waited until.
Diabetes are by insulopathic or the abnormal a kind of metabolic disorder disease caused of insulin active, are caused with the disorderly hyperglycaemia of sugar, fat and protein metabolism by insulin deficiency.Diabetes are mainly divided into 4 types: type 1 diabetes, diabetes B, gestational diabetes mellitus and specific type diabetes, wherein, diabetes B (Type2diabetesmellitus, T2DM) increase of the insulin secretion fault that insulin resistance causes is derived from, account for diabetes proportion maximum, diabetes B patient accounts for 90% ~ 95%.For a long time, focus mostly in in the analysis of Leaf proteins to the research of diabetes B.
In recent years, there is many research to the saliva of diabetes B patient, urine, serum and tissue protein, in succession set up the measuring technologies such as multidimensional liquid chromatography/tandem mass spectrum (2D-LC-MS/MS), high performance liquid chromatography (HPLC), MALDI-TOF/TOF time-of-flight mass spectrometry, bidirectional electrophoresis technique and enzyme linked immunosorbent assay (ELISA) and diabetes B is studied.At present usually all will with reference to blood test results to the earlier evaluations of diabetes B.
Some researchs in biological process and molecular function find, the change of salivary component can reflect the change of multiple proteins composition in blood to a certain extent, such as, also the biomarker with the disease association such as infectious disease, angiocardiopathy, cancer, auto-immune disease, diabetes is there is in sialoprotein matter, therefore, there is the possibility replacing blood testing health with saliva detection.Compared with blood, the safer convenience of collection of saliva, hurtless measure, frequently can monitor the real-time change that saliva index observes health more accurately.Compared with urine, saliva having can the advantage of real-time sampling.Therefore, in recent years, saliva detect delay also receives very large concern.
But at present, less to the research of diabetes B patient SGP sugar chain structure, also not yet occur that a kind of detection method can detect the N-glycosylation of diabetic's saliva holoprotein and O-glycosylation sugar chain spectrum fast.
Summary of the invention
The invention provides a kind of agglutinin test chip for saliva sample, cost of manufacture is lower, test with strong points, the Inherent advantage of binding lectin chip, can detect the information (the N-glycosylation of saliva holoprotein and O-glycosylation sugar chain structure information) of the intermediate result obtained as diabetes B assessment fast, simply, efficiently.
The solution of the present invention is as follows:
Should for the agglutinin test chip of saliva sample, adopt epoxidation sheet base, its special character is: on epoxidation sheet base, only point sample is fixed with at least one agglutinin in PTL-I, LCA and SBA.
The factor such as considering cost and accuracy, the two kinds of agglutinins preferably chosen wherein make agglutinin test chip product.
By a large amount of experiments and data analysis, the present invention is also optimized the preparation of above-mentioned agglutinin test chip and disposal route, thus set up for generating the optimal reaction system detecting holoprotein N-glycosylation and O-glycosylation information in saliva sample, provide convenience for detecting diabetes B patient SGP sugar chain structure fast and efficiently.
Scheme is as follows:
For preparation and the disposal route of the lectin chip of saliva sample, for generating the reaction system detecting holoprotein N-glycosylation and O-glycosylation sugar chain structure information in saliva sample, comprise the following steps:
1) epoxidation sheet base is got for subsequent use;
2) agglutinin is mixed with the sampling liquid that concentration is 1mg/mL;
3) sampling liquid of preparation is added in 384 orifice plates; Use brilliant core 48 spotting system point sample on epoxidation sheet base again;
4) chip made by point is overnight incubation in the environment of 55%-65% in humidity;
5) 3 hours are vacuumized in the vacuum dryer of 37 DEG C to the chip of hatching, make agglutinin be fixed on chip;
6) lectin chip fixed is placed in exsiccator, keeps in Dark Place;
7) weighing 0.02g/mLBSA and 500mM glycocoll dissolves in the phosphate buffer-polysorbas20 of pH7.4, dissolves the membrane filtration with 0.2 μm after mixing, the lectin chip prepared is placed in confining liquid 1 hour;
8) the lectin chip phosphate buffer-polysorbas20 after closing and phosphate buffer are cleaned 1 time respectively successively, each 5 minutes, more for subsequent use after drying;
9) lectin chip after drying is added to the incubation buffer of 750-800 μ L, as the reaction environment with sample to be tested;
Described phosphate buffer pH7.4 is with 1L total amount, and component is: NaCl8.0g, KCl0.2g, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g;
Described phosphate buffer-polysorbas20 is the described phosphate buffer also containing massfraction 0.05-0.5% polysorbas20;
Described incubation buffer is the described phosphate buffer-polysorbas20 also containing massfraction 1% bovine serum albumin(BSA) and massfraction 10-20% azanol.
The above-mentioned chip made by point is hatch 12 hours in the environment of 60% for best in humidity.
Above-mentioned incubation buffer lectin chip after drying being added to 4 μ g samples to be tested and 700 μ L, and within 3 hours, be best in incubated at room.
The present invention has the following advantages:
Agglutinin test chip of the present invention, establish the agglutinin that PTL-I, LCA and SBA these three kinds detects for diabetes B patient saliva sample, test with strong points, cost of manufacture is lower; And set up for generating the optimal reaction system detecting holoprotein N-glycosylation and O-glycosylation sugar chain structure information in saliva sample, the Inherent advantage of binding lectin chip, provides convenience for detecting diabetes B patient SGP sugar chain structure fast and efficiently.
Accompanying drawing explanation
Fig. 1 is the testing result figure of diabetes B patient young man mixing sample of the present invention and normal healthy controls young man mixing sample;
Fig. 2 is the testing result figure of diabetes B patient elderly men mixing sample of the present invention and normal healthy controls elderly men mixing sample;
Fig. 3 is the testing result figure of diabetes B patient old women mixing sample of the present invention and normal healthy controls old women mixing sample;
Fig. 4 is the testing result figure of diabetes B patient young man list of the present invention example sample;
Fig. 5 is the testing result figure of diabetes B patient elderly men list of the present invention example sample;
Fig. 6 is the point sample matrix design of the lectin chip adopted in R&D process of the present invention.
Embodiment:
Contemplated by the invention the difference of different sexes, age groups, mark off young man, elderly men and old women tree-span continue, application lectin chip technology has carried out a large amount of experiments and analysis respectively.
The lectin chip result of previous experiments to the loading gradient of 2 μ g, 4 μ g, 6 μ g and 8 μ g is analyzed, and show that the signal value of 4 μ g applied sample amounts obviously raises than the signal value of 2 μ g applied sample amounts; And when 4 μ g and 6 μ g applied sample amount, the signal value difference of each agglutinin is little, some raises a little at 6 μ g signal values, a little reduction had; The signal value of 8 μ g applied sample amounts substantially all raises than the signal value of 6 μ g applied sample amounts, only has the reduction of indivedual agglutinin.Do not make too saturated false positive or the loading of causing of loading cause false negative very little, we select 4 μ g as the applied sample amount of experiment sample lectin chip.
For young man mixing saliva sample, major experimental step is:
1) lectin chip is prepared;
1.1) epoxidation sheet base is got for subsequent use;
1.2) agglutinin is mixed with the sampling liquid that concentration is 1mg/mL;
1.3) sampling liquid of preparation is added in 384 orifice plates; Use brilliant core 48 spotting system point sample on epoxidation sheet base again;
1.4) chip made by point is hatch 12 hours in the environment of 60% in humidity;
1.5) 3 hours are vacuumized in the vacuum dryer of 37 DEG C to the chip of hatching, make agglutinin be fixed on chip;
1.6) lectin chip fixed is placed in 4 DEG C of exsiccators, keeps in Dark Place with for subsequent use.
2) biological specimen is prepared;
2.1) saliva sample needed for collection;
2.2) centrifugal filtration purifying sample holoprotein;
2.3) carry out concentrating quantitatively to the holoprotein sample of purifying, make final concentration be greater than 1mg/mL;
2.4) with fluorescent reagent, concentrated sample is marked, remove unreacted fluorescent reagent;
2.5) by frozen for the sample packing that marked in the refrigerator of-20 DEG C or-80 DEG C.
3) biological specimen is loaded on lectin chip to obtain in sample sugar chain structure on glycoprotein;
3.1) by 0.02g/mLBSA(bovine serum albumin(BSA)) and 500mM glycocoll dissolve in the phosphate buffer-polysorbas20 of pH7.4 and be mixed with confining liquid, the lectin chip prepared is placed in confining liquid 1 hour;
3.2) by close after lectin chip phosphate buffer-polysorbas20 and phosphate buffer clean 1 time respectively, each 5 minutes, then through dry after for subsequent use;
3.3) lectin chip after drying is added to the incubation buffer of sample that 4 μ g mark and 700 μ L, and incubated at room 3 hours;
3.4) the lectin chip phosphate buffer-polysorbas20 after hatching and phosphate buffer are cleaned each 2 times, each 5 minutes respectively, then dry;
3.5) to the GenePix4000B chip scanner scanning of the lectin chip after drying, the fluoroscopic image of Glycoprotein binding after obtaining agglutinin and marking; See Fig. 1: diabetes B young man compound sample and normal healthy controls young man compound sample.
3.6) by GenePix3.0 software, interim analysis is carried out to fluoroscopic image, diabetes B patient SGP sugar chain spectrum result is drawn after sugar chain structure in image corresponding to agglutinin is checked, and compare with the normal healthy controls group at corresponding age, find out the sugar chain structure of diabetes B patient-specific.
Step 3.6) concrete operations are: from lectin chip scanning result figure, obtain the fluorescence signal value that each agglutinin and sialoprotein combine, will the value of 2 times of background standard deviations be greater than as effective value.In each district on a slice, thin piece, each agglutinin has 3 to repeat a little, and the intermediate value choosing the fluorescence signal value of often kind of agglutinin is normalized analysis, and 3 differentiations are analysed and obtained 3 normalization intermediate values, try to achieve mean value and standard deviation value.The relatively SGP sugar chain structure difference of diabetes B patient and normal healthy controls.Calculate the ratio R atio of diabetes B patient's saliva sample fluorescence signal and normal healthy controls saliva sample fluorescence signal, fluorescence signal value used is all the numerical value that intermediate value normalization obtains.General situation, statistical theory using the value of Ratio value between 0.66 to 1.5 as the value without significant difference, and Ratio value be greater than 1.5 and be less than 0.66 as the value that there are differences.Draw the glycoprotein candy chain textural difference that diabetes B patient young man mixing saliva sample is compared with the normal healthy controls group at corresponding age.
Several main agglutinin and corresponding test result has been arranged with following table one, table two, table three choosing.
The glycoprotein candy chain textural difference that table one is compared with the normal healthy controls group at corresponding age for diabetes B patient young man mixing saliva sample of the present invention.
Table one
| Lectin | Specificity | T2DM |
| HHL | Non-substitutedα-1,6Man | － |
| WFA | Terminal GalNAc | － |
| GSL-Ⅱ | GlcNAc and galactosylated N-glycans | － |
| MAL-Ⅱ | Sia2-3Galβ1-4Glc(NAc) | － |
| PHA-E | Bisecting GlcNAc and biantennary N-glycans | － |
| PTL-Ⅰ | αGalNAc and Gal | － |
| SJA | terminal in GalNAc and Gal | － |
| PNA | Galβ1-3GalNAcα-Ser/Thr(T) | － |
| AAL | Fucose | ＋ |
| MPL | αGalNAc | － |
| LEL | Poly-LacNAc and(GlcNAc)n | － |
| GSL-Ⅰ | αGalNAc,GalNAcα-Ser/Thr(Tn)andαGal | － |
| DBA | GalNAcα-Ser/Thr(Tn)and GalNAcα1-3Gal | － |
| LCA | Fucoseα-1,6GlcNAc(core fucose) | － |
| STL | GlcNAc | － |
| PTL-Ⅱ | Gal | － |
| DSA | GlcNAc | ＋ |
| SBA | Terminal GalNAc(especially GalNAcα1-3Gal) | ＋ |
| NPA | Non-substitutedα-1,6Man | － |
Note :+represent high expressed in T2DM patient ,-represent low expression in T2DM patient.
The glycoprotein candy chain textural difference that table two is compared with the normal healthy controls group at corresponding age for diabetes B patient elderly men mixing saliva sample of the present invention;
Table two
| Lectin | Specificity | T2DM |
| Jacalin | Galβ1-3GalNAcα-Ser/Thr(T)and GalNAcα-Ser/Thr(T) | － |
| ECA | Galβ-1,4GlcNAc | ＋ |
| HHL | Non-substitutedα-1,6Man | ＋ |
| GSL-Ⅱ | GlcNAc and galactosylated N-glycans | ＋ |
| MAL-Ⅱ | Sia2-3Galβ1-4Glc(NAc) | ＋ |
| PHA-E | Bisecting GlcNAc and biantennary N-glycans | － |
| PTL-Ⅰ | αGalNAc and Gal | － |
| SJA | terminal in GalNAc and Gal | － |
| PNA | Galβ1-3GalNAcα-Ser/Thr(T) | － |
| EEL | Galα1-3(Fucα1-2)Gal | － |
| AAL | Fucose | － |
| LTL | Fucoseα-1,3GlcNAc(core fucose)，Sia-Lex and Lex | ＋ |
| MPL | αGalNAc | ＋ |
| GSL-Ⅰ | αGalNAc,GalNAcα-Ser/Thr(Tn)andαGal | ＋ |
| DBA | GalNAcα-Ser/Thr(Tn)and GalNAcα1-3Gal | － |
| LCA | Fucoseα-1,6GlcNAc(core fucose) | － |
| STL | GlcNAc | ＋ |
Note :+represent high expressed in T2DM patient ,-represent low expression in T2DM patient.
The glycoprotein candy chain textural difference that table three is compared with the normal healthy controls group at corresponding age for diabetes B patient old women mixing saliva sample of the present invention;
Table three
| Lectin | Specificity | T2DM |
| ECA | Galβ-1,4GlcNAc | ＋ |
| HHL | Non-substitutedα-1,6Man | ＋ |
| WFA | Terminal GalNAc | － |
| GSL-Ⅱ | GlcNAc and galactosylated N-glycans | ＋ |
| PTL-Ⅰ | αGalNAc and Gal | － |
| EEL | Galα1-3(Fucα1-2)Gal | ＋ |
| AAL | Fucose | － |
| MPL | αGalNAc | ＋ |
| LEL | Poly-LacNAc and(GlcNAc)n | ＋ |
| GSL-Ⅰ | αGalNAc,GalNAcα-Ser/Thr(Tn)andαGal | ＋ |
| LCA | Fucoseα-1,6GlcNAc(core fucose) | － |
| RCA120 | β-gal | ＋ |
| STL | GlcNAc | － |
| BS-Ⅰ | α-gal andα-GalNAc | ＋ |
| SBA | Terminal GalNAc(especially GalNAcα1-3Gal) | ＋ |
| VVA | GalNAc and GalNAcα-Ser/Thr(Tn) | ＋ |
| NPA | Non-substitutedα-1,6Man | ＋ |
| PSA | Fucoseα-1,6GlcNAc(core fucose) | － |
| ACA | Galβ1-3GalNAcα-Ser/Thr(Tn) | － |
| UEA-Ⅰ | Fucoseα1-2Galβ1-4Glc(NAc) | ＋ |
| PWM | GlcNAc | ＋ |
| MAL-Ⅰ | Galβ-1,4GlcNAc | ＋ |
| BPL | Galβ1-3GalNAc | ＋ |
| SNA | Sia2-6Galβ1-4Glc(NAc) | ＋ |
Note :+represent high expressed in T2DM patient ,-represent low expression in T2DM patient.
From the sialoprotein sugar chain structure comparative result of above young man, elderly men and old women three groups of T2DM patients and corresponding normal healthy controls, due to sex difference and the difference at age, the glycosylation of people's sialoprotein is expressed also very big-difference, some sugar chain structures express rising in patients, and some sugar chain structures express reduction in patients.
Through statistics, have the sugar chain structure corresponding to 3 kinds of agglutinins 3 groups relatively in be consistent, namely PTL-I, LCA identify all low expression of sugar chain structure, SBA identify sugar chain structure all high expresseds.
When testing sample to be tested, consider larger difference between individuality, the lectin chip result of the single routine sample of T2DM patient's saliva and the lectin chip result of T2DM patient's saliva mixing sample can not be completely the same, so in analyzing using T2DM patient over half routine discrepant value as effective discrepant value, obtain the sugar chain structure of differential expression in T2DM patient.As shown in following table four, table five, by the sugar chain structure of the differential expression of young man T2DM example and the individual routine gained of elderly men T2DM, mix with young man T2DM respectively and compare with the sugar chain structure of the differential expression of elderly men T2DM mixing gained, can find out that the sugar chain structure of differential expression has very high consistance.Have the sugar chain structure corresponding to indivedual agglutinin to express inconsistent, this may be because limited sample size, does not reach good statistics.
Table four is the contrast of young man compound sample and single routine sample.Table five is the contrast of elderly men compound sample and single routine sample.
Table four
Note :+represent at T2DM patient's high expressed ,-represent in the low expression of T2DM patient
Table five
note :+represent at T2DM patient's high expressed ,-represent in the low expression of T2DM patient
Therefore, T2DM can be selected to lower the agglutinin PTL-I corresponding to sugar chain structure expressed, and the agglutinin SBA corresponding to the sugar chain structure of up-regulated expression does sialoprotein chip, carries out authenticate reverse to lectin chip result.Can find out low than Healthy People sample of the signal value of T2DM mixing sample from the interpretation of result of PTL-I, it is low that the mono-routine sample of T2DM has height to have, and great majority are on the low side than Healthy People sample.Can find out the height of signal value than Healthy People sample of T2DM mixing sample from the interpretation of result of SBA, it is low that the mono-routine sample of T2DM has height to have, and great majority are higher than Healthy People sample.The sialoprotein chip that these two kinds of agglutinins carry out all describes the reliability of lectin chip result, effectively demonstrates lectin chip result.
Claims (2)
- At least one agglutinin in 1.PTL-I, LCA and SBA is preparing the purposes in the lectin chip in order to detect diabetes B appreciation information, the feature of described purposes is to take saliva sample as detected object, and on epoxidation sheet base only point sample be fixed with at least one agglutinin in PTL-I, LCA and SBA.
- 2. purposes according to claim 1, is characterized in that: only point sample fixes two kinds of agglutinins wherein.
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| CN104678103A (en) * | 2014-08-05 | 2015-06-03 | 首都医科大学附属北京佑安医院 | Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid |
| CN105652002B (en) * | 2016-01-07 | 2018-05-04 | 西北大学 | A kind of lectin chip and its method based on sialoprotein detection sugar chain marker |
| CN105675893A (en) * | 2016-03-04 | 2016-06-15 | 西北大学 | Lectin chip for detecting carbohydrate chain markers based on blood serum and commonly based on protein in blood serum and saliva as well as kit and application of lectin chip |
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