CN108469519A - It is a kind of for the composition of enzyme linked immunological kit and diabetes antibody repertoire detection kit and preparation method thereof - Google Patents
It is a kind of for the composition of enzyme linked immunological kit and diabetes antibody repertoire detection kit and preparation method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention relates to Enzyme-multiplied immune technique field, a kind of composition for enzyme linked immunological kit and diabetes antibody repertoire detection kit and preparation method thereof are disclosed.Composition of the present invention includes confining liquid and enzyme mark dilution；The confining liquid contains BSA, polyvinyl alcohol, mannitol, Sodium azide and disodium hydrogen phosphate, and the enzyme mark dilution contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, glycine betaine and Proclin300.The present invention starts with from the confining liquid and enzyme mark dilution of enzyme linked immunological kit, by selecting suitable compositions so that enzyme linked immunological kit can keep the stability of detection the long period.Meanwhile the diabetes detection kit prepared with the composition is on the basis of with preferable stability, additionally it is possible to carry out Accurate Diagnosis to diabetes and parting has higher positive rate especially in the detection of IAA.
Description
Technical field
The present invention relates to Enzyme-multiplied immune technique fields, and in particular to a kind of composition for enzyme linked immunological kit and
Diabetes antibody repertoire detection kit and preparation method thereof.
Background technology
Diabetes (diabetes) are by inherent cause, immunologic function disorder, microorganism infection and its toxin, free radical poison
Element, the various virulence factors of mental element etc. act on body lead to hypoinsulinism, insulin resistance etc. and cause sugar,
A series of metabolic disorder syndromes such as protein, fat, water and electrolyte, clinically using hyperglycemia as main feature, typical disease
Example such as may occur in which diuresis, more drink, eat more, become thin at the performances, i.e. " three-many-one-little " symptom.Diabetes in the cause of disease, pathogenesis and from
Right course of disease etc. display height heterogeneity, so diabetes are the general names of one group of hyperglycemic disorder.Diabetes be based on the cause of disease and
Pathogenesis is divided into Type I diabetes, type II diabetes and other types.Type I diabetes is with beta Cell of islet autoimmune destruction
For a kind of disease of Important cause of disease, patient's body is directed to beta Cell of islet insulin synthesis, transhipment and secretion there are one or more
Key enzyme autoantibody, mainly include islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamate decarboxylase
Antibody (GADA), tyrosine phosphatase antibody (IA-2A), carboxypeptidase antibody (CPH-A) and 8 antibody of Zinc transporter (ZnT8-A)
Deng 6 kinds of autoantibodies.85%~90% patient usually exists one or more above-mentioned anti-when detecting hyperglycemia for the first time
Body.
Protein-chip is a kind of protein-function assays technology of high throughput, is biology inspection developed in recent years
Survey technology, this technology are detected using fluorescence or enzyme colour developing, finally on the analysis micro to small chip by protein
It is analyzed with specific computer software.By elisa technique, protein-chip can quickly, high-throughput, quantitative analysis is big
The advantages such as the protein sample of amount and required reagent and sample are less, and price is less expensive after commercialization, make it rapidly become research
Hot spot.But the stability of such kit and bad at present.In addition, such kit is in detection diabetes insulin itself
Shortcomings on the positive rate of antibody (IAA), positive rate is relatively low, is easy missing inspection.
Invention content
In view of this, the purpose of the present invention is to provide a kind of compositions for enzyme linked immunological kit so that described
Composition can significantly improve the stability of kit in low temperature and at room temperature when being used to prepare enzyme linked immunological kit, when preservation
Between extend；
Another object of the present invention is to provide application of the above-mentioned composition in preparing enzyme linked immunological kit, special
It is not the related kit for detecting diabetes；
Another object of the present invention is to provide a kind of diabetes antibody repertoire detection reagent comprising above-mentioned composition
Box and preparation method thereof so that the kit has the stability of long period, while testing result in low temperature and at room temperature
With higher specificity and sensibility；
Another object of the present invention is to provide a kind of diabetes antibody repertoire detection reagent comprising above-mentioned composition
Box and preparation method thereof so that the kit can significantly improve the positive of detection diabetes insulin autoantibody (IAA)
Rate.
To achieve the goals above, the present invention provides the following technical solutions：
A kind of composition for enzyme linked immunological kit, including confining liquid and enzyme mark dilution；The confining liquid contains
BSA, polyvinyl alcohol, mannitol, Sodium azide and disodium hydrogen phosphate, the enzyme mark dilution contain Tris, citric acid, BSA, poly- second
Glycol, gum arabic, glycine betaine and Proclin300.
For existing enzyme linked immunological kit stablize defect poor, that the holding time is shorter, present inventors have unexpectedly found that from
The confining liquid and enzyme mark dilution of reagent preparation box are started with (for diluting enzyme-labelled antigen or antibody use), suitable by selecting
Component improves the composition of the two, can significantly improve stability and the holding time of enzyme linked immunological kit.
Preferably, the confining liquid contains 0.6%-1%BSA, polyvinyl alcohol, the 0.8%- of 0.8%-1.5%
1.5% mannitol, 0.05% Sodium azide and 0.01M disodium hydrogen phosphates, pH value 7.4, surplus are water, and the percentage is quality
Percentage (w/v)；In the specific embodiment of the invention, the confining liquid contains 1%BSA, 1% polyvinyl alcohol, 0.8% sweet
It is ultra-pure water to reveal alcohol, 0.05% Sodium azide and 0.01M disodium hydrogen phosphates, pH value 7.4, surplus.Wherein, polyvinyl alcohol is preferably
Polyvinyl alcohol 2W.
Preferably, the enzyme mark dilution contains BSA, 1%- of the citric acid of Tris, 0.05M of 0.1M, 2%-3%
3% polyethylene glycol, 0.01% gum arabic, the glycine betaine of 0.8%-1.5% and 0.05% Proclin300,
Surplus is water, and in the percentage in addition to Proclin300 is percent by volume, remaining is all mass percent (w/v)；
In the specific embodiment of the invention, the enzyme mark dilution contain the citric acid of Tris, 0.05M of 0.1M, 2.5% BSA,
2% polyethylene glycol, 0.01% gum arabic, 1% glycine betaine and 0.05% Proclin300.Wherein, poly- second
Glycol is preferably polyethylene glycol 1W.
The present invention carries out the preparation of diabetes antibody ELISA immune reagent kit using above-mentioned composition, with the envelope using routine
It closes liquid to compare with enzyme mark dilution diabetes antibody ELISA immune reagent kit, under low temperature (2-8 DEG C), kit of the present invention exists
Be maintained to prepare stability when completing after placing 24 months, and the kit compareed after placing 18 months just
There is significantly unstability；Under room temperature (18-28 DEG C), kit of the present invention remains able to protect after placing 6 months
The stability prepared when completing is held, higher stability is maintained to after placing 8 months, and the kit compareed is being put
Just occurs significantly unstability after setting 4 months.
Based on above-mentioned excellent technique effect, the present invention proposes the composition in preparing enzyme linked immunological kit
Using the especially application in preparing diabetes enzyme-linked immunologic detecting kit.
Meanwhile the present invention also provides a kind of diabetes antibody repertoire detection kits, including following components：
Be coated with diabetes autoantigen protein chip, with the enzyme labelled antibody of enzyme mark diluted, sample diluting liquid,
Cleaning solution and developing solution；Wherein, it is closed using confining liquid after the protein chip coating diabetes autoantigen, the confining liquid
Containing BSA, polyvinyl alcohol, mannitol, Sodium azide and disodium hydrogen phosphate, the enzyme mark dilution contain Tris, citric acid, BSA,
Polyethylene glycol, gum arabic, glycine betaine and Proclin300, the confining liquid and enzyme mark dilution and aforementioned composition scheme
It is identical.
Preferably, affiliated coating is mutual by the protein chip of Avidin and biotinylated diabetes autoantigen
In conjunction with coating.Wherein, Avidin is preferably Streptavidin.
Preferably, the diabetes autoantigen is selected from one or both of IA-2, GAD, IC, CPH, ZnT8, IA
More than；In the specific embodiment of the invention, the diabetes autoantigen be IA-2, GAD, IC, CPH, ZnT8 and IA (
IA and insulin mentioned in the present invention indicate insulin).
In the preferred scheme, the protein chip of kit of the present invention includes at least biotinylated IA as sugar
The sick autoantigen of urine, in order to improve the positive rate of IA antibody Is AA, the present invention changes the biotinylation method of IA
Into, multiple insulin molecules are coupled to by SATA and SMCC on high molecular weight protein amino, while high molecular weight protein be coupled
Upper biotin, the Biotin- high molecular weight protein-insulin conjugates of formation, in conjunction with multiple insulin molecules and biotin point
Sub (schematic diagram is shown in Fig. 1), then through coating Avidin amplification in advance, the coating efficiency of antigen insulin is substantially increased, from
And the sensitivity with the insulin antibody tests reagent of its preparation or kit can be significantly improved, it is greatly improved insulin
The positive rate of antibody test reduces the probability of missing inspection or flase drop.It is as follows：
It is step 1, insulin and S- acetylthio acetate succinates imide ester (SATA, be purchased from Thermo) room temperature is anti-
It answers, (by-product is small molecule can penetrate bag filter to dialysis purification, and macromolecular insulin-SH-P cannot penetrate bag filter, lead to
By-product can be removed by crossing the mode repeatedly dialysed) insulin-SH-P is obtained afterwards, reaction equation is as follows：
In insulin-SH-P, insulin indicates that insulin, SH indicate that reactive thiol group, P indicate blocking group, i.e.,In insulin structural formulas, circle indicates that insulin amine acid chain, n indicate the number of amino on insulin amine acid chain
Mesh, 1≤x≤n, x and n are integer, and x is preferably 1.
Insulin-SH-P with containing EDTA (metal ion in chelate solution removes the interference of metal ion in solution) and
Blocking group is removed in the PBS room temperature reactions of hydroxylamine hydrochloride, is formed after purification containing reactive thiol group through sephadex column
Insulin-SH, spare, reaction equation is as follows：
High molecular weight protein, Sulfo-N- succinimidos 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts
(SMCC, be purchased from Thermo) and-ten two polyethylene glycol of succinic acid Asia amide-biotin room temperature reaction, it is pure by PBS dialysis 3 times
(by-product, which is small molecule, can penetrate bag filter, and high molecular weight protein cannot penetrate bag filter, can by way of repeatedly dialysing for change
To remove by-product) obtain Biotin- high molecular weight proteins-SMCC, the high molecular weight protein be HBA, BSA, OVA or casein,
Reaction equation is as follows：
It, both can be by high molecular weight protein, Sulfo-N- succinimides when adding reactant according to above-mentioned reaction process
Base 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts (SMCC) and succinic acid Asia amide-ten two polyethylene glycol-biotin
Reaction is completed in addition together, can also first by high molecular weight protein and succinic acid Asia amide-ten two polyethylene glycol-biotin reaction,
Then it adds SMCC again to complete in two steps, reaction essence will not change.In step reaction, ellipse represents high molecular weight protein
Amino acid chain, m indicate the number of amino groups on high molecular weight protein amino acid chain, and y+z≤m, z >=10, y >=1, y, z and m are whole
Number, if z/x is non-integer, z/x round numbers and in integer-bit plus 1.
Step 2 reacts spare insulin-SH and Biotin- high molecular weight protein-SMCC mixed room temperatures, poly- by Portugal
Sugared gel column obtains Biotin- high molecular weight protein-Insulin after purification, has structure shown in formula 1, as biotinylated
Insulin antigens, reaction equation are as follows：
In Biotin- high molecular weight proteins-Insulin, if z/x is non-integer, z/x round numbers and in integer-bit plus 1.
For the special biotinylated insulin antigens of the present invention, no matter x is 1 or to be number of amino groups n on insulin, not shadows
The effect that multiple insulin are connected on the same high molecular weight protein is rung, but the amount of insulin connected when x=1 is most.
Wherein, IA and S- acetylthio acetate succinate imide esters respectively preferably with PBS (preferably pH values be 7.0,
The PBS of 0.01M, following PBS are preferable over this) and dimethyl sulfoxide (DMSO) be that solvent prepares reaction solution, the i.e. PBS solution of IA, IA
A concentration of 2-4mg/mL；The dimethyl sulphoxide solution of S- acetylthio acetate succinate imide esters, S- acetylthio acetate succinates
A concentration of 40-80mmol/L of imide ester.The PBS solution of IA is sub- with the dimethyl of S- acetylthio acetate succinate imide esters
The volume ratio of sulfolane solution is 1mL：10μL.
In PBS containing EDTA and hydroxylamine hydrochloride, a concentration of 20mmol/L of EDTA, hydroxylamine hydrochloride a concentration of 0.5mol/L, pH
Value is 7.0；The volume ratio of insulin-SH-P and PBS containing EDTA and hydroxylamine hydrochloride is (6-10):1.
HBA and Sulfo-N- succinimidos 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts preferably use
PBS prepares reaction solution as solvent, and-ten two polyethylene glycol of succinic acid Asia amide-biotin is matched by solvent of dimethyl sulfoxide (DMSO)
The PBS solution of reaction solution processed, i.e. human serum albumin (HBA), a concentration of 10-20mg/mL of HBA；Sulfo-N- succinimides
The PBS solution of base 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts, Sulfo-N- succinimidos 4- (Malaysia acyls
Formimino group) a concentration of 10-15mg/mL of hexamethylene -1- carboxylic acid sodium salts；- ten two polyethylene glycol of succinic acid Asia amide-biotin
Dimethyl sulphoxide solution, a concentration of 0.25-0.4mol/L of-ten two polyethylene glycol of succinic acid Asia amide-biotin.Sulfo-N-
The PBS solution of succinimido 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts and-ten dimerization of succinic acid Asia amide
The volume of the dimethyl sulphoxide solution equal proportion mixed solution of ethylene glycol-biotin and the PBS solution of human serum albumin (HBA)
Than for (10-20 μ L)：1mL.
In the specific embodiment of the invention, above-mentioned biotinylated method can be specific as follows：
Prepare the insulin PBS solution of 2-4mg/mL, the S- acetylthio acetic acid ambers that configuration concentration is about 40-80mmol/L
The dimethyl sulphoxide solution (solution A) of amber imide ester, 10 microlitres of solution A is added in 1 milliliter of insulin PBS solution
Mixing reacts at room temperature 1-4 hours, and reaction product recycles after dialysing in PBS 3 times and obtains insulin-SH-P, and 4 degree temporary standby
With；
The hydroxylamine hydrochloride (EDTA containing 20mmol/L, pH7.0) that 0.5mol/L is configured using PBS as solvent is used as B solution,
By insulin-SH-P and the volume ratio of the PBS containing EDTA and hydroxylamine hydrochloride is (6-10):1, mixed room temperature reacts 1-2 hours
Blocking group is sloughed, forms the insulin-SH containing reactive thiol group after purification through sephadex column；
The PBS solution for preparing the human serum albumin (HBA) of 10-20mg/mL, configures the Sulfo-N- ambers of 10-15mg/mL
The PBS solution of amber imide 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts makees C solution, and another configuration concentration is
The dimethyl sulphoxide solution of-ten two polyethylene glycol of 0.25-0.4mol/L succinic acids Asia amide-biotin makees solution D, by C solution
1 is pressed with solution D：It takes in the 10-20 microlitres of human serum albumin PBS solution for being added to 1mL 10-20mg/mL and mixes after the mixing of 1 ratio
Even room temperature reaction 1-2 hours obtains Biotin-HBA-SMCC with PBS 3 purifying of dialysis；
By the insulin-SH of above-mentioned purifying and Biotin-HBA-SMCC by volume 10:(1-4) mixed room temperature reacts
Biotin- high molecular weight protein-Insulin are obtained after 30-60 minutes, as biotinylated insulin antigens.
By verification experimental verification, the biotinylated IA obtained using conventional method is obtained with according to biotinylation method of the present invention
The IA obtained, when for detecting, the present invention can obtain higher IAA positive rates, and sample is avoided to be missed because of low concentration
Or misjudgement.
In coating, the diabetes autoantigen use CB buffer solutions or Tris buffer solutions for antigen diluent buffer solution into
Row coating；Preferably, the buffer solution is selected from the CB buffer solutions of PH9.6 or the Tris buffer solutions of PH8.5, it is highly preferred that
PEG or PVP, Proclin300 are added in buffer solution, while being added to water-soluble cyclodextrin, and coating can be made more stable, anti-
Primordial covering point is more regular, more round, CV smallers.
Wherein, water soluble Beta-cyclodextrin can be Captisol, 2- hydroxy-beta-cyclodextrin or carboxymethyl-beta-cyclodextrin etc., dense
Degree is 0.02%；A concentration of 5%, the Proclin300 a concentration of 0.05% of PEG or PVP is removed in the percentage
Proclin300 is except percent by volume, remaining is all mass percent (w/v).
In the specific embodiment of the invention, the dilution buffer of GAD and CPH are the 0.02M Tris buffer solutions of PH8.5
(containing 5% PEG, 0.05% Proclin300, the glycerine of 0.02%Captisol and 15%), final concentration is respectively
8ug/ml、30ug/ml。
The dilution buffer of IA-2, IC, IA, ZnT8 be PH9.6 CB buffer solutions, final concentration be respectively 10ug/ml,
10ug/ml、80ug/ml、15ug/ml。
In addition, the protein chip in kit of the present invention further include negative Quality Control point, positive quality control point, sample Quality Control point,
It is more than one or two of enzyme mark Quality Control point, reference curve point and position reference point；More specifically, at least one is cloudy
Property control point (NC) and a positive quality control point (PC)；At least one sample spot Quality Control point (SC) and an enzyme mark Quality Control point
(EC)；At least three reference curve point (S1-S3) and a coated position reference point of chip (Loc) itself.
In a specific embodiment, on protein chip of the present invention also include a negative Quality Control point (NC) and a positive matter
Control point (PC)；One sample spot Quality Control point (SC) and an enzyme mark Quality Control point (EC)；3 reference curve points (S1-S3) and one
The coated position reference point of a chip itself (Loc).
Wherein, positive quality control point can be human IgG, then the corresponding enzyme labelled antibody used is exactly the enzyme mark of anti-human igg.It is positive
Quality Control point can also be the DNP for being coated with BSA couplings, then the corresponding enzyme labelled antibody used is exactly that the enzyme of anti-human igg is marked with and anti-DNP
Enzyme target mixed liquor.
And negative Quality Control point can be less than the human IgG of the micro-concentrations of reaction signal value, or using other unrelated eggs
It is white to substitute；Sample Quality Control point can be the IgG or other anti-human igg of goat-anti people；Enzyme mark Quality Control point can be human IgG, or
The antibody (enzyme mark is rabbit anti-human igg) of other anti-rabbit, such as goat anti-rabbit igg antibody.The reference curve point is specific
It is the human IgG of basic, normal, high three kinds of concentration in implementation process.
The position reference point of protein chip itself is human IgG solution, mainly to the positioning on protein chip when array value
Effect.
Preferably, above-mentioned negative Quality Control point, positive quality control point, sample Quality Control point, enzyme mark Quality Control point, reference curve point and
Position reference point is coated with also by the bottom plate of advance biotinylation and Avidin.
Conventional enzyme and corresponding developing solution may be selected in enzyme marker in enzyme labelled antibody of the present invention, such as horseradish peroxide
Compound enzyme and TMB color developing agents.
The also corresponding preparation method for providing the kit of the present invention, including：
Diabetes autoantigen is coated on protein chip, is washed after coating, confining liquid closing is then added, obtains
It must be coated with the protein chip of diabetes autoantigen, the confining liquid contains BSA, polyvinyl alcohol, mannitol, Sodium azide and phosphorus
Sour disodium hydrogen；
Prepare the enzyme mark containing Tris, citric acid, BSA, polyethylene glycol, gum arabic, glycine betaine and Proclin300
Dilution simultaneously dilutes enzyme labelled antibody, the enzyme labelled antibody of acquisition enzyme mark diluted, then prepares sample diluting liquid, cleaning solution
And developing solution, obtain diabetes antibody repertoire detection kit.
Serum sample is detected using diabetes antibody repertoire detection kit of the present invention, total antibody is in T1MD positive rates
98.21%, T2DM positive rate are 10.58%, normal person's positive rate only 2.56%, and difference, which has, significantly learns meaning (P<
0.01)。
By above technical scheme it is found that the present invention starts with from the confining liquid and enzyme mark dilution of enzyme linked immunological kit, lead to
Cross selection suitable compositions so that enzyme linked immunological kit can keep the stability of detection the long period.Meanwhile with the combination
Object prepare diabetes detection kit on the basis of with preferable stability, additionally it is possible to diabetes carry out Accurate Diagnosis and
Parting has higher positive rate especially in the detection of IAA.
Description of the drawings
Fig. 1 show the structural schematic diagram of biotinylated insulin antigens of the present invention.
Specific implementation mode
The invention discloses a kind of composition for enzyme linked immunological kit and diabetes antibody repertoire detection kits
And preparation method thereof, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular
, all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in
The present invention.Composition, kit and application of the present invention are described by preferred embodiment, and related personnel is apparent
The content of present invention can be not being departed from, composition as described herein, kit and application is being modified or is fitted in spirit and scope
When changing and combining, to realize and apply the technology of the present invention.
Just a kind of composition and diabetes antibody repertoire for enzyme linked immunological kit provided by the present invention is examined below
Test agent box and preparation method thereof is described further.
Embodiment 1：The preparation of diabetes antibody repertoire detection kit of the present invention
1, the pretreatment of die substrate and diabetes autoantigen
(1) pretreatment of chip：
6K times of Streptavidin (dilution is the PBS of the 0.01M of PH7.4) will be diluted, is added in 96 orifice plates,
Then the holes 50ul/ stand 2h in 37 DEG C of insulating boxs.
96 orifice plates are taken out, is washed 3 times with PBS, finally be washed once, blot with purified water.Wash conditions：The holes 300ul/,
It stands 30sec/ times.
37 DEG C of constant temperature blast drying oven drying for standby.
(2) pretreatment of diabetes autoantigen：
With Thermo'sSulfo-NHS-LC-Biotin biotinylation kits to IA-2, GAD, IC,
CPH, ZnT8 carry out biotinylation respectively.
For the biotinylation of insulin (IA) antigen using the technology improved, concrete scheme is as follows：
Prepare the insulin PBS solution of 2mg/mL, the S- acetylthio acetate succinate acyls that configuration concentration is about 50mmol/L
10 microlitres of solution A, is added to mixing in 1 milliliter of insulin PBS solution by the dimethyl sulphoxide solution (solution A) of imines ester
Room temperature reaction 1 hour, reaction product recycle after dialysing in PBS 3 times and obtain insulin-SH-P, and the detection of infrared and nuclear-magnetism is aobvious
Show consistent with expected structure, 4 degree temporary spare；
The hydroxylamine hydrochloride (EDTA containing 20mmol/L, pH7.0) that 0.5mol/L is configured using PBS as solvent is used as B solution,
100 microlitres of B solution is added in the insulin-SH-P of above-mentioned 1mL, mixed room temperature reacts 1 hour, desalting column desalting and purifying
Insulin-SH is obtained, the detection display of infrared and nuclear-magnetism is consistent with expected structure.
The PBS solution for preparing the human serum albumin (HBA) of 10mg/mL, the Sulfo-N- succinyls for configuring 10mg/mL are sub-
The PBS solution of amido 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts makees C solution, and another configuration concentration is 0.25mol/L
The dimethyl sulphoxide solution of-ten two polyethylene glycol of succinic acid Asia amide-biotin makees solution D, and C solution and solution D are pressed 1：1 ratio
It takes mixing in 20 microlitres of human serum albumin PBS solutions for being added to 1mL 10mg/mL to react at room temperature after example mixing 1 hour, uses PBS
3 purifying of dialysis obtain Biotin-HBA-SMCC, and the detection display of infrared and nuclear-magnetism is consistent with expected structure；
By the insulin-SH of above-mentioned purifying and Biotin-HBA-SMCC by volume 10:1 mixed room temperature reacts 30 minutes
After obtain Biotin- high molecular weight protein-Insulin, have 1 structure of formula, as biotinylated insulin antigens, it is infrared and
Nuclear-magnetism detection display is consistent with expected structure.
2, the coating of diabetes autoantigen and GAP-associated protein GAP
PC, NC, S1, S2, S3, EC in ProteinChip array coated respectively are 2ug/ml, 0.01ug/ml, 0.5ug/
Biotinylation (using aforementioned biological element kit, similarly hereinafter) human IgG of ml, 2ug/ml, 4ug/ml, can be slow with the CB of PH9.6
Fliud flushing (Captisol containing 5% PEG, 0.05% Proclin300 and 0.02%) is diluted.
It is the CB buffer solutions of PH9.6 that SC points, which use 2ug/ml biotinylation goat anti-human igg antibodies, dilution buffer,.
It is the CB buffer solutions of PH9.6 that Loc points, which use the biotinylation human IgG solution of 2ug/ml, dilution buffer,.
The 20mM Tris buffer solutions that the dilution buffer of GAD and CPH is PH8.5 are (containing 5% PEG, 0.05%
Proclin300,0.02%Captisol and 15% glycerine), final concentration is respectively 8ug/ml, 30ug/ml.
The dilution of IA-2, IC, IA, ZnT8 are the CB buffer solutions of PH9.6, and final concentration is respectively 10 ug/ml, 10ug/
ml、80ug/ml、15ug/ml。
The membrane filtration that the antigen diluted is used to 0.22um respectively, then carries out array with BioDot precisions point sample instrument
Coating.After whole arrays complete point sample, chip is placed in 2-8 DEG C, is coated with 24-30h overnight.ProteinChip array can refer to
Such as the array that following table is presented, it can also adjust, not be limited according to actual needs：
1 ProteinChip array of table
| PC | NC | IC | NC |
| S1 | GAD | IA | SC |
| S2 | CPH | ZnT8 | EC |
| S3 | IA-2 | PC | Loc |
3, it closes
Coated chip is taken out, is cleaned 3 times with the PBST cleaning solutions of PH7.4, the confining liquid of 150ul is then added per hole
(PH7.4, the disodium phosphate soln containing 1%BSA, wherein be added to 0.8% mannitol, 1% PVA2W, 0.05% it is folded
Nitrogen sodium preservative), room temperature closes 1h, then pats dry, and in humidity 15% hereinafter, being placed at room temperature for, dry 4h is sealed, 2-8 DEG C afterwards
It preserves.
4, enzyme mark dilution, enzyme labelled antibody, developing solution, sample diluting liquid and concentrated cleaning solution are prepared
Enzyme mark dilution：The citric acid of Tris, 50mM containing 100mM, 2.5% BSA, 2% PEG1W, 0.01%
Gum arabic, 1% glycine betaine and 0.05% Proclin300；
Enzyme labelled antibody：Rabbit anti-human igg's antibody of horseradish peroxidase-labeled；
In use, rabbit anti-human igg's antibody of horseradish peroxidase-labeled is diluted to 4K times of (enzyme with enzyme mark dilution
Labeling antibody concentration).
Developing solution：Sedimentation type TMB.
Sample diluting liquid：0.02M Tris, 0.15M NaCl, 0.05%Tween20,0.01% caseins, pH7.4.
10 times of concentrated cleaning solutions：0.2M Tris, 1.5M NaCl, 0.5%Tween20, pH7.4.
The above-mentioned protein chip for being coated with diabetes autoantigen, the enzyme labelled antibody of enzyme mark diluted and developing solution,
And sample diluting liquid and 10 times of concentrated cleaning solutions form diabetes antibody repertoire detection kit of the present invention.
5, detection method
(1) protein chip, balance to room temperature are taken out；
(2) it is loaded：101 times of sample to be tested is diluted by negative and positive control serum and with sample diluting liquid, often
Hole 100uL, which is added in chip hole to be measured, to react.
(3) it incubates：It is stored at room temperature reaction 30min.Add 300uL cleaning solutions (being used after diluting 10 times with ultra-pure water), washing
3 times, 1min is stood every time.
(4) enzyme labeling antibody：50uL enzyme labelled antibodies are added per hole.
(5) it incubates：It is stored at room temperature reaction 30min.Add 300uL cleaning solutions, washs 3 times, stand 1min every time.
(6) it develops the color：TMB color developing agent 50uL are added per hole, is stored at room temperature, is protected from light 30min.
(7) it measures：In 30min, signal value and concentration that each reacting hole corresponds to antibody are read and calculated with detector；Its
In, signal detection system can also be chemiluminescent mode to realize, can use chemiluminescent substrate, such as luminol, then
The reading of result is carried out by fluorescence detection device.
Embodiment 2：The detection of clinical sample
Samples sources：Diabetic is all from the patient made a definite diagnosis recently, meets diabetes diagnostic criterion in 1999, wherein
T1DM patient 56, the age, T2DM patient 104, the age was all from health in 20-56 Sui, normal person 78 at 6-52 Sui
Examinee, equal non-diabetic family history, the age is at 15-55 Sui.Venous blood samples respectively detach serum, in -20 DEG C after packing
It freezes, it is to be checked.
Specific experimental result and the following table of data：
Table 2
| Group | n | IA-2A | GADA | ICA | CPH-A | ZnT8-A | IAA | Total positives rate |
| T1DM | 56 | 26 | 42 | 17 | 3 | 15 | 14 | 55/56 (98.21%) |
| T2DM | 104 | 2 | 10 | 1 | 3 | 0 | 2 | 11/104 (10.58%) |
| Normal group | 78 | 0 | 0 | 0 | 2 | 0 | 0 | 2/78 (2.56%) |
Type 1 diabetes (T1DM) cause islet cells to be damaged by autoantibody is immune, belong to the absolute deficiency of insulin,
And diabetes B (T2DM) is generated by insulin resistance, it is generally related with living habit diet, wherein can detect from
Body antibody is less.The autoantibody of diabetes should have significant difference in the positive rate of these three crowds.
In terms of experimental result of the present invention, serum sample is detected with the kit of the present invention, total antibody is in T1MD positive rates
It is 10.58% for 98.21%, T2DM positive rates, normal person's positive rate only 2.56%, three groups of difference, which have, significantly learns meaning (P<
0.01), meet the rule and standard clinically detected.
Embodiment 3：Stabilization of kit detection of the present invention
Contrast agents box：It is prepared according to the method for embodiment 1, difference lies in confining liquids and enzyme mark dilution to be all made of
Conventional 0.01MPBS (PH7.4)+10%BSA；
Test kit：1 kit of embodiment；
Detection method：By two kinds of kits be respectively placed under room temperature (18-28 DEG C) and low temperature (2-8 DEG C) place one section when
Between, it is then detected according to 1 detection method of embodiment using identical serum, counts instrument signal value, the results are shown in Table 3-6.
1, the stability data under 1 kit low temperature of embodiment
Table 3
As can be seen from Table 3, kit of the present invention has detected the inspection placed at low temperature 0,6,12,18,24 month respectively
Signal value is surveyed, while having counted the ratio of each signal value, the results show that placed 24 months kits, detects signal
Value is almost in 90% or more, it was demonstrated that kit of the present invention compared with placing 0,6,12,18 month detected signal value
It places 24 months under cryogenic and still has higher detection stability.
2, the stability data of 1 kit of embodiment at room temperature
Table 4
As can be seen from Table 4, kit of the present invention has detected the detection placed at normal temperatures 0,2,4,6,8 month respectively
Signal value, while having counted the ratio of each signal value, the results show that placed 8 months kits, detected signal value with
The detected signal value placed 0,2,4,6 month is compared, and is in 80% or more, it was demonstrated that kit of the present invention is in normal temperature condition
Lower place 8 months still has higher detection stability.
3, the stability data comparison under 1 kit of embodiment and contrast agents box low temperature
Table 5
As can be seen from Table 5, kit and contrast agents box of the present invention have detected respectively at low temperature place 6,12,18,
24 months detected signal values, while having counted under same time, the ratio of the signal value of two kits, the results show that putting
18 months contrast agents boxes are set, detected signal value starts to occur significantly declining, can be apparent from conjunction with 3 data of table
The stability of contrast agents box is not so good as the conclusion of kit of the present invention, and the difference of the two is only that confining liquid and the dilution of enzyme mark
Liquid.
4, the stability data comparison under 1 kit of embodiment and contrast agents box room temperature
Table 6
As can be seen from Table 6, kit and contrast agents box of the present invention has detected respectively places 3,6,9,12 at normal temperatures
A month detected signal value, while having counted under same time, the ratio of the signal value of two kits, the results show that placing
4 months contrast agents boxes, the detected signal value of certain indexs start to occur significantly declining, can be in conjunction with 4 data of table
The stability for being apparent from contrast agents box is not so good as the conclusion of kit of the present invention, and the difference of the two is only that confining liquid and enzyme
Mark dilution.
Embodiment 4：Kit of the present invention detects experiment to the positive rate of IAA
Contrast agents box：It is prepared according to the method for embodiment 1, difference lies in the biotinylations of IA equally to useSulfo-NHS-LC-Biotin biotinylation kits；
Test kit：1 kit of embodiment；
Detection method：8 positive samples, 8 weakly positive samples and 5 negative samples are chosen, are respectively adopted above-mentioned two
Kind kit is detected according to 1 detection method of embodiment, is counted instrument signal value, be the results are shown in Table 7.
Table 7
As can be seen from Table 7, in the present invention to the biotinylated method of IA compared with, using general biotinylation try
Agent box carries out method of the biotinylation to the detection result of positive sample not as good as being used in the present invention to IA, this using the present invention
The positive rate of IAA can be greatly improved to the biotinylation method of IA, effect ratio is apparent using general biotinylation kit
It is better.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (14)
1. a kind of composition for enzyme linked immunological kit, which is characterized in that including confining liquid and enzyme mark dilution；The envelope
Close liquid and contain BSA, polyvinyl alcohol, mannitol, Sodium azide and disodium hydrogen phosphate, the enzyme mark dilution contain Tris, citric acid,
BSA, polyethylene glycol, gum arabic, glycine betaine and Proclin300.
2. composition according to claim 1, which is characterized in that the confining liquid contains 0.6%-1%BSA, 0.8%-
1.5% polyvinyl alcohol, 0.8%-1.5% mannitol, 0.05% Sodium azide and 0.01M disodium hydrogen phosphates, pH value 7.4 are remaining
Amount is water.
3. composition according to claim 1, which is characterized in that the enzyme mark dilution contains Tris, 0.05M's of 0.1M
The glycine betaine of citric acid, the polyethylene glycol of BSA, 1%-3% of 2%-3%, 0.01% gum arabic, 0.8%-1.5%
And 0.05% Proclin300, surplus is water.
4. application of the composition in preparing enzyme linked immunological kit described in claim 1-3 any one.
5. applying according to claim 4, which is characterized in that the enzyme linked immunological kit is diabetes immunologic function test reagent
Box.
6. a kind of diabetes antibody repertoire detection kit, which is characterized in that including following components：
Protein chip, the enzyme labelled antibody, sample diluting liquid, washing with enzyme mark diluted for being coated with diabetes autoantigen
Liquid and developing solution；Wherein, it is closed using confining liquid after the protein chip coating diabetes autoantigen, the confining liquid contains
BSA, polyvinyl alcohol, mannitol, Sodium azide and disodium hydrogen phosphate, the enzyme mark dilution contain Tris, citric acid, BSA, poly- second
Glycol, gum arabic, glycine betaine and Proclin300.
7. kit according to claim 6, which is characterized in that the confining liquid contains 0.6%-1%BSA, 0.8%-
1.5% polyvinyl alcohol, 0.8%-1.5% mannitol, 0.05% Sodium azide and 0.01M disodium hydrogen phosphates, pH value 7.4 are remaining
Amount is water.
8. kit according to claim 6, which is characterized in that the enzyme mark dilution contains Tris, 0.05M's of 0.1M
The glycine betaine of citric acid, the polyethylene glycol of BSA, 1%-3% of 2%-3%, 0.01% gum arabic, 0.8%-1.5%
And 0.05% Proclin300, surplus is water.
9. kit according to claim 6, which is characterized in that the protein chip and biology that the coating passes through Avidin
The diabetes autoantigen of elementization be combined with each other coating.
10. according to the kit of claim 6 or 9, which is characterized in that the diabetes autoantigen be selected from IA-2, GAD,
One or more of IC, CPH, ZnT8, IA.
11. kit according to claim 9, which is characterized in that the biotinylated diabetes autoantigen includes life
The IA of object element, biotinylation proce are as follows：
Step 1 reacts at room temperature insulin and S- acetylthio acetate succinate imide esters (SATA), is obtained after dialysis purification
insulin-SH-P：N indicates number of amino groups on insulin, and 1≤x≤n, x and n are integer；
Blocking group is removed in the PBS of insulin-SH-P and hydroxylamine hydrochloride room temperature reactions, is formed after purification through sephadex column
Insulin-SH containing reactive thiol group, it is spare；
High molecular weight protein, Sulfo-N- succinimidos 4- (maleimidomehyl) hexamethylene -1- carboxylic acid sodium salts (SMCC)
With-ten two polyethylene glycol of succinic acid Asia amide-biotin room temperature reaction, Biotin- macromolecular eggs are obtained by PBS dialysis purifications
- SMCC in vain；M indicates the number of amino groups on high molecular weight protein, and 2≤y+z≤m, y, z and m are integer
- ten two polyethylene glycol of succinic acid Asia amide-biotin
Step 2 reacts spare insulin-SH and Biotin- high molecular weight protein-SMCC mixed room temperatures, solidifying by glucan
Rubber column gel column obtains Biotin- high molecular weight protein-Insulin after purification, has structure shown in formula 1, as biotinylated insulin
Antigen；
Wherein, round to indicate that insulin, 1≤x≤n, n indicate the number of amino on insulin, x and n are integer；Ellipse represents
High molecular weight protein, y+z≤m, z >=10, y >=1, m indicate the number of amino groups on high molecular weight protein, and y, z and m are integer, if z/x
For non-integer, then z/x round numbers and in integer-bit plus 1.
12. according to the kit of claim 6 or 9, which is characterized in that the diabetes autoantigen using CB buffer solutions or
Tris buffer solutions are that antigen diluent buffer solution is coated with.
13. according to kit described in claim 6-12 any one, which is characterized in that the protein chip further includes negative matter
Control one or two of point, positive quality control point, sample Quality Control point, enzyme mark Quality Control point, reference curve point and position reference point
More than.
14. the preparation method of kit described in claim 6, which is characterized in that including：
Diabetes autoantigen is coated on protein chip, is washed after coating, confining liquid closing is then added, is wrapped
There are the protein chip of diabetes autoantigen, the confining liquid to contain BSA, polyvinyl alcohol, mannitol, Sodium azide and phosphoric acid hydrogen
Disodium；
Prepare the enzyme mark dilution containing Tris, citric acid, BSA, polyethylene glycol, gum arabic, glycine betaine and Proclin300
Liquid simultaneously dilutes enzyme labelled antibody, the enzyme labelled antibody of acquisition enzyme mark diluted, then prepares sample diluting liquid, cleaning solution and shows
Color liquid obtains diabetes antibody repertoire detection kit.
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| CN201810188203.6A CN108469519B (en) | 2018-03-07 | 2018-03-07 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof |
| PCT/CN2018/093039 WO2019169798A1 (en) | 2018-03-07 | 2018-06-27 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody repertoire detection kit and preparation method therefor |
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|---|---|---|---|
| CN201810188203.6A CN108469519B (en) | 2018-03-07 | 2018-03-07 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof |
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| WO2019169798A1 (en) | 2019-09-12 |
| CN108469519B (en) | 2020-03-24 |
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