CN109959794A - A kind of ELISA kit of detection metabolism element - Google Patents
A kind of ELISA kit of detection metabolism element Download PDFInfo
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- CN109959794A CN109959794A CN201711409132.XA CN201711409132A CN109959794A CN 109959794 A CN109959794 A CN 109959794A CN 201711409132 A CN201711409132 A CN 201711409132A CN 109959794 A CN109959794 A CN 109959794A
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Abstract
The present invention relates to polypeptide detection fields, in particular to a kind of ELISA kit of detection metabolism element, including antibody, antibody is any one or more in the plain immune antibody obtained of polypeptide artificial antigen warp of metabolism obtained as the polypeptide as shown in SEQ ID NO:1 and the carrier protein couplet of activation.Antibody is any one or more in antibody of the plain polypeptide artificial antigen of metabolism through immune acquisition obtained by the carrier protein couplet of polypeptide and activation, the plain polypeptide of metabolism therein contains cysteine, the artificial antigen obtained is comlete antigen, it is obtained through test, the polypeptide and the macro-molecular protein KLH of activation are coupled to form the comlete antigen with immunogenicity, and the intracorporal immune system of mouse can be excited to generate antimetabolic element monoclonal antibody.ELISA kit provided by the invention provides a kind of specific serological index for the diagnosis and identification of adult's sclerotin and related disease, to improve the accuracy of diagnosis.
Description
Technical field
The present invention relates to polypeptide detection fields, in particular to a kind of ELISA kit of detection metabolism element.
Background technique
Metabolism element (metabolism element) is one section of conserved sequence in Mouse Bone calcium cellulose protein molecule, analyzes this section by sequence
Polypeptide has antigenicity.It includes albumen (bonegamma-carboxyglutamic- that osteocalcin, which is also known as bone γ-carboxylic glutamic acid,
acid-containingproteins,BGP).The albumen just gathers after the bone mineralising peak phase.Use vitamin K antagonism
Agent can be such that content of this albumen in bone reduces, but have no effect on the content of its proline, nor affect on the mechanical strength of bone.
Osteocalcin is secreted by osteoblast, and content is different with the variation at age, can be with by the content of serum osteocalcin
The state for understanding osteoblast, has great importance for the judgement etc. of osteoporosis.
Clinical manifestation, imageological examination and pathology are relied primarily on for the diagnosis of adult's sclerotin and related disease at present
Phenomenon etc..Still lack specific serological diagnosis index at present.It is the special of one antibody of antigen used by enzyme-linked immunization
Property immune response, relatively at present clinically used in diagnostic method specificity it is higher, reproducible, stable reagent, sensibility is good,
It is not required to special instruments and equipment, is currently an important method of clinical detection.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of ELISA kits of detection metabolism element, are adult's sclerotin and related disease
The diagnosis and identification of disease provide a kind of specific serological index, to improve the accuracy of diagnosis.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of ELISA kit of detection metabolism element, including antibody, the antibody are as more as shown in SEQID NO:1
Any of the antibody of the plain polypeptide artificial antigen of the metabolism that peptide and the carrier protein couplet of activation obtain through immune acquisition is more
It is a.
Detection provided by the invention is metabolized the ELISA kit of plain content of peptides, and antibody used is by such as SEQ ID
The metabolism element polypeptide artificial antigen that the plain polypeptide of metabolism shown in NO:1 and the carrier protein couplet of activation obtain obtains through immune,
In metabolism element polypeptide contain cysteine, the artificial antigen obtained is comlete antigen, is obtained through test, the polypeptide and activation
Macro-molecular protein KLH be coupled to form the comlete antigen with immunogenicity, the intracorporal immune system of mouse can be excited to produce
Raw antimetabolic element monoclonal antibody.ELISA kit provided by the invention is diagnosis and the mirror of adult's sclerotin and related disease
Indescribably for a kind of specific serological index, to improve the accuracy of diagnosis.
Further, the compound of the activation is maleimide.
Further, the carrier protein is spoon sky hemocyanin.
Through screening, these three antibody and the plain polypeptide identification specificity of metabolism are high, and sensitivity is strong.
Further, the antibody is that following three kinds of antibody that screening obtains is any one or more of:
Antibody 1: the hypotype of monoclonal antibody is G2a, potency 4000；
Antibody 2: the hypotype of monoclonal antibody be G2b, potency 8000, also with polypeptide shown in SEQ ID NO:2
In conjunction with；
Antibody 3: the hypotype of monoclonal antibody be G2a, potency 32000, also with polypeptide shown in SEQ ID NO:3
In conjunction with.
These three antibody are monoclonal antibody.
In the present invention, the preparation method of the plain polypeptide artificial antigen of the metabolism, comprising the following steps:
The plain polypeptide dissolution of the metabolism as shown in SEQ ID NO:1, obtains being metabolized the polypeptide that plain peptide concentration is 6-9mg/ml
Solution；
The carrier protein of activation dissolves, and the carrier protein that the carrier protein concentration for obtaining the activation is 4-6mg/ml is molten
Liquid；
The polypeptide solution is that 1:2-3 is mixed with volume ratio with the carrier protein solution, is coupled；
The product of the coupling is isolated and purified to obtain the plain polypeptide artificial antigen of the metabolism.
The preparation method provided by the invention for being metabolized plain polypeptide artificial antigen, it is simple and easy to do, and be coupled and imitate through this method
Rate is high, 90% or more.
Further, the carrier protein of the activation is the spoon sky hemocyanin of maleimide activation.
Further, the solvent for being metabolized plain polypeptide dissolution is 20 ± 1mM sodium phosphate buffer, wherein also containing 230
± 5mM NaCl, 2 ± 0.2mM EDTA, 80 ± 2mM sucrose, pH 6.5-6.7.
Further, the solvent of the carrier protein dissolution of the activation is 20 ± 1mM sodium phosphate buffer, wherein also containing
100 ± 5mM EDTA, 80 ± 2mM sucrose, pH 6.5-6.7.
Further, further, coupling condition is to continue 2 hours or 2-8 DEG C overnight in room temperature under stiring.
The plain polypeptide artificial antigen of metabolism obtained above is immunized mouse and screens to obtain the stronger antibody of specificity.
Further, the ELISA kit further includes ELISA Plate, yin and yang attribute referring to product, two corresponding anti-solution, developing solution, anti-
Answer terminate liquid, concentrated cleaning solution, sample diluting liquid, sealing plate film and Fresco Bag, product description and product quality report in appoint
It is one or more.
Further, the antibody is coated on the ELISA Plate.
Further, the ELISA Plate is to react capillary strip by the enzyme mark on outer frame support and insertion support frame as described above
Composition.In this way, each removable enzyme mark reaction capillary strip itself constitutes independent detection unit.Each reaction capillary strip has more
A reacting hole, the pre-coated specific antibody of each reacting hole.According to the enzyme mark reaction of how much selection corresponding numbers of detection sample
Capillary strip.Can the detection of complete fulfillment large sample size, capillary strip repeated detection in batches can also be reacted by detachable enzyme mark.By
How much different in user's clinical sample amount, this feature facilitates condition for what client provided repeated detection.
Further, the ELISA Plate is 24-96 hole elisa Plates, and the ELISA Plate is using one end glue envelope zip mode opening
Fresco Bag is vacuum-packed.
Such as ELISA Plate can be 96 common hole elisa Plates, 48 hole elisa Plates, 36 hole elisa Plates.
Glue envelope zip mode in Fresco Bag one end heals up and can operate repeatedly, thus it is guaranteed that being not used in the validity period of lath
Storage.
Further, the secondary antibody is marked with horseradish peroxidase.
In this way, antigen-antibody reaction occurs for target antibody and envelope antigen in serum to be checked when detection, addition is marked with peppery
The secondary antibody of root peroxidase forms antigen antibody complex, is developed the color with tmb substrate.
Further, the sealing plate film is stickup property plastic foil.Sealing plate film is mutually fitted with ELISA Plate surface size.
In the present invention, positive reference substance, negative controls, ELIAS secondary antibody liquid, sample diluting liquid, concentrated cleaning solution and termination
Liquid is attached separately to the composite plastic bottle being characterized with white body: developing solution A liquid and developing solution B liquid are attached separately to brown lid
With brown bottle as the composite plastic bottle of feature.
Wherein, positive reference substance, negative controls, the plain polypeptide standard items of metabolism, ELIAS secondary antibody liquid, concentrated cleaning solution are
1ml solution/bottle: sample diluting liquid is 15ml solution/bottle: developing solution A liquid and developing solution B liquid are 8ml solution/bottle: terminate liquid
For 10ml solution/bottle.
Kit provided by the invention uses direct competive ELISA method, and when detection, yin is added in corresponding reacting hole
Sun control and sample to be tested, if it is multiple to form antigen-antibody in conjunction with the antibody of micropore surface containing plain polypeptide is metabolized in sample
Object is closed, enzyme mark secondary antibody sufficiently is added after washing；The zymolyte that the enzymatic being connected in secondary antibody is added makes reaction solution
Blue is presented, blue liquid changes into yellow in the presence of terminate liquid, and the content of antibody is at just in the depth and sample of color
It is related.This kit has many advantages, such as easy to operate, high specificity, high sensitivity.
Compared with prior art, the invention has the benefit that
(1) detection provided by the invention is metabolized the ELISA kit of plain content of peptides, is adult's sclerotin and related disease
Diagnosis and identification a kind of specific serological index is provided, with improve diagnosis accuracy.
(2) detection provided by the invention is metabolized the ELISA kit of plain content of peptides, kit standard product concentration and OD value
Correlation curve linear regression coeffficient (R2Value) it is greater than or equal to 0.990, sensitivity is less than or equal to 0.5U/ml, has behaviour
The advantages that making simple, high specificity, high sensitivity.
(3) ELISA kit of detection metabolism element provided by the invention, detection is quick and can quantify, and batch internal difference is less than
10%, stability is good, can detect simultaneously to multiple samples, and the time required for detection sample is short, easy to operate, does not need
Special experiment instrument and equipment, can be widely applied to laboratory testing.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the KLH structural schematic diagram of maleimide activation in the embodiment of the present invention 1；
Fig. 2 is the potency result linear graph that number is 21 monoclonal antibodies in the embodiment of the present invention 3；
Fig. 3 is the potency result linear graph that number is 24 monoclonal antibodies in the embodiment of the present invention 3；
Fig. 4 is the potency result linear graph that number is 35 monoclonal antibodies in the embodiment of the present invention 3；
Fig. 5 is the result figure of monoclonal antibody (21#) and the plain polypeptide identification specificity of metabolism in the embodiment of the present invention 4；
Fig. 6 is the result figure of monoclonal antibody (24#) and the plain polypeptide identification specificity of metabolism in the embodiment of the present invention 4；
Fig. 7 is the result figure of monoclonal antibody (35#) and the plain polypeptide identification specificity of metabolism in the embodiment of the present invention 4；
Fig. 8 is the result figure of monoclonal antibody (24#) and OCN polypeptide identification specificity in the embodiment of the present invention 5；
Fig. 9 is the result figure of monoclonal antibody (35#) and oc-22 polypeptide identification specificity in the embodiment of the present invention 5.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
In order to better understand the present invention, special to be stated the preparation process of monoclonal antibody, unless otherwise specified,
For conventional method.Agents useful for same material is conventional biochemical reagent unless otherwise specified in case study on implementation.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.Used in embodiment
Phosphate buffer is the PBS buffer solution of pH 7.4,0.01M.Carbonate buffer solution used in embodiment be pH 9.6,
The sodium carbonate buffer of 0.05M.Bovine serum albumin(BSA) abbreviation BSA, spoon sky hemocyanin are referred to as KLH.
Embodiment 1
A, maleimide activation KLH and polypeptide are coupled
1. opening the KLH addition 1ml 20mM sodium phosphate buffer (as shown in Figure 1) of a pipe maleimide activation
(230mM NaCl, 2mM EDTA, 80mM sucrose, pH 6.6), which is that the KLH of maleimide activation is reconstructed,
Obtain the solution of 5mg/ml, matching while using；
2. preparing 20mM sodium phosphate buffer (100mM EDTA, 80mM sucrose, pH 6.6), as coupling buffer；
3. 4mg polypeptide is dissolved in 0.5ml coupling buffer, polypeptide solution is obtained, 50 μ l polypeptide solutions is left and taken and is used for really
Determine coupling efficiency (total cys), saves to 2-8 DEG C.
4. immediately mixing the KLH solution that 0.5ml polypeptide solution and 1ml maleic amide activate in reaction tube, stir, room
Warm (15-25 DEG C) continues 2 hours or 2-8 DEG C overnight (8-15 hours), and the polypeptide after obtaining coupling KLH is named as later
NH2-KLH；
5. retaining 100 μ l coupling reaction liquid for determining coupling efficiency (free cys).
B, the separation of KLH conjugate
1. 0.01% Sodium azide, long-term preservation is added in 1L PBS；
2. Sephadex G-25M gel column is fixed on suitable beaker；
3. removing the lid of gel column, the bottom end of gel column is cut off, allows excessive liquid to flow out, forbids liquid flow in column
It is dry；
4. 30ml PBS balanced gel column is used, if after upper and lower lid please not cover using gel column immediately, saved extremely
2-8℃；
5. the liquid after reaction is added to gel column, lower end trickle is collected；
6. being washed with total volume 10ml PBS, eluate is collected, 0.5ml-1.0ml is collected each time, is measured at 280nm
Absorbance value；
Contain protein eluate 7. collecting, -20 DEG C freeze.
C cysteine standard curve
1.DTNB analytical solution (0.1mM): add the Tris buffer of 99 volume 0.25M by 1 volume 10mMDTNB titer
It is formulated, matching while using；
2. one water object of 32mg L-cysteine hydrochloride is dissolved in 1ml distilled water, gradient dilution range 0.4-
0.04mg/ml；
3. prediluted 50 μ l of cysteine standard liquid is added in testing tube, 50 μ l distilled waters are added in blank tube；
4. 0.1ml distilled water is added in all pipes, 0.1ml DTNB examination is added in 0.75ml pH8.0DTNB buffer immediately
Agent solution makes total volume 1ml, mixes；
5. measuring absorbance value at 412nm, standard curve is made, in triplicate.
The measurement of D coupling efficiency
1. 50 μ l polypeptide solutions (total cys) are added in 0.1ml distilled water；
2. by following 50 μ l solution be added testing tube in: DTNB buffer (blank), diluted polypeptide sample (total cys,
KLH combination), polypeptide-K LH (free cys, KLH combination), diluted polypeptide sample (total cys, BSA combination), polypeptide-BSA (trip
From cys, BSA combination)；
3. 0.1ml distilled water is added into all test tubes, 0.1ml is added in the DTNB buffer of 0.75ml pH8.0 immediately
DTNB reagent solution, final volume 1ml are mixed；
4. absorbance value is measured at 412nm, if absorbance value is greater than 1.4, dilute sample retest；
5. calculation formula: coupling efficiency (%)=(total cys- dissociate cys)/total cys ╳ 100%
Obtained coupling efficiency is 90% or more.
Embodiment 2
Polypeptide is metabolized the preparation of plain monoclonal antibody
BALB/c female mice is purchased from Guangdong Province's Experimental Animal Center.
A animal immune
The NH2-KLH immunogene prepared in embodiment 1 is used for the immune of BALB/c female mice, initial immunity presses 60 μ g
Albumen/mouse amount, 4 SPF Balb/c female mices of subcutaneous inoculation, number 1-4 is primary every 2 weeks booster immunizations,
It is specific as follows:
Subcutaneous first time booster immunization, the amount of being immunized are 30 μ g albumen/only；
Subcutaneous second of booster immunization, be immunized amount be 30 μ g albumen/only；
Subcutaneous third time booster immunization, the amount of being immunized are 30 μ g albumen/only；
Subcutaneous 4th booster immunization, be immunized amount be 30 μ g albumen/only.
Each mouse orbit takes blood, surveys serum titer.
Immunizing potency detection:
Step: using " NH2-BSA ", and 2 μ g/ml, 4 DEG C of coatings are overnight；2% milk, 37 DEG C of closing 2h；Serum is opened from 200 times
Begin 2 times of gradient dilutions, and blank control (blank) is PBS, and negative control (negative) is 200 times of negative serum dilutions.Potency
For the corresponding dilution of the minimum OD reading greater than maximum OD/2.
The results are shown in Table 1 for potency.
1 potency result of table
| Extension rate | Number 1 | Number 2 | Number 3 | Number 4 |
| 200 | 1.162 | 1.253 | 1.096 | 1.24 |
| 400 | 1.139 | 1.226 | 1.253 | 1.327 |
| 800 | 1.132 | 1.186 | 1.164 | 1.189 |
| 1600 | 0.889 | 0.999 | 1.075 | 0.994 |
| 3200 | 0.723 | 0.813 | 0.833 | 0.92 |
| 6400 | 0.577 | 0.612 | 0.593 | 0.634 |
| 12800 | 0.379 | 0.409 | 0.506 | 0.425 |
| 25600 | 0.264 | 0.326 | 0.347 | 0.306 |
| 51200 | 0.162 | 0.19 | 0.183 | 0.153 |
| 102400 | 0.119 | 0.116 | 0.139 | 0.111 |
| Blank | 0.036 | 0.037 | 0.033 | 0.028 |
| It is negative | 0.034 | 0.042 | 0.037 | 0.033 |
From determination data as can be seen that the Mouse titers of number 4 are excellent, therefore, chooses impact 4# mouse and do cell fusion reality
It tests.
B cell fusion experiment
Mouse boosting cell and SP2/0 cell are taken, is merged using PEG method.Cell semisolid culturemedium has been merged (to contain
HAT screening and culturing) is carried out.
1. experiment equipment
The surgical instrument of sterilizing: three scissors, three tweezers, a cell sieve, the inner core of syringe, one it is flat
Ware.Wet box, 2 500ml beakers, 2 50ml centrifuge tubes, 3 15ml centrifuge tubes.
2. experiment reagent
IMDM culture medium；IMDM complete medium (contains 15% serum)；2.2% methylcellulose, producer SIGMA, article No.:
M0262-100G；Newborn bovine serum: 10ml；PEG1500, producer Roche, article No.: 78364；HAT: producer Sigma, article No.:
H0262-10VL。
3. fusion experiment step
A) it is blown and beaten from culture bottle wall by sp2/0 cell in good condition is soft, is drawn into 50ml centrifuge tube.
B) 50 μ g of NH2-KLH immunogene is used, #4 mouse is impacted in abdominal cavity, and the neck that breaks is put to death, and is put into 75% alcohol and is impregnated
5min。
C) IMDM that a small amount of serum-free is poured into plate, cell sieve and plunger are put into plate.Use scissors
The spleen that mouse is removed with tweezers, is put into cell sieve.Lightly spleen is sufficiently pulverized with the inner core of syringe, it is thin by what is ground
Born of the same parents are drawn into the centrifuge tube of dress sp2/0, are centrifuged 1500r/min, 5min.
D) thymus gland that mouse is removed with scissors and tweezers, pulverizes.The thymocyte ground is put into 15ml centrifuge tube, then
The HAT of 1ml is added, is placed on spare in incubator.
E) cell that will be centrifuged outwells supernatant, and cell is carefully gently blown to even, centrifugation with the IMDM of serum-free,
1500r/min, 5min.
F) cell conditioned medium being centrifuged is outwelled as far as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37
In DEG C warm water, it is slowly added to the PEG of 1ml in 1 minute, after adding, 1min is stood in warm water.Then it is slowly added in 2min
The IMDM of the serum-free of 2ml is then slowly added to the IMDM of 8ml serum-free in 2min.It is centrifuged 1000r/min, 5min.
G) supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours into the ready thymocyte in front.
The sterilized semisolid culturemedium of 25ml is added, is mixed well.Then it uniformly pours into 30 Tissue Culture Dish.Cell is trained
Feeding ware is put into wet box, is then placed in incubator and is cultivated.
C chooses clone
10 plate × 93 cell monoclonals are chosen, are incubated at 96 porocyte culture plates (in advance with thymocyte bed board, 100 μ l/
Hole).
The screening of D monoclonal cell
(1) experiment reagent
Coating buffer: sodium carbonate-bicarbonate buffer, pH 9.6；
Confining liquid: 2% milk in PBS washing lotion；
Developing solution: 1%A liquid+10%B liquid (A liquid: 1%TMB in DMSO；B liquid: 0.1%H2O2In citrate buffer solution)；
Terminate liquid: 2M sulfuric acid；
Secondary antibody: goat anti-mouse IgG/HRP
(2) experimental procedure
1) " NH2-BSA " is diluted with coating buffer, final concentration of 2 μ g/ml, 100 holes μ l/, 4 DEG C, overnight；It is washed afterwards with washing lotion
3 times.
2) 2% milk confining liquid is closed, 200 holes μ l/, 37 DEG C of incubators, 2h；It is washed 3 times with washing lotion afterwards.
3) addition primary antibody (cells and supernatant), negative control (SP2/0 culture supernatant), blank control (PBS), the positive are right
It is 100 holes μ l/, 37 DEG C of incubators, 1h according to (200 times of positive serum PBS dilutions)；It is washed 3 times with washing lotion afterwards.
4) secondary antibody that PBS (pH7.4) dilutes 20000 times, 100 holes μ l/, 37 DEG C of incubators, 1h is added；It is washed after taking-up with washing lotion
It washs 3 times.
5) it develops the color, 100 hole μ l/ of developing solution, developing time is 5min or so.
6) every hole is added 50 μ l terminate liquids and terminates.
7) dual wavelength (450,630) surveys light absorption value, and record saves data, as shown in table 2.In table 2, different culture plate marks
Number NO1-10.
The OD numerical value of the different culture plate measurements of table 2
With " NH2-BSA " wrapper sheet, ELISA method is used to the clone selected, does and screens for the first time, in addition to positive control
Group, OD value share 36 plants of positive hybridoma cell strains greater than 0.15.
E monoclonal cell programmed screening
By 36 plants of positive cell strains, with " NH2-BSA ", wrapper sheet using ELISA method does programmed screening again, obtains
To 17 plants of positive hybridoma cell strains.
F monoclonal cell subgroup identification
Steps are as follows:
1. experiment reagent
Coated antibody: Southern Biotech；Confining liquid: 2%BSA+3% sucrose in PBS；
Developing solution :+10 μ l 30%H of 0.2ml A liquid2O2In 10ml B liquid (A liquid: 15mg/mlABTS in H2O；B liquid:
Citrate buffer solution, pH4.0)；Various subclass secondary antibody.
2. experimental procedure
A) with 100mM PBS (pH7.4) dilution coated antibody to 0.5 μ g/ml, every hole adds 0.1ml, 4 DEG C, stays overnight.
B) PBS-T is washed 2 times, and 200 μ l confining liquids, 37 DEG C of incubation 2h are added in every hole.
C) PBS-T is washed 3 times, and 100 μ l hybridoma supematants, 37 DEG C of incubation 1h are added in every hole.
D) PBS-T is washed 3 times, with the antibody of the diluted HRP label of confining liquid 1:1000 (κ, λ) or 1:2000 (others)
The every hole 0.1ml, is separately added into hole appropriate, 37 DEG C of incubation 1h.
E) PBS-T is washed 3 times；Every hole adds 50 μ l substrate solutions, and 10-20min is interior to survey light absorption value in dual wavelength (450,630),
Record saves data.
The 17 plants of positive cell strains screened carry out subgroup identification, finally obtain the positive hybridoma cell of 14 plants of IgG
Strain.It is specific as shown in Table 3 and Table 4.
The subtype data of 3 17 plants of positive cell strains of table measurement
The positive hybridoma cell strain of 4 14 plants of IgG of table
Embodiment 3
Antibody titer measurement-ELISA method
1. antigen coat: polypeptide antigen is diluted to 2 μ g/ml of concentration, is added 96 orifice plates, every 100 μ l of hole, in 4 DEG C of wet box
Coating is overnight；
2. washing: washing coated ELISA Plate, 200 holes μ l/, washing 3 with the PBS buffer solution containing 0.05% Tween-20
It is secondary, 5min/ times；
3. closing: being closed with 1%BSA solution to 96 orifice plates, 250 holes μ l/, 37 DEG C of wet box inner sealing 2h；
4. washing: ibid washing methods；
5. primary antibody be incubated for: by rabbit anteserum polyclonal antibody according to 1:1000,1:2000,1:4000,1:8000,1:16000,
1:32000,1:64000 are diluted with PBS buffer solution, while blank control and negative serum control is arranged, 100 holes μ l/, and 37
1h is incubated in DEG C wet box；
6. washing: ibid washing methods；
7. secondary antibody is incubated for: the HRP goat anti-mouse igg antibody marked being diluted with PBS buffer solution according to 1:5000,100 μ l/
Hole, 37 DEG C of wet box are interior to be incubated for 1h；
8. washing: ibid washing methods；
9. adding substrate: 96 orifice plates are added according to 100 holes μ l/ in TMB, are protected from light 20min in 37 DEG C of wet box；
10. terminating reaction: using 2M H2SO496 orifice plates are added according to 50 holes μ l/ and terminate reaction, with microplate reader in 450nm wave
Absorbance value is measured when long, the OD positive/OD feminine gender > 2.1 is serum titer criterion.
11. it is as in Figure 2-4 to screen the potency result that obtained number is 21,24 and 35 monoclonal antibodies.
The antibody titer that obtained number is 21 is 1:4000, and the antibody titer that number is 24 is 1:
8000, the antibody titer that number is 35 is 1:32000.
Embodiment 4
Monoclonal antibody identifies specificity-Western blot method
1. the preparation of protein sample:
Will after the pipe high pressure sterilization of silanization be added 0.1%BSA and 0.9%NaCl mixed solution after soaked overnight,
Solution was sucked out to and was made pipe natural air drying in second day, polypeptide is added in air-dried pipe and freezes.Pipette tips are equally used 0.1%
The mixed solution soaked overnight of BSA and 0.9%NaCl uses ddH in second day2O rinses (3 times) well high pressure sterilization and dries standby afterwards
With.
2.SDS-PAGE
It selects stand-by after suitable glass plate wiped clean.First with prepared separation gel is added after 1.5% agar edge sealing,
Space needed for reserving concentration glue, is added on 1ml deionized water rubber cover, is allowed to and air exclusion, in favor of the cohesion of separation gel.
After the completion of separation gel polymerization, coating water is poured out, rinses the top of glue for several times to remove unpolymerized acryloyl with deionized water
Amine sucks remaining liquid at the top of glue with filter paper.Glue is concentrated in preparation, and careful injection separation gel upper layer rapidly is inserted into completely at once
Comb cannot generate bubble, place to solidifying.
After glue polymerization completely to be concentrated, electrophoretic buffer is added, carefully extracts comb.The total protein of cell sample that will be prepared
Product loading in order.1 × sample-loading buffer is added in the sample well not being loaded.With constant pressure from cathode to positive electrophoresis, bromophenol blue
Voltage is 80V in concentration glue, and voltage increases to 120V after bromophenol blue enters separation gel, continues electrophoresis and divides until bromophenol blue reaches
From glue bottom, power supply is closed, gel is unloaded.
3.Western-blot
1) a clip pvdf membrane sum number filter paper identical with gel size, electricity consumption, which turns liquid, which impregnates filter paper and electrophoresis, terminates
Gel afterwards, pvdf membrane are first impregnated 10 seconds with methanol, and deionized water is rinsed, and are then soaked in electricity again and are turned in liquid；
2) plastic stent of protein electrophoresis instrument is opened, one piece of sponge is respectively placed in two sides, according to from cathode to anode successively
It is placed for filter paper-gel-pvdf membrane-filter paper order, removes bubble, plastic stent clamping is put into electric turn trough, is powered on,
Turn 1h in ice bath constant current 400mA electricity；
3) sample film that electricity takes a turn for the better is placed in room temperature in the TBST containing 5% skimmed milk power and closes 1h；
4) film is placed in room temperature jog 2-3h in the primary antibody of appropriate extension rate, or overnight in 4 DEG C of reactions；
5) film is washed twice with TBST；
6) film is placed in room temperature jog 1-2h in secondary antibody, or overnight in 4 DEG C of reactions；
7) film is washed twice with TBST；
4. development
Suck excessive moisture on film, after film is reacted 1min in ECL reaction solution, suck moisture content on film, by sample film in
Developed with X-ray tabletting in darkroom.
I.e. above-mentioned metabolism element polypeptide is special using Western blot method verifying monoclonal antibody (21#, 24#, 35#) identification
The result of property is as illustrated in figs. 5-7.
Embodiment 5
Method same as Example 4, unlike, 24# antibody can also specific bond OCN, OCN amino acid sequence such as
Shown in SEQ ID NO:2, as shown in Figure 8.35# antibody can also specific bond oc-22, oc-22 amino acid sequence such as SEQ ID
Shown in NO:3, as shown in Figure 9.
Embodiment 6
A kind of ELISA kit of detection metabolism element, 96 hole elisa Plates, the normal concentration of specific antibody are coated with by one piece
Antibody-solutions, yin and yang attribute referring to product, two corresponding anti-solution, developing solution A and B, reaction terminating liquid, concentrated cleaning solution, sample diluting liquid,
Sealing plate film and Fresco Bag, product description and product quality report.
Wherein, ELISA Plate is made of outer frame support and the removable 12 enzyme mark being placed on it reaction capillary strip, often
A removable enzyme mark reaction capillary strip itself constitutes independent detection unit.Each reaction capillary strip has 8 reacting holes, each
The pre-coated specific antibody of reacting hole.
Sealing plate film is Pasting plastic foil, and size and ELISA Plate cross section are in the same size.
Pre-filled reagent in kit can carry out meeting 96 sample sizes measurement (including standard items and yin and yang attribute reference substance).
Can the detection of complete fulfillment large sample size, detachable micropore lath repeated detection in batches can also be passed through.Due to user's clinic sample
How much different this amount is, this feature facilitates condition for what client provided repeated detection.Fresco Bag one end glue seals zip mode simultaneously
Heal up to operate repeatedly, therefore ensure that the interior storage of the effect phase of unused lath.
Kit uses the reaction principle of direct competive ELISA method: when detection, yin-yang is added in corresponding reacting hole
Control and sample to be tested, if it is compound to form antigen-antibody in conjunction with the antibody of micropore surface containing plain polypeptide is metabolized in sample
Object；The secondary antibody formation antigen antibody complex for being marked with horseradish peroxidase is sufficiently added after washing；Zymolyte is added
TMB, the zymolyte that the enzymatic being connected in secondary antibody is added make reaction solution that blue be presented.It is blue in the presence of terminate liquid
Liquid changes into yellow.The content of antibody is positively correlated in the depth and sample of color.The standard curve for including according to kit
The OD value of sample to be tested is substituted into the content of peptides calculated in sample to be tested according to the equation that standard curve obtains.
Specific detecting step is as follows:
1. collecting whole blood sample using conventional method, setting blood sample, absorption serum is stand-by after 30 minutes at room temperature.It is to be measured
Blood is clean such as to be used in 24 hours, can be saved in 28 DEG C of conditions, if need to store for a long time, should be stored in 20 DEG C hereinafter, and avoiding anti-
Multiple freeze thawing.
2. blood serum sample to be checked is added into the micropore of the ELISA Plate of coating specific antibody, standing makes its antigen-antibody reaction
In conjunction with；
1) test serum is diluted in proportion with sample diluting liquid, 100 μ l are added in every hole, and shaken at room temperature is incubated for；
2) it washs: removing solution in hole, cleaning solution is added, stand, remove cleaning solution, and be repeated 3 times；
3) ELIAS secondary antibody: being added ELIAS secondary antibody, every 100 μ l of hole, and shaken at room temperature is incubated for；
4) it washs: same to step 2；
5) it develops the color: adding developing solution A, developing solution B, shaken at room temperature colour developing 15min, under the action of enzyme, zymolyte is converted into
Substance that show color；
6) terminate reaction: terminate liquid is added in every hole, and OD value is then measured under 450nm optical wavelength；
7) according to the ratio of the concentration of standard items known in standard curve and OD value, public affairs calculating antibody concentration value: can be passed through
Formula calculates the concentration values of test antibodies.Then value (Cut-off value) is defined according to the kit indicated in specification not determine
Antibody concentration in surveyed blood sample belongs to normal range (NR) or abnormal ranges.
According to method validation program, the linear regression coeffficient (R of the correlation curve of this kit standard product concentration and OD value2
Value) it is greater than or equal to 0.990, sensitivity is less than or equal to 0.5U/ml, and batch internal difference is less than 10%.The operation of this kit is without spy
Different laboratory apparatus, it is only necessary to the laboratories such as microplate reader, pipettor, oscillator underlying instrument.Kit has operational manual, operation
It is simple and efficient.
The ELISA kit of detection metabolism element provided by the invention, high sensitivity, detection is quick and can quantify, stability
It is good, multiple samples can be detected simultaneously, the time required for detection sample is short, and it is easy to operate, do not need special experiment instrument
Device equipment, can be widely applied to laboratory testing.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen Xianjin Technology Academe
<120>a kind of ELISA kit of detection metabolism element
<130> 2010
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213>artificial sequence
<400> 1
Cys Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr
1 5 10 15
<210> 2
<211> 46
<212> PRT
<213>artificial sequence
<400> 2
Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr Arg
1 5 10 15
Glu Gln Cys Glu Leu Asn Pro Ala Cys Asp Glu Leu Ser Asp Gln Tyr
20 25 30
Gly Leu Lys Thr Ala Tyr Lys Arg Ile Tyr Gly Ile Thr Ile
35 40 45
<210> 3
<211> 22
<212> PRT
<213>artificial sequence
<400> 3
Glu Gln Cys Glu Leu Asn Pro Ala Cys Asp Glu Leu Ser Asp Gln Tyr
1 5 10 15
Gly Leu Lys Thr Ala Tyr
20
Claims (10)
1. a kind of ELISA kit of detection metabolism element, which is characterized in that including antibody, the antibody is by such as SEQ ID
The plain polypeptide artificial antigen of the metabolism that the carrier protein couplet of polypeptide and activation shown in NO:1 obtains is through in the immune antibody obtained
Any one or more.
2. ELISA kit according to claim 1, which is characterized in that the compound of the activation is maleimide.
3. ELISA kit according to claim 2, which is characterized in that the carrier protein is spoon sky hemocyanin.
4. ELISA kit according to claim 3, which is characterized in that the antibody is following three kinds that screening obtains
Antibody is any one or more of:
Antibody 1: the hypotype of monoclonal antibody is G2a, potency 4000；
Antibody 2: the hypotype of monoclonal antibody is G2b, potency 8000, also with SEQ ID NO:2 shown in conjunction with polypeptide；
Antibody 3: the hypotype of monoclonal antibody be G2a, potency 32000, also with polypeptide knot shown in SEQ ID NO:3
It closes.
5. ELISA kit according to claim 1-4, which is characterized in that the ELISA kit further includes
ELISA Plate, yin and yang attribute referring to product, two corresponding anti-solution, developing solution, reaction terminating liquid, concentrated cleaning solution, sample diluting liquid, sealing plate film and
Fresco Bag, product description and product quality report are any one or more of.
6. ELISA kit according to claim 5, which is characterized in that be coated with the antibody on the ELISA Plate.
7. ELISA kit according to claim 5, which is characterized in that the ELISA Plate be by outer frame support and
The enzyme mark reaction capillary strip composition being embedded on support frame as described above.
8. ELISA kit according to claim 7, which is characterized in that the ELISA Plate is 24-96 hole elisa Plates, institute
ELISA Plate is stated to be vacuum-packed using the Fresco Bag of one end glue envelope zip mode opening.
9. ELISA kit according to claim 5, which is characterized in that the secondary antibody is marked with horseradish peroxidase.
10. ELISA kit according to claim 5, which is characterized in that the sealing plate film is stickup property plastic foil.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711409132.XA CN109959794B (en) | 2017-12-22 | 2017-12-22 | ELISA kit for detecting metabolin |
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| CN109959794B CN109959794B (en) | 2022-05-27 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040082018A1 (en) * | 2002-10-29 | 2004-04-29 | Athersys, Inc. | Methods for using osteocalcin |
| CN102154217A (en) * | 2011-01-25 | 2011-08-17 | 南京农业大学 | Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
| CN106353506A (en) * | 2016-08-12 | 2017-01-25 | 江苏泽成生物技术有限公司 | Kit for detecting osteocalcin content and testing method thereof |
| CN107011427A (en) * | 2017-03-16 | 2017-08-04 | 深圳先进技术研究院 | Adjust polypeptide of energetic supersession and application thereof |
-
2017
- 2017-12-22 CN CN201711409132.XA patent/CN109959794B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040082018A1 (en) * | 2002-10-29 | 2004-04-29 | Athersys, Inc. | Methods for using osteocalcin |
| CN102154217A (en) * | 2011-01-25 | 2011-08-17 | 南京农业大学 | Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
| CN106353506A (en) * | 2016-08-12 | 2017-01-25 | 江苏泽成生物技术有限公司 | Kit for detecting osteocalcin content and testing method thereof |
| CN107011427A (en) * | 2017-03-16 | 2017-08-04 | 深圳先进技术研究院 | Adjust polypeptide of energetic supersession and application thereof |
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