CN106153935B - A kind of enzyme linked immunological kit for quantitatively detecting CD79 α - Google Patents
A kind of enzyme linked immunological kit for quantitatively detecting CD79 α Download PDFInfo
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Abstract
The present invention relates to a kind of enzyme linked immunological kit for quantitatively detecting CD79 α.The enzyme linked immunological kit of the present invention for quantitatively detecting CD79 α includes：It is coated with ELISA Plate, confining liquid, cleaning solution, CD79 α standard solutions, substrate nitrite ion, terminate liquid, anti-CD79 α monoclonal antibodies detection antibody, the streptavidin of capsicum peroxidase labelling of joint acquisition antibody；Wherein, the joint acquisition antibody is included as the anti-CD79 α polyclonal antibodies obtained by fusion protein immunization experimental animal and anti-CD79 alpha monoclonal antibodies.Enzyme linked immunological kit of the present invention is easy to operate, takes few, high sensitivity, is adapted to the quick detection of a large amount of samples.
Description
Technical field
The invention belongs to biological technical field, is related to enzyme-linked immunologic detecting kit and its preparation of a kind of detection CD79 α
Method.
Background technology
CD79 α belong to immunoglobulin superfamily (IgSF) member.In B cell lymphoma, most acute B lymphocyte
Positive expression in leukaemia and myeloma, one as CD20 aids in detecting factor, is widely used in B cell and its source tumour
Identification.The positive position of CD79 alpha expressions is in after birth and endochylema.CD79 α are the broad-spectrum markers of B cell, since preceding B into
Ripe thick liquid cell can mark.CD79 α can have partial immunity reaction to hand over during being adjusted to B cell terminal differentiation with CD79 β
Fork.CD79 α are available for B cell lymphoma CD79 α (+), pre B cell acute lymphoblastic leukemia (ALL) CD79 α (+) and suddenly
The discriminating of property T cell lymphocytic leukemia CD79 α (-).
The method for being clinically used to detect CD79 α mainly uses immunohistochemistry, but this methods experiment process is grasped
Make more complicated, required time length, sensitivity is low, it is impossible to carry out quantitative analysis, agents useful for same endangers experimenter larger；It is required
Instrument and equipment is expensive, and technical merit is more demanding, is unfavorable for clinical quick detection.Moreover, need to be into using immunohistochemical method
Row biopsy, it is time-consuming and laborious to collect sample.The requirement of biopsy tumor biopsy skillfully with careful technology, makes extraordinary section
It is difficult to which it is required, thickness is uniform, section is complete；Tumour cell complex shape, differentiation degree is different, to the experience of pathologist and
It is high to operate level requirement.In addition, CD79 α albumen is penetrated into the circulatory system after tumour cell can be secreted or ruptured.Therefore, have
The kit of the new quantitative detection CD79 α of necessity exploitation.
The content of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of easy, quick, practical, sensitive, efficient inspection
Survey the enzyme linked immunological kit of CD79 α.
A kind of enzyme linked immunological kit for quantitatively detecting CD79 α of the present invention, the kit include：Coating joint
Capture ELISA Plate, confining liquid, cleaning solution, CD79 α standard solutions, substrate nitrite ion, terminate liquid, the anti-CD79 α monoclonal antibodies of antibody
Detect antibody, the streptavidin of horseradish peroxidase-labeled；Wherein, the joint acquisition antibody includes being exempted from by CD79 α albumen
Anti- CD79 α polyclonal antibodies and anti-CD79 alpha monoclonal antibodies obtained by epidemic disease experimental animal.
The further feature of enzyme linked immunological kit according to the present invention, the coating concentration of the joint acquisition antibody
For every kind of antibody 10-100ng/100ul, the concentration for detecting antibody is 200ng/ml.
The further feature of enzyme linked immunological kit according to the present invention, the anti-CD79 α monoclonal antibodies are to be with sequence
SEQ ID NO:It is prepared by 1 polypeptide.
The further feature of enzyme linked immunological kit according to the present invention, the anti-CD79 α monoclonal antibodies are according to following
Prepared by method：It is SEQ ID NO by the sequence of purifying:For 1 polypeptide with SPF grades of Balb/c mouse are immunized after KLH couplings, gained is small
Screening obtains 3 strain of hybridoma strains after mouse spleen is merged with myeloma cell SP2/0, and hybridoma secretion gained antibody is used
ProteinG/A affinity columns are further purified, and obtain anti-CD79 α monoclonal antibodies.
The further feature of enzyme linked immunological kit according to the present invention, the confining liquid are pH7.2 phosphate
0.01% polysorbas20 is added in buffer solution, 5% skimmed milk power and polysaccharide, wherein the concentration range of polysaccharide is 0.5 to 10mg/ml
Between, polysaccharide is selected from：One or more kinds of combinations of sucrose, mannose, lactose, trehalose.
The CD79 α albumen is lured with the Isopropyl β-D-1-thiogalactopyranoside (IPTG) of 0.1mM
Lead CD79 α
The concentration of the joint acquisition antibody is every kind of antibody 10-100ng/100ul, and the concentration for detecting antibody is
200ng/ml。
The coating buffer with the addition of to guarantee to preserve for a long time in the carbonate buffer solution of 0.05M PH 9.6
0.01% thimerosal is as preservative.
It with sequence is SEQ ID NO that the anti-CD79 α monoclonal antibodies, which are,:It is prepared by 1 polypeptide.
The anti-CD79 α monoclonal antibodies are prepared according to following methods：It is SEQ ID NO by the sequence of purifying:1 polypeptide with
SPF grades of Balb/c mouse are immunized after KLH couplings, gained mouse spleen screens after being merged with myeloma cell SP2/0 obtains 3 plants of hybridization
Tumor cell strain, hybridoma secretion gained antibody are further purified with ProteinG/A affinity columns, obtain anti-CD79 α monoclonal antibodies.
The confining liquid is that 0.01% polysorbas20,5% skimmed milk power and a branch are added in pH7.2 phosphate buffers
Hold the polysaccharide of protective effect, wherein the concentration range of polysaccharide is 0.5 between 10mg/ml, and polysaccharide type can be sucrose, sweet dew
Sugar, lactose, trehalose one or more combination therein.
The content of the kit antigen standard items is 1.5 between 100mg/ml.
The enzyme linked immunological kit of the present invention for quantitatively detecting CD79 α, its advantage are：
(1) the defects of present invention overcomes memebrane protein to be not suitable as ELISA reagents, being obtained using special way of purification can
Dissolubility memebrane protein, then animal is immunized using memebrane protein and obtains capture antibody of the polyclonal antibody as kit of the present invention, make examination
Agent is more stablized in the reaction system.
(2) employ joint acquisition antibody, as the anti-CD79 α polyclonal antibodies obtained by fusion protein immunization experimental animal with
Anti- CD79 alpha monoclonal antibodies composition, thus increases the binding site of antigen-antibody, increases the sensitivity of kit, background value is low.
Because the memebrane protein characteristic of CD79 α and being not easy to be coated in solid phase carrier, the sensitivity of detection must be increased, and add
.01% polysorbas20 is in order to reduce non-specific adsorption, just with compensate for the issuable false positive of joint antibody.
(3) kit of the present invention overcomes many disadvantages of traditional ImmunohistochemistryMethods Methods, its this kit can
The CD79 alpha levels of as low as 15mg/L are measured to, can be used in detection and the danger forecasting of the neoplastic hematologic disorder disease such as leukaemia.
(4) common antigen antibody response ELISA systems are using the tradition envelope such as 5% skimmed milk power and 1% bovine serum albumin(BSA)
Liquid is closed, the sealing effect to Membrane protein antigen antibody response system is difficult often satisfactory, and kit of the present invention
It is by using the confining liquid for having added polysaccharide and Tween-20, there is certain protective effect for coating protein, reduce non-spy
Opposite sex absorption, has certain supporting function especially for the conformation of this kind of memebrane proteins of CD79 α.
Brief description of the drawings
Fig. 1 is independent spirit of the anti-coated ELISA kit of CD79 Alpha antibodies with combining the coated ELISA kit of antibody
Sensitivity contrasts.
Fig. 2 is the contrast and experiment for using different confining liquids under the same conditions.
Embodiment
To make the present invention easier to understand with reference to specific embodiments the present invention is further explained.It is to be understood that this
A little embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
Embodiment 1：The preparation of polyclonal antibody
1. the source of gene and protein sequence
People's CD79 α gene orders can be found in XM_002829281, from NCBI.
According to the gene order of CD79 α, synthesis is respectively positioned at the primer in reading frame and downstream.
Sense primer：caccatggctgggg gtccaggagt cctcca
Anti-sense primer：ttctcgagtcacggcttc tccagctgga c
People CD79 α full length genes are 681bp, encode 226 amino acid, wherein 1 to 32 amino acids of N- ends are signal peptide.
Protein sequence source：BAD97091, from NCBI.
The polypeptide sequence for being used to prepare monoclonal antibody is CSRGLQGTYQDVGSLNIGDVQLEK (SEQ ID NO.1).
2. the acquisition of the structure and high expression engineering strain of expression plasmid
Expression vector establishment method：Purpose people's CD79 α genes are obtained by PCR amplification, with the double enzyme cutting PCR of NdeI+XhoI
Product and pET28a plasmids are by two fragment T4DNA ligase is attached, and connection product conversion enters bacillus coli DH 5 alpha,
The selected clone on the LB tablets containing ampicillin, prepares plasmid in a small amount, goes out the positive by double digestion/PCR evaluation and screenings
Clone.The pET28a/CD79 α plasmids of a large amount of amplification confirmations, are identified with the double enzyme cuttings of NdeI+XhoI；PET28a/CD79 α plasmids pass through
After sequence verification, conversion enters Escherichia coli DE3, and positive colony is selected on the LB tablets containing ampicillin and carries out matter
Granzyme cuts identification, prepares plasmid in a small amount, goes out positive colony by double digestion/PCR evaluation and screenings, obtains the restructuring containing CD79 α
Plasmid engineering bacterium.
3.CD79 the expression and purification of α albumen
Above-mentioned CD79 α restructuring is induced with the Isopropyl β-D-1-thiogalactopyranoside (IPTG) of 0.1mM
Plasmid engineering bacterium DE3-pET28a, 18 DEG C overnight, and low temperature induction avoids the poison that the expression of a large amount of foreign proteins produces host strain
Evil.Bacterium is collected, using ultrasonic disruption, supernatant acquisition crude protein is collected for (12,000g, 4 DEG C) through centrifugation.Crude protein uses 0-
The reducing agent of 0.02M, the denaturant of 4-8M are resuspended.The reducing agent can be mercaptoethanol, TCEP or DTT, and denaturant can be
Urea or guanidine hydrochloride.
After the fusion protein that slightly carries runs SDS-PAGE electrophoresis, PAGE glue is with the 0.1 of 4 degrees Celsius of precoolings to 0.5mol/L's
KCL dyes 3min, cuts purpose band and smashs to pieces and is put into bag filter, and bag filter is placed on Horizontal electrophoresis tank, adds PBS bufferings
Liquid run 1 it is small when, by positive and negative umpolung run again 1 it is small when after recycle bag filter in solution, place into freeze dryer freeze.This method is grasped
Make simple, without changing dialyzate repeatedly, time and more efficiently is more saved than conventional dialysis renaturation method.
Preparation more anti-4.CD79 α
With SPF grades of new zealand rabbits of CD79 α protein immunizations of above-mentioned purifying：By Freund's complete adjuvant and antigen during initial immunity
150 μ l (50 μ g) are mixed in equal volume, after fully emulsified, subcutaneous multi-point injection.After 2 weeks, new zealand rabbit is immunized again, and
Use incomplete Freund's adjuvant instead, volume injected and method are constant, hereafter new zealand rabbit are continued to be immunized every 2 weeks, and 3 are immunized altogether
It is secondary.Culling heart blood is ready for after new zealand rabbit serum titer reaches requirement, new zealand rabbit is injected intraperitoneally in first three day of taking a blood sample
It is immune that 50 μ g antigens carry out impact.
5.CD79 the preparation purifying of Alpha antibodies
Above-mentioned anti-CD79 α antiserums using the HiTrap Protein G/A of GE Healthcare companies and will be purified
It is lyophilized that antibody afterwards carries out packing.
The preparation of embodiment 2.CD79 α monoclonal antibodies
Monoclonal antibody is prepared using conventional method：It is used to prepare the polypeptide sequence CSRGLQGTYQDVGSLNIGDVQLEK of monoclonal antibody
(SEQ ID NO.1) and immune SPF grades of Balb/c mouse after KLH couplings, after gained mouse spleen is merged with myeloma cell SP2/0
Screening obtains 3 strain of hybridoma strains, and hybridoma secretion gained antibody is further purified with ProteinG/A affinity columns, obtained
To anti-CD79 α monoclonal antibodies.1 plant is picked out from 3 strain of hybridoma strains using indirect ELISA anti-to be combined as joint acquisition with more and resist
Body.
Embodiment 3：Detect the enzyme linked immunological kit of CD79 α
The enzyme linked immunological kit of detection CD79 α is set up, makes its contained following component：
1. the more anti-and anti-CD79 α monoclonal antibodies of the anti-CD79 α of coating and the ELISA Plate as capture antibody；
2.pH values are 7.2, containing 5% skimmed milk power, 0.01% polysorbas20, trehalose, the phosphate buffer of 0.1mol/L
For confining liquid；
3.pH values are 7.2, containing 0.5% polysorbas20, the cleaning solution of the phosphate buffer of 0.1mol/L；
4.CD79 α standard solutions；
5. substrate nitrite ion is made of nitrite ion A and nitrite ion B, nitrite ion A liquid is hydrogen peroxide, and nitrite ion B liquid is neighbour
Phenylenediamine or tetramethyl benzidine；
6. terminate liquid is 2M sulfuric acid solutions；
7. the anti-CD79 α monoclonal antibodies (article No. 130-10122) of biotinylation are as detection antibody
8. the streptavidin of horseradish peroxidase-labeled.
Embodiment 4：Detect the application process of the enzyme linked immunological kit of CD79 α
CD79 α are detected using double antibody sandwich method using the enzyme linked immunological kit constructed by embodiment 3, specific detection
Step is：
(1) it is coated with：Capture antibody is fixed on solid phase polystyrene supporter, dilutes (PH9.6 carbonate with coating buffer
Solution), when 4 degrees Celsius of coatings 12 are small.Wherein the concentration range of joint acquisition antibody is 10-100ng per 100ul；
(2) close：PH value is used as 7.2, containing 5% skimmed milk power, 0.01% polysorbas20, polysaccharide, 0.1mol/L phosphoric acid
The concentration range of salt buffer, wherein polysaccharide is 0.5 between 10mg/ml.
(3) standard items and test serum are added：The standard items of concentration known are diluted in proportion and are added in microwell plate, are set up
Blank control.Test serum sample is added into other microwell plates again.
(4) detection antibody is added：Add the detection antibody of biomarker and streptavidin is respectively incubated half an hour, detection is anti-
The concentration range of body is 200ng/ml.The detection antibody is purchased from Rui Boao bio tech ltd of the U.S., article No. 130-
10122。
(5) substrate and terminate liquid are added.
Embodiment 5：Detect the quality analysis of the enzyme linked immunological kit of CD79 α
1. the sensitivity of kit of the present invention
Each component is added according to the step of embodiment 3, independent anti-CD79 α polyclonal antibodies will be used, independent anti-CD79 α are mono-
The coated reagent of clonal antibody carries out contrast experiment with using the coated kit of joint antibody.
By CD79 α antigen standards be configured to 1.5625,3.125,6.25,12.5,25,50,100ng/100ul 7 it is dense
Gradient is spent, is measured according to the ELISA conditions after optimization, draws standard curve.As shown in Figure 1, the standard items of ELISA detection method
The range of linearity is 1.5625~100mg/L, using the result of joint antibody than the high (minimum detected value of OD values using independent antibody
1/2>Control value), prompt to add sensitivity.
As a result such as table 1 below：
| Standard concentration (mg/L) | OD450 values (joint antibody) | OD450 values (individually how anti-) | OD450 values (independent monoclonal antibody) |
| 100 | 2.03 | 2.664 | 0.944 |
| 50 | 1.661 | 2.357 | 0.794 |
| 25 | 1.106 | 1.706 | 0.455 |
| 12.5 | 0.575 | 1.175 | 0.196 |
| 6.25 | 0.334 | 0.843 | 0.079 |
| 3.125 | 0.211 | 0.719 | 0.067 |
| 1.5625 | 0.195 | 0.561 | 0.051 |
| NC | 0.061 | 0.371 | 0.049 |
2. the specificity of kit of the present invention
The ability (non-false positive) for the analyte being not present correctly is examined and determine, depending on joint acquisition antibody and detection antibody
Purity and specificity.
(1) it is coated with：Capture antibody is fixed on solid phase polystyrene supporter, dilutes (PH9.6 carbonate with coating buffer
Solution), when 4 degrees Celsius of coatings 12 are small.Wherein catch and combine the concentration range for obtaining antibody for the every 100ul of 10-100ng；
(2) close：PH value is used as 7.2, containing 5% skimmed milk power, 0.01% polysorbas20, polysaccharide, the phosphoric acid of 0.1mol/L
The concentration range of salt buffer, wherein polysaccharide is 0.5 between 10mg/ml.
(3) each antigen protein is added：By the antigen protein of concentration known (CD79 α, CD38, IGF1, CD23, CEBPB,
Keratin) dilute in addition microwell plate.
(4) detection antibody is added：The detection antibody and streptavidin for adding biomarker are incubated each half an hour, wherein examining
Survey antibody concentration range is 200ng/ml.
(5) substrate and terminate liquid are added.
Cross reaction result such as table 2 below：
| Cross reaction thing | CD79α | CD38 | IGF1 | CD23 | CEBPB | Keratin 8 |
| OD detected values | 1.823 | 0.165 | 0.149 | 0.157 | 0.212 | 0.161 |
Embodiment 5：Confining liquid determines
Each component is added according to the step of embodiment 3, under the same conditions using the contrast experiment of different confining liquids.
Confining liquid A, component are：PH value is 7.2, containing 5% skimmed milk power, 0.01% polysorbas20, polysaccharide, the phosphorus of 0.1mol/L
The concentration range of phthalate buffer, wherein polysaccharide is 0.5 between 10mg/ml.
Confining liquid B, component are：PH value is 7.2, containing 5% skimmed milk power, polysaccharide, and the phosphate buffer of 0.1mol/L, its
The concentration range of middle polysaccharide is 0.5 between 10mg/ml.
Confining liquid C, component are：PH value is 7.2, containing 5% skimmed milk power, the phosphate buffer of 0.1mol/L,.
Confining liquid D, component are：PH value is 7.2, containing 1% casein sodium salt, the phosphate buffer of 0.1mol/L.
CD79 α antigen standards are configured to 1.5625,3.125,6.25,12.5,25,50,100ng/100ul 7 it is dense
Gradient is spent, is measured according to the ELISA conditions after optimization, draws standard curve.
Comparison result such as table 3 below：
| Standard concentration (mg/L) | Component A | B component | Component C | D components |
| 100 | 2.03 | 1.823 | 0.227 | 0.396 |
| 50 | 1.661 | 1.3 | 0.255 | 0.374 |
| 25 | 1.106 | 1.093 | 0.243 | 0.266 |
| 12.5 | 0.575 | 0.842 | 0.177 | 0.212 |
| 6.25 | 0.334 | 0.827 | 0.105 | 0.197 |
| 3.125 | 0.211 | 0.71 | 0.114 | 0.174 |
| 1.5625 | 0.195 | 0.687 | 0.128 | 0.161 |
| NC | 0.061 | 0.305 | 0.128 | 0.14 |
As shown in Fig. 2, adding identical standard items, detection is using the ELISA of different confining liquids the results show that A groups are closed
Liquid figure viewed from behind value is low, detection sensitivity highest, and does not use C, D component detection sensitivity of polysaccharide low.
Claims (3)
1. a kind of enzyme linked immunological kit for quantitatively detecting CD79 α, it is characterised in that the kit includes：Join for being coated with
Close the capture ELISA Plate of antibody, confining liquid, cleaning solution, CD79 α standard solutions, substrate nitrite ion, terminate liquid, biotinylated
Anti- CD79 α monoclonal antibodies detection antibody, the streptavidin of horseradish peroxidase-labeled, joint acquisition antibody；Wherein, it is described combine catch
Obtaining antibody is included as the anti-CD79 α polyclonal antibodies obtained by CD79 α protein immunization experimental animals and anti-CD79 alpha monoclonal antibodies；
It with sequence is SEQ ID NO that anti-CD79 alpha monoclonal antibodies in the joint acquisition antibody, which are,:It is prepared by 1 polypeptide；The envelope
Liquid is closed to add 0.01% polysorbas20, the concentration model of 5% skimmed milk power and polysaccharide, wherein polysaccharide in pH7.2 phosphate buffers
0.5 is trapped among between 10mg/ml, polysaccharide is selected from：One or more kinds of combinations of sucrose, mannose, lactose, trehalose.
2. enzyme linked immunological kit according to claim 1, it is characterised in that：The coating concentration of the joint acquisition antibody
For every kind of antibody 10-100ng/100ul, the concentration for detecting antibody is 200ng/ml.
3. enzyme linked immunological kit according to claim 1, it is characterised in that the anti-CD79 in the joint acquisition antibody
Alpha monoclonal antibodies are prepared according to following methods：It is SEQ ID NO by the sequence of purifying:1 polypeptide is exempted from after being coupled with KLH
SPF grades of Balb/c mouse of epidemic disease, gained mouse spleen screens after being merged with myeloma cell SP2/0 and obtains 3 strain of hybridoma strains, miscellaneous
Hand over oncocyte secretion gained antibody to be further purified with ProteinG/A affinity columns, obtain anti-CD79 alpha monoclonal antibodies；Between
Meet ELISA and the anti-CD79 alpha monoclonal antibodies of 1 strain of hybridoma strain secretion are picked out from 3 strain of hybridoma strains as institute
State the anti-CD79 alpha monoclonal antibodies in joint acquisition antibody.
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| CN108535076A (en) * | 2018-03-19 | 2018-09-14 | 广州江元医疗科技有限公司 | A kind of confining liquid and preparation method thereof applied to immunocytochemistry P16 protein stainings |
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| CN1420987A (en) * | 1999-11-16 | 2003-05-28 | 杰南技术公司 | ELISA for VEGF |
| CN102168072A (en) * | 2010-12-20 | 2011-08-31 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
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| CN1420987A (en) * | 1999-11-16 | 2003-05-28 | 杰南技术公司 | ELISA for VEGF |
| CN102168072A (en) * | 2010-12-20 | 2011-08-31 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
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| Title |
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| CD79a Expression in Acute Myeloid Leukemia High Frequency of Expression in Acute Promyelocytic Leukemia;Daniel A. Arber et al.;《American Journal of Pathology》;19961031;第149卷(第4期);1105-1110 * |
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