CN109942703A - The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody - Google Patents
The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody Download PDFInfo
- Publication number
- CN109942703A CN109942703A CN201910230425.4A CN201910230425A CN109942703A CN 109942703 A CN109942703 A CN 109942703A CN 201910230425 A CN201910230425 A CN 201910230425A CN 109942703 A CN109942703 A CN 109942703A
- Authority
- CN
- China
- Prior art keywords
- microglobulin
- reagent
- polyclonal antibody
- added
- acid chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000003966 Alpha-1-microglobulin Human genes 0.000 title claims abstract description 54
- 101800001761 Alpha-1-microglobulin Proteins 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 65
- 239000004816 latex Substances 0.000 claims abstract description 38
- 229920000126 latex Polymers 0.000 claims abstract description 38
- 239000002245 particle Substances 0.000 claims abstract description 33
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 15
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 5
- 230000005593 dissociations Effects 0.000 claims abstract description 5
- 230000001235 sensitizing effect Effects 0.000 claims abstract description 3
- 206010070834 Sensitisation Diseases 0.000 claims description 23
- 230000008313 sensitization Effects 0.000 claims description 23
- 210000002966 serum Anatomy 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- 239000000725 suspension Substances 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 230000036961 partial effect Effects 0.000 claims description 7
- 150000001718 carbodiimides Chemical class 0.000 claims description 6
- 230000003053 immunization Effects 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 5
- 239000004005 microsphere Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000004793 Polystyrene Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000003763 carbonization Methods 0.000 claims description 4
- 229920002223 polystyrene Polymers 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 150000001412 amines Chemical class 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 210000001165 lymph node Anatomy 0.000 claims description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 230000003042 antagnostic effect Effects 0.000 claims description 2
- 238000010612 desalination reaction Methods 0.000 claims description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims 1
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical class COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims 1
- LLJZKKVYXXDWTB-UHFFFAOYSA-N acetic acid;sodium Chemical compound [Na].[Na].CC(O)=O LLJZKKVYXXDWTB-UHFFFAOYSA-N 0.000 claims 1
- 230000029087 digestion Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 239000000872 buffer Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 230000009103 reabsorption Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000011017 operating method Methods 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 1
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation of α 1- microglobulin polyclonal antibody and α 1- microglobulin detection kits；The kit is made of R1 reagent, R2 reagent two parts, and wherein R1 reagent is the phosphate buffer containing dissociation protein agent, and R2 reagent is the buffer containing the sensitizing latex particle for being coated with α 1- microglobulin polyclonal antibody.The present invention uses a kind of dissociation protein agent, the antigen site in sample is exposed to greatest extent, increase the Percentage bound of antigen-antibody, and a kind of specific α 1- microglobulin polyclonal antibody is coated with using a kind of more stable, amplified signal effect more preferably latex particle, through these means, to realize the high sensitivity of reagent of the present invention；Solves currently on the market the problems such as α 1-MG reagent detection sensitivity is not high, expensive.
Description
Technical field
The present invention relates to a kind of medical immunology in-vitro diagnosis fields, and in particular to a kind of α 1- microglobulin polyclonal antibody
Preparation and α 1- microglobulin detection kit.
Background technique
α 1-MG is synthesized by the liver and lymphocyte of human body, and molecular weight is about the glycoprotein of 33000 dalton.It by
167 amino acid compositions, with human leucocyte antigen-A 11, the antigenic determinants such as HLA-B20 and HLA-51 have intersection anti-
It answers.
α 1-MG is widely present in the various body fluid of human body and lymphocyte film surface, and the α 1-MG in blood is deposited in two forms
In the i.e. free α 1-MG and α 1-MG (α 1MG-1gA) in conjunction with IgA.Under normal circumstances, α 1-MG-1gA accounts for about total in blood
The 40~70% of α 1-MG, immunoglobulin level has an impact to the ratio between α 1-MG and α 1-MG-1gA in blood.In blood
Free α 1-MG can pass freely through glomerular filtration membrane, and 95%~99% in kidney proximal tubule reabsorption and metabolism, only micro
From urine discharge eventually；And the α 1-MG of mating type then cannot be by glomerulus, the concentration in urine is zero.
It is generally acknowledged that α 1-MG has the reason of increasing in serum and urine:
1. the ability of reabsorption and metabolism α 1-MG reduce；
2. detection of glomeruli filtration function is impaired；
3. synthesis is excessive in vivo；
4. lymphocyte destruction discharges.
Two o'clock and a large amount of clinical and experimental study are confirmed based on above-mentioned 1., 2., at present it has been recognized that serum and urine
The measurement of α 1-MG can be used as an impaired sensitive indexes of reaction reabsorption function in liquid, and to a certain extent
It is also advantageous over β2-microglobulin.
The current detection method of α 1- microglobulin has ELISA, radioimmunology, colloidal gold method, latex immunoturbidimetry
Deng.ELISA, radioimmunology, colloidal gold method operation are relative complex in these methods, are easy to appear the deviation of result, and valence
Lattice are relatively expensive, and detection time and cost are all higher.And latex immunoturbidimetry can clinical examination department it is indispensable it is complete from
It is used on Automatic Biochemical Analyzer, it is easy to detect, examining report fast can be rapidly issued, and to light Medium hemolysis and slightly
Yellow subcutaneous ulcer, piarhemia have certain anti-interference ability, available to be widely applied.
The problems such as low problem of the generally existing sensitivity of α 1- microglobulin detection reagent currently on the market, poor repeatability,
And import reagent price is more expensive.
By the retrieval discovery to existing patent document, the Chinese invention patent application of application number 201410193619.9 is public
A kind of α 1- microglobulin detection kit is opened, the kit is based on colloid gold immune turbidimetry, including reagent R2, described
Reagent R2 is the solution containing the gold nano grain that α 1- microglobulin antibody is marked, and the partial size of the gold nano grain is
62.2nm-79.1nm, the gold nano grain and antibody mass ratio are 50:20~60.Kit of the present invention has high sensitivity,
High specificity is swift in response, the good feature of stability.However, the test operation of colloidal gold method is cumbersome, accuracy is not high, cost
It is expensive.
The Chinese invention patent application of application number 201510847828.5 discloses one kind can detect urine and blood simultaneously
The kit of α 1- microglobulin in final proof sheet, including reagent 1 and reagent 2, the reagent 1 are to contain anti-human rheumatoid factor
The buffer of antibody and coagulant；The reagent 2 is to contain the big partial size for being coated with anti-human α 1- microglobulin monoclonal antibody
The buffer of polystyrene nanoparticles.With high sensitivity, the wide line, specific more preferable, strong antijamming capability, and can examine
The advantages of surveying human urine and serum.However, monoclonal antibody is nothing like polyclonal antibody in stability and sensitivity.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of systems of α 1- microglobulin polyclonal antibody
Preparation Method and more detection kits of α 1- microglobulin containing the antibody, the kit are used to detect containing for α 1-MG in serum
Amount, it is good to reach easy to operate, high sensitivity, specificity, quickly, measurement result accurately and reliably purpose.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of α 1- microglobulin polyclonal antibody, the α 1- microglobulin is polyclonal
Antibody is prepared by the method comprised the steps of:
The preparation of S1, immunogene: protected amino acid chain 6-His and 10- is added in recombinant alpha 1- microglobulin amino acid chain
Cys, and 2 restriction enzyme site Hind III and BamH I are added, recombinant expression obtains antigen；
S2, immune animal: selecting rabbit immunization route, is for the first time 15 with second of immunization interval using lymph node immunization
It, is spaced 10 days after second；
S3, sero-fast purifying and preservation: affine and purifying, antibody after purification are carried out using affinity column antagonistic Serum and is removed
Glycerol is added after salt to save.
Preferably, step S1 specifically: in recombinant alpha 1- microglobulin amino acid chain, protected amino acid is added
Protected amino acid chain 10-Cys is added in the tail portion of original acid chain in the head of original acid chain in chain 6-His, and in head 6-
III restriction enzyme site of Hind is added in His amino acid chain, I restriction enzyme site of BamH is added in the 10-Cys amino acid chain of tail portion.
Preferably, in step S3, the affinity column used is Ago-Gel affinity column, purification solution is PBS slow
Fliud flushing.
Second aspect, the present invention also provides a kind of α 1- microglobulin detection kit, including R1 reagent and R2 reagent,
The R1 reagent is the phosphate buffer containing dissociation protein agent, and the reagent R2 is to contain α 1- microglobulin above-mentioned
The buffer system of the coated sensitizing latex particle of polyclonal antibody.
Preferably, the R1 reagent includes following ingredient: pH is 7.0~7.6, concentration is 10~100mmol/L
Phosphate buffer, concentration be 5~20mmol/L disodium ethylene diamine tetraacetate, concentration be 200~500mmol/L hydrochloric acid
Guanidine, the sodium chloride that concentration is 10~50mmol/L, the polyethylene glycol and volume fraction that concentration is 10~100mmol/L are
0.01%~0.05% preservative.
Preferably, the R2 reagent includes following ingredient: pH is 7.0~7.6, concentration is 10~100mmol/L
Phosphate buffer, concentration be 10~100ml/L the coated sensitization latex of α 1- microglobulin polyclonal antibody
Grain.
Preferably, the sensitization latex particle is poly (methyl methacrylate) micro-sphere or polystyrene microsphere.
Preferably, the coated sensitization latex particle of α 1- microglobulin polyclonal antibody is by the inclusion of such as
The method of lower step is prepared:
A1, α 1- microglobulin polyclonal antibody is added in the sensitization latex particle that partial size is 150~300nm, controls institute
The volume ratio for stating α 1- microglobulin polyclonal antibody and sensitization latex particle is 0.5~1:1, is incubated at room temperature 1.5-2.5 hours,
Obtain solution；
A2, carbodiimides is added in the solution, controls the corresponding 5~10mg carbonization two of every milliliter of sensitization latex particle
Imines is incubated at room temperature 1.5-2.5 hours, obtains suspension；
A3, after being separated by solid-liquid separation the suspension, solid portion is taken to wash dispersion with phosphate buffer.
Preferably, the sensitization latex particle is poly (methyl methacrylate) micro-sphere or polystyrene microsphere.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the present invention obtains a kind of special α 1- microballoon by a kind of preparation method of α 1- microglobulin polyclonal antibody
Protein polyclone antibody, the Antibody stability is good, high specificity, can largely improve antigen-antibody Percentage bound, make reagent
Sensitivity greatly improves, and still can detect that when sample concentration is down to 0.1mg/L as a result, and the minimum inspection of the reagent of the prior art
Survey is limited to 0.5mg/L；
2, the antibody in the present invention and latex binding ability are strong, and waste is few during coating, is applicable to existing market
The latex particle of upper majority, it is applied widely.And combine rear sensitization particle more stable with latex, so that the stabilization of reagent
Property improve, the range of linearity is wider；Reagent stability can reach 18 months, and the range of linearity can arrive 200mg/L；During coating
Loss reduce, cost is greatly saved, so that the price of kit is more competitive；
3, kit of the invention is suitable for full automatic biochemical apparatus, easy to operate, and accuracy is high, and cost is relatively low；
4, the present invention dissociates protein agent guanidine hydrochloride in reagent R1, the antigen site in sample can be made to obtain adequately sudden and violent
Dew, improves the Percentage bound of antigen-antibody；Stabilization of kit box sensitivity of the invention is far superior to existing market level.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is the standard curve of the α 1-MG reference standard of 5 kinds of different contents；
Fig. 2 is that the α 1-MG reagent of reagent of the present invention and German Roche Holding Ag is respectively adopted to 50 parts of human serums (comprising normal
And monstrosity) measurement structure correlation analysis figure.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1
The present embodiment provides a kind of α 1- microglobulin polyclonal antibodies, are obtained by being prepared via a method which:
(1) head in original α 1- microglobulin amino acid chain (carrying amino acid chain sequence in commercial antibody)
It joined one section of protective amino acid chain 6-His, joined one section of protective amino acid chain 10-Cys in tail portion.Add simultaneously
Added 2 groups of restriction enzyme site Hind III and BamH I (specifically: III restriction enzyme site of Hind is added in the 6-His amino acid chain of head,
I restriction enzyme site of BamH is added in the 10-Cys amino acid chain of tail portion)；Using recombinant protein expression technology, expresses and extract antigen；
(2) it will extract after immunologic adjuvant is added in antigen, it is immune to carry out lymph node to rabbit, 0.1ml is measured every time, for the first time with the
Secondary immunity time interval be 15 days, it is secondary after between be divided into 10 days；
(3) after detection antibody titer is up to standard, antibody blood is acquired, and affinity purification, glycerol is added after desalination and is saved.
Embodiment 2
The present embodiment provides a kind of α 1-MG detection kits, form as follows:
1. reagent R1 are as follows:
2. reagent R2 are as follows:
Phosphate buffer (PH 7.0) 10mmol/L
α 1- microglobulin polyclonal antibody
Coated sensitization latex particle 10ml/L；
The preparation process of the above-mentioned coated sensitization latex particle of α 1- microglobulin polyclonal antibody is as follows:
S1: the α 1- microglobulin polyclonal antibody of embodiment 1 is added in the sensitization latex particle that partial size is 150nm, control
Antibody processed and the volume ratio of latex particle are 0.5:1, are incubated at room temperature 2 hours, obtain solution；
S2: being added carbodiimides in the above solution, controls and 5mg carbodiimides is added in every milliliter of latex particle,
Incubation at room temperature 2 hours, obtains suspension；
S3: after above-mentioned suspension is separated by solid-liquid separation, solid portion is taken to wash dispersion with phosphate buffer.
The α 1-MG detection kit of the present embodiment description, is suitable for various types of full automatic biochemical apparatus, with Hitachi 7170
For full automatic biochemical apparatus, operation such as table 1.Analysis method: Two point end assay, the i.e. dosage of reagent R1, R2 are respectively 240 μ l
With 60 μ l, 2 μ l of sample size；2 μ l samples are added after 37 DEG C of incubation 5min in 240 μ l reagent R1,60 μ l reagent R2 are added, 37 DEG C incubate
30s is educated, absorbance is read, reads another point after being then incubated for 4.5min again；Detection dominant wavelength is 600nm.
Using this reagent and said determination method, using the α 1- for 5 kinds of different contents that 7170 Biochemical Analyzer of Hitachi measures
The curve (as shown in Figure 1) of MG calibration object, each point represent the reference calibrations product an of content, and wherein X-axis indicates α 1-MG content
(mg/L)；Y-axis indicates absorbance.
Table 1
Embodiment 3
The present embodiment provides a kind of α 1-MG detection kits, form as follows:
1. reagent R1 are as follows:
2. reagent R2 are as follows:
Phosphate buffer (PH 7.6) 50mmol/L
α 1- microglobulin polyclonal antibody
Coated sensitization latex particle 50ml/L.
The preparation process of the above-mentioned coated sensitization latex particle of α 1- microglobulin polyclonal antibody is as follows:
S1: the α 1- microglobulin polyclonal antibody of embodiment 1 is added in the sensitization latex particle that partial size is 200nm, control
Antibody processed and the volume ratio of latex particle are 0.8:1, are incubated at room temperature 2 hours, obtain solution；
S2: being added carbodiimides in the above solution, controls and two Asia of 7.5mg carbonization is added in every milliliter of latex particle
Amine is incubated at room temperature 2 hours, obtains suspension；
S3: after above-mentioned suspension is separated by solid-liquid separation, solid portion is taken to wash dispersion with phosphate buffer.
Kit concrete operation method is the same as embodiment 1.
Embodiment 4
The present embodiment provides a kind of α 1-MG detection kits, form as follows:
1. reagent R1 are as follows:
2. reagent R2 are as follows:
Phosphate buffer (PH 7.2) 100mmol/L
α 1- microglobulin polyclonal antibody
Coated sensitization latex particle 100ml/L；
The preparation process of the above-mentioned coated sensitization latex particle of α 1- microglobulin polyclonal antibody is as follows:
S1: the α 1- microglobulin polyclonal antibody of embodiment 1 is added in the sensitization latex particle that partial size is 300nm, control
Antibody processed and the volume ratio of latex particle are 1:1, are incubated at room temperature 2 hours, obtain solution；
S2: being added carbodiimides in the above solution, controls and 5~10mg carbonization, two Asia is added in every milliliter of latex particle
Amine is incubated at room temperature 2 hours, obtains suspension；
S3: after above-mentioned suspension is separated by solid-liquid separation, solid portion is taken to wash dispersion with phosphate buffer.
Kit concrete operation method is the same as embodiment 1.
Embodiment 5: the correlation test of detection reagent
Using the BMG reagent of this law invention kit (specific formula is with embodiment 1) and Roche Holding Ag, contrast agents Germany,
Using automatic 7170 automatic clinical chemistry analyzer to 50 parts of human serums (including normal and monstrosity) by each autoregressive parameter simultaneously into
Row measurement carries out correlation analysis to measured value.It is measured according to the parameter in above-mentioned " table 1 "；Measurement result is shown in Fig. 2,
X, Y-axis are measured value (the content mg/L of α 1-MG),
Found out by the result of Fig. 2, the phase relation of two kinds of reagents is R2=0.99, regression equation y=0.9997x+
0.3277.The result shows that this reagent and import reagent measurement patients serum's correlation are good, have specific and accurate well
Property.In addition, the above experiment is to be carried out using 7170 full automatic biochemical apparatus of Hitachi, Ltd's manufacture, but reagent of the invention is unlimited
In above-mentioned instrument, other full-automatic or semi-automatic biochemical analyzers are applied also for.
Test example 6: minimum detection limit test
This experiment purpose is minimum check-up inducing degree of the detection reagent when testing clinical sample.
Using 1 reagent of experimental example, contrast agents (Ningbo Meikang biology), calibration object, blank solution (normal saline solution), just
Ordinary person's serum sample, low value sample.
Machine: 7170 automatic biochemistry analyzer of Hitachi.
Operating procedure: using normal saline solution or deionized water dissolving low value sample, then 50% be diluted to 5 points,
With zero point together each test sample 5 times, average value is calculated, SD numerical value is acquired.
As a result it parses: according to detection data, calculating SD numerical value and CV numerical value, calculate separately 1SD, 2SD, opened from the smallest
Begin, the numerical value of average value -2SD more than zero point average value+2SD be exactly reagent minimum check-up inducing degree.The display of table 2, this
When invention kit reagent measurement 1/16,1/8,1/4,1/2 serum of dilution, the numerical value of dilution average value -2SD is all larger than zero point
Average value+2SD shows that kit reagent minimum detection limit of the present invention at least can achieve 0.1mg/L.The display of table 3, Ningbo Meikang
Biological reagent measurement 1/16,1/8,1/4,1/2 serum of dilution, and compare serum average value -2SD and zero point average value+2SD is big
Small, the numerical value of 1/8 and 1/16 dilute serum average value -2SD is respectively less than zero point average value+2SD, shows that Ningbo Meikang biology tries
Agent lowest detection is limited to 0.5mg/L or so.
Table 2
Table 3
Test example 7: sensitivity experiment
This experiment purpose is absorbance change of the kit reagent when testing physiological saline and certain density serum
Value.
Using 1 reagent of experimental example, contrast agents (Ningbo Meikang), calibration object, blank solution, 0.9% normal saline solution,
Absorbance change value when the serum of concentration.
Machine: 7170 automatic biochemistry analyzer of Hitachi.
Operating procedure: using normal saline solution, low value sample, and each test sample 5 times calculates absorbance.
Table 4,5 shows that absorbance change is -0.0092 when reagent of the present invention measures physiological saline, theoretical concentration 10mg/
Absorbance change is 0.0157 when L serum sample；Absorbance change is -0.0047 when Ningbo Meikang reagent measures physiological saline,
Absorbance change is 0.0135 when theoretical concentration is 10mg/L serum sample.Table 4,5 respectively indicates invention kit reagent, Ningbo
The sensitivity of Meikang reagent, kit reagent sensitivity of the present invention, which is slightly better than, is better than Ningbo Meikang biological reagent.
Table 4
Table 5
In conclusion using a kind of dissociation protein agent in system of the invention, the antigen position in sample is exposed to greatest extent
Point increases the Percentage bound of antigen-antibody, and using a kind of more stable, amplified signal effect more preferably latex particle coating one
The specific α 1- microglobulin polyclonal antibody of kind, through these means, to realize the high sensitivity of reagent of the present invention；It solves
The problems such as α 1-MG reagent detection sensitivity is not high currently on the market, expensive.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (8)
1. a kind of α 1- microglobulin polyclonal antibody, which is characterized in that the α 1- microglobulin polyclonal antibody be by comprising
The method of following steps is prepared:
The preparation of S1, immunogene: being added protected amino acid chain 6-His and 10-Cys in recombinant alpha 1- microglobulin amino acid chain,
And 2 restriction enzyme site Hind III and BamH I are added, recombinant expression obtains antigen；
S2, immune animal: selecting rabbit immunization route, is for the first time 15 days with second of immunization interval using lymph node immunization, the
Interval 10 days after secondary；
S3, sero-fast purifying and preservation: it carries out affine using affinity column antagonistic Serum and purifies, after antibody desalination after purification
Glycerol is added to save.
2. α 1- microglobulin polyclonal antibody as described in claim 1, which is characterized in that step S1 specifically: in recombinant alpha
In 1- microglobulin amino acid chain, protected amino acid chain 6-His is added in the head of original acid chain, protected amino acid chain is added
Hind III digestion site is added in the tail portion of original acid chain in 10-Cys in the 6-His amino acid chain of head, in tail portion
BamH I restriction enzyme site is added in 10-Cys amino acid chain.
3. α 1- microglobulin polyclonal antibody as described in claim 1, which is characterized in that in step S3, the affinity column that uses
It is PBS buffer solution for Ago-Gel affinity column, purification solution.
4. a kind of α 1- microglobulin detection kit, which is characterized in that including R1 reagent and R2 reagent；The R1 reagent be containing
There is the phosphate buffer of dissociation protein agent, the R2 reagent is to contain more grams of α 1- microglobulin as described in claim 1
The buffer system of the grand coated sensitizing latex particle of antibody.
5. α 1- microglobulin detection kit as claimed in claim 4, which is characterized in that the R1 reagent include as follows at
Point: the phosphate buffer that pH is 7.0~7.6, concentration is 10~100mmol/L, concentration are the ethylenediamine tetraacetic of 5~20mmol/L
Sodium chloride that guanidine hydrochloride that acetic acid disodium, concentration are 200~500mmol/L, concentration are 10~50mmol/L, concentration is 10~
The preservative that the polyethylene glycol and volume fraction of 100mmol/L is 0.01%~0.05%.
6. α 1- microglobulin detection kit according to claim 4, which is characterized in that the R2 reagent includes as follows
Ingredient: the α that pH is 7.0~7.6, concentration is 10~100mmol/L phosphate buffer, concentration are 10~100ml/L
The coated sensitization latex particle of 1- microglobulin polyclonal antibody.
7. α 1- microglobulin detection kit according to claim 6, which is characterized in that more grams of the α 1- microglobulin
The grand coated sensitization latex particle of antibody is prepared by the inclusion of the method for following steps:
A1, α 1- microglobulin polyclonal antibody is added in the sensitization latex particle that partial size is 150~300nm, controls the α
The volume ratio of 1- microglobulin polyclonal antibody and sensitization latex particle is 0.5~1: 1, is incubated at room temperature 1.5-2.5 hours, obtains
Solution；
A2, carbodiimides is added in the solution, it is sub- controls the corresponding 5~10mg carbonization two of every milliliter of sensitization latex particle
Amine is incubated at room temperature 1.5-2.5 hours, obtains suspension；
A3, after being separated by solid-liquid separation the suspension, solid portion is taken to wash dispersion with phosphate buffer.
8. α 1- microglobulin detection kit according to claim 6 or 7, which is characterized in that the sensitization latex
Grain is poly- methylacrylic acid methyl esters microballoon or polystyrene microsphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910230425.4A CN109942703A (en) | 2019-03-25 | 2019-03-25 | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910230425.4A CN109942703A (en) | 2019-03-25 | 2019-03-25 | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109942703A true CN109942703A (en) | 2019-06-28 |
Family
ID=67011860
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910230425.4A Pending CN109942703A (en) | 2019-03-25 | 2019-03-25 | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109942703A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6153192A (en) * | 1990-08-06 | 2000-11-28 | Boehringer Mannheim Gmbh | Peptides with a characteristic antigenic determinant of α1-microglobulin |
| CN105699655A (en) * | 2016-01-26 | 2016-06-22 | 宁波天康生物科技有限公司 | Alpha 1-microglobulin detection kit |
| CN106324251A (en) * | 2016-08-08 | 2017-01-11 | 上海睿康生物科技有限公司 | Preparation method of small-fragment BMG antibody and beta2-microglobulin detection kit |
| CN108659132A (en) * | 2017-03-29 | 2018-10-16 | 周珂 | A kind of combined protein prepares the methods and applications of anti-human cystatin C polyclonal antibody |
-
2019
- 2019-03-25 CN CN201910230425.4A patent/CN109942703A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6153192A (en) * | 1990-08-06 | 2000-11-28 | Boehringer Mannheim Gmbh | Peptides with a characteristic antigenic determinant of α1-microglobulin |
| CN105699655A (en) * | 2016-01-26 | 2016-06-22 | 宁波天康生物科技有限公司 | Alpha 1-microglobulin detection kit |
| CN106324251A (en) * | 2016-08-08 | 2017-01-11 | 上海睿康生物科技有限公司 | Preparation method of small-fragment BMG antibody and beta2-microglobulin detection kit |
| CN108659132A (en) * | 2017-03-29 | 2018-10-16 | 周珂 | A kind of combined protein prepares the methods and applications of anti-human cystatin C polyclonal antibody |
Non-Patent Citations (2)
| Title |
|---|
| BOUIC, P等: "LOCALIZATION OF ALPHA-1-MICROGLOBULIN (HC PROTEIN) IN NORMAL HUMAN-TISSUES - AN IMMUNOHISTOCHEMICAL STUDY USING MONOCLONAL-ANTIBODIES", 《HISTOCHEMICAL JOURNAL》 * |
| 蒋泽军等: "胶乳增强免疫比浊法检测尿液中α 1 -微球蛋白的含量", 《中国医学装备》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108362688B (en) | Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles | |
| CN101769932B (en) | Full-range C-reactive protein detection kit | |
| CN102628865B (en) | Latex enhanced immunoturbidimetry kit for detection of myoglobin content | |
| CN109085333A (en) | A kind of preparation, detection kit and the preparation method of rheumatoid factor antigen | |
| CN108414748A (en) | A kind of test strip and detection method of THSD7A antibody | |
| CN105467131B (en) | The kit of the microglobulins of α 1 in urine and serum sample can be detected simultaneously | |
| CN104215770A (en) | Two-particle-based retinol binding protein detection kit | |
| CN107942069A (en) | A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof | |
| CN105510604A (en) | Method for improving sensitivity and linearity of latex reagent | |
| CN101310186B (en) | Method for determining antigen and kit therefor | |
| CN101699287B (en) | Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof | |
| CN104198725A (en) | Anti-cyclic citrullinated peptide (CCP) antibody detection kit | |
| CN106093426A (en) | A kind of test kit measuring bladder chalone C and preparation method thereof | |
| CN102680698A (en) | Neutrophil gelatinase-associated lipocalin (NGAL) assay kit (latex-enhanced immunoturbidimetry) | |
| CN106324251B (en) | The preparation method and beta 2-microglobulin detecting kit of small fragment BMG antibody | |
| CN102421799B (en) | Novel monoclonal antibody for analyzing high-molecular weight adiponectin and utilization of same | |
| CN102590497A (en) | Cysteine protease inhibitor C test kit | |
| CN102662064A (en) | Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof | |
| CN105486875A (en) | Retinol conjugated protein detection kit | |
| CN106290910A (en) | The test kit of a kind of omnidistance C reactive protein and detection method thereof | |
| CN114295827A (en) | Magnetic particle acridinium ester chemiluminescence detection kit and preparation method and application thereof | |
| CN109142743A (en) | A kind of multinomial detection kit for exempting from liver antibody certainly | |
| CN111239404B (en) | Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample | |
| CN111239403B (en) | Beta 2 microglobulin latex enhanced immunoturbidimetry kit and application | |
| CN109942703A (en) | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190628 |
|
| RJ01 | Rejection of invention patent application after publication |