CN106324251A - Preparation method of small-fragment BMG antibody and beta2-microglobulin detection kit - Google Patents
Preparation method of small-fragment BMG antibody and beta2-microglobulin detection kit Download PDFInfo
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- CN106324251A CN106324251A CN201610643873.3A CN201610643873A CN106324251A CN 106324251 A CN106324251 A CN 106324251A CN 201610643873 A CN201610643873 A CN 201610643873A CN 106324251 A CN106324251 A CN 106324251A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
Abstract
The invention provides a preparation method of a small-fragment BMG antibody and a high-sensitivity beta2-microglobulin detection kit comprising the antibody. The kit is composed of three parts, namely a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 is a phosphate buffer system which contains a protein dissociation agent, the reagent R2 is a buffer solution which contains a certain quantity of latex particles coated with the small-fragment BMG antibody, and the calibrator is mainly a bovine serum matrix. By the adoption of the protein dissociation agent, an antigen site in a sample is exposed to the maximum extent, and the combination rate of an antigen and the antibody is increased; the latex particles which are more stable and ideal in signal amplification effect are coated with the special small-fragment BMG antibody; by the two means, high sensitivity of the kit is realized; the problems that BMG reagents on the current market are not high in sensitivity, high in price and the like are solved.
Description
Technical field
The present invention relates to the preparation method of a kind of small fragment BMG antibody and the B2M detection examination containing this antibody
Agent box, especially a kind of medical immunology in-vitro diagnosis field.
Background technology
B2M is Sweden scholar Berggard in nineteen sixty-eight first from the Wilson ' s that can cause tamm-Horsfall protein urine
The twenty-four-hour urine of the patients such as sick and chronic cadium poisoning separates and finds, be molecular weight be the serum proteins of 11815Da, its molecule
Including a pair disulfide bond, the most sugary, serum protein electrophoresis is positioned at β 2 region.
The synthesis of B2M is more stable, and its average generating rate is estimated as 2.4mg/kg/day under normal circumstances.
The metabolism of this albumen only relies upon kidney, has cell surface to come off or is released into the B2M of blood, from the free mistake of glomerule
Filter, 99.9% heavily absorbs also catabolism, no longer reflux at nearly kidney end tubule enters blood, thus serumβ 2-is micro-under normal circumstances
Globulin remains maintenance level.
B2M molecular weight is little, it is thus possible to freely filter from glomerule, and B2M except by
The rarely catabolism outside kidney, its generation constant rate in vivo, age, sex, body muscular tissue outside renal excretion
Do not interfere with its serum levels content.In the case of B2M generates and does not increases in vivo, serum beta-2-microglobulin water
Flat rising is the pole sensitive indexes of reflection Glomerular filtration function injury, and than serum urea nitrogen, creatinine to glomerular filtration
Evaluation more sensitive.Serum beta-2-microglobulin level raises, but Urine β2-microglobulin level keeps normal, and this is due to kidney
The filtering function damage of dirty glomerule is caused, usually sees acute, chronic nephritis, renal failure etc..Proximal tubular is β 2-
Unique place that microglobulin processes in vivo, Urine β2-microglobulin is the specific parameters evaluating proximal tubular function, when closely
End renal tubules slight impaired time, Urine β2-microglobulin can substantially increase, it is possible to accurately reflect proximal tubular damage degree.
Serum beta-2-microglobulin is normal and Urine β2-microglobulin raises that to be common in congenital proximal convoluted tubule functional defect, Fanconi comprehensive
Levy, acute tubular necrosis, tubulointerstitial injury, Wilson ' s are sick.
The detection method of B2M has RIA, ELISA, TRFIA, Microparticle enzyme immunoassay, capillary electrophoresis
Immunoassay, micro-fluid control chip electrophoretic immunization, immunoturbidimetry etc..The β 2-carried out on automatic clinical chemistry analyzer is micro-
Globulin immunoturbidimetry measures, and experimenter will not cause radioactive pollution, and stability, precision and accuracy performance are good
Good, can be in routine clinical mensuration and Clinical detection requirement can be met.At the automatic clinical chemistry analyzer that Clinical Laboratory section office are indispensable
Upper use, easy to detect, it is possible to fast to send examining report rapidly, and light Medium hemolysis and slight yellow cellulitis, lipidemia are had
Certain capacity of resisting disturbance.Generally there is the problem that sensitivity is low in B2M detectable in the market, import tries
Agent price is the most costly.
Summary of the invention
For defect of the prior art, it is an object of the invention to provide a kind of small fragment BMG antibody preparation method and
Containing the beta 2-microglobulin detecting kit of this antibody, this test kit is for detecting the content of BMG in serum, to reach operation
Easy, highly sensitive, specificity is good, quickly, measurement result purpose accurately and reliably.
The present invention is achieved by the following technical solutions:
First aspect, the invention provides the preparation method of a kind of small fragment BMG antibody, and it comprises the steps of
With protease, BMG antibody is hydrolyzed, obtains Fab fragment；
Adding dithiothreitol, DTT under conditions of PH8.5, room temperature effect opens the disulfide bond of described Fab fragment for 5 hours, cruelly
After exposing sulfydryl, carry out coupling with artificial polypeptide, obtain described small fragment BMG antibody.
The small fragment BMG antibody of preparation in the present invention, is to eliminate Fc fragment in antibody, resisting by polypeptide coupling
Body.
Preferably, one during described protease is pepsin, papain, subtilisin or
Several.
Preferably, one or more during described artificial polypeptide is tripeptides, tetrapeptide, pentapeptide.
Second aspect, present invention also offers the detection examination of a kind of B2M containing aforesaid small fragment BMG antibody
Agent box, it is characterised in that include that R1 reagent, R2 reagent and calibration object, described R1 reagent are the phosphate containing the protein agent that dissociates
Buffer system, described reagent R2 is the buffer system containing the latex particle being coated described small fragment BMG antibody, described calibration
Product include the Ox blood serum substrate of 5 different BMG concentration.
Preferably, described R1 reagent comprise following composition: pH be 7.0～7.6, concentration be 10～100mmol/L
Phosphate buffer, concentration is 200～500mmol/L to dissociate protein agent, and concentration is 5～20mmol/L ethylenediaminetetraacetic acid two
Sodium, concentration is 10～50mmol/L sodium chloride and preservative that volume fraction is 0.01%～0.05%.
Preferably, the protein agent that dissociates described in is one or more in carbamide, triton x-100, guanidine hydrochloride；Institute
Stating preservative is Proclin 300.
Preferably, described R2 reagent comprise following composition: pH be 7.0～7.6, concentration be 10～100mmol/L
Phosphate buffer, small fragment BMG antibody coated sensitization latex particle.
Preferably, described sensitization latex particle is poly (methyl methacrylate) micro-sphere.
Preferably, the preparation method of described small fragment BMG antibody coated sensitization latex particle includes walking as follows
Rapid:
S1: small fragment BMG antibody is added in the emulsion of sensitization latex particle that particle diameter is 100～200nm, controls small pieces
Section BMG antibodies Antibodies is 0.2～1:1 with the volume ratio of latex particle, and incubated at room 1 hour obtains solution；
S2: added by carbodiimides in step S1 gained solution, controls to add in every milliliter of latex particle 5mg carbonization two
Imines, incubated at room 2 hours, obtain suspension；
S3: after step S2 gained suspension is carried out solid-liquid separation, takes the washing dispersion of solid portion phosphate buffer
?.
Preferably, the BMG concentration of 5 described Ox blood serum substrate calibration objects be respectively 1.25mg/L, 2.5mg/L,
5mg/L, 10mg/L and 20mg/L.
Compared with prior art, the present invention has a following beneficial effect:
1, the present invention is by the preparation method of a kind of BMG antibody, obtains a kind of special small fragment coupling BMG antibody, should
Antibody stability is good, high specificity；
2, in reagent R1, add albumen to dissociate agent, make the BMG antigen site in sample fully be exposed, improve antigen
Antibodies rate.Meanwhile, during being coated with small fragment coupling BMG antibody, the poly-first more more stable than polystyrene microsphere is used
Base benzene e pioic acid methyl ester microsphere, plays the effect that signal amplifies, makes reagent sensitivity be greatly improved, at sample concentration as little as
Still can detect that result during 0.05mg/L, and the lowest detection of the reagent of prior art is limited to 0.2mg/L；
3, test kit of the present invention compared with prior art sensitivity significantly improves, when measuring normal saline such as reagent of the present invention
Absorbance is changed to 13.4 (1/10000A), and when theoretical concentration is 1mg/L serum sample, absorbance is changed to 289.2 (1/
10000A)；Absorbance change generally 22.2 (1/10000A) when the reagent of prior art measures normal saline, theoretical concentration is
During 1mg/L serum sample, absorbance is changed to 130.2 (1/10000A)；Meanwhile, R1 does not adds albumen dissociate agent reagent measure
Absorbance during normal saline is changed to 10.8 (1/10000A), and when theoretical concentration is 1mg/L serum sample, absorbance is changed to
111.4(1/10000A).Show in reagent add albumen dissociate agent to improve reagent sensitivity effect obvious.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the standard curve of the BMG reference standard of 5 kinds of different contents；
Fig. 2 be the BMG reagent being respectively adopted reagent of the present invention and Roche Holding Ag of Germany to 50 parts of human serums (comprise normal and
Monstrosity) measure structure correlation analysis figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in the technology of this area
Personnel are further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into the present invention
Protection domain.
Embodiment 1
The present embodiment provides the preparation method of a kind of small fragment BMG antibody, specifically comprises the following steps that
With protease, BMG antibody is hydrolyzed, obtains Fab fragment；
Adding dithiothreitol, DTT under conditions of PH8.5, room temperature effect opens the disulfide bond of described Fab fragment for 5 hours, cruelly
After exposing sulfydryl, carry out coupling with artificial polypeptide, obtain described small fragment BMG antibody.
Embodiment 2
The present embodiment provides a kind of highly sensitive BMG detection kit, forms as follows:
1. reagent R1 is:
2. reagent R2 preparation process is:
Step 1: BMG antibody is added in the latex particle that particle diameter is 100nm, incubated at room 1 hour, wherein antibody and breast
The volume ratio of micelle is 0.2:1；
Step 2: being added by carbodiimides in step 1 gained solution, incubated at room 2 hours, in every milliliter of latex particle
Add 5mg carbodiimides；
Step 3: after centrifugal for step 2 gained aaerosol solution, take precipitation 10mmol/L phosphate buffer (PH7.0) and wash
Wash and disperse and get final product.
The BMG detection kit that the present embodiment describes, it is adaptable to various types of full automatic biochemical apparatus is complete with Hitachi 7170
As a example by automatic biochemical analyzer, its operation is such as table 1.Analysis method: Two point end assay, the i.e. consumption of reagent R1, R2 be respectively 240ul and
60ul, sample size 3ul (serum)/12ul (urine)；240ul reagent R1 adds 3ul serum sample or 12ul urine specimen in 37
After DEG C hatching 5min, add 60ul reagent R2, hatch 30s for 37 DEG C, read absorbance, after hatching 3min the most again, read another
Point；Detection dominant wavelength is 546nm.
Use this reagent and said determination method, the BMG of 5 kinds of different contents that employing Hitachi 7170 biochemistry analyzer records
The curve (as shown in Figure 1) of calibration object (making by oneself), each point represents the reference calibrations product of a content, and wherein X-axis represents that BMG contains
Amount (mg/L)；Y-axis represents absorbance.
Table 1
Embodiment 3
The present embodiment provides a kind of highly sensitive BMG detection kit, forms as follows:
1. reagent R1 is:
2. reagent R2 preparation process is:
Step 1: BMG antibody is added in the latex particle that particle diameter is 200nm, incubated at room 1 hour, wherein antibody and breast
The volume ratio of micelle is 1:1；
Step 2: being added by carbodiimides in step 1 gained solution, incubated at room 2 hours, in every milliliter of latex particle
Add 5mg carbodiimides；
Step 3: after centrifugal for step 2 gained aaerosol solution, take precipitation 100mmol/L phosphate buffer (PH7..4)
Washing disperses and get final product.
Test kit concrete operation method is with embodiment 1.
Embodiment 4:
The present embodiment provides a kind of highly sensitive BMG detection kit, forms as follows:
1. reagent R1 is:
2. reagent R2 preparation process is:
Step 1: BMG antibody is added in the latex particle that particle diameter is 150nm, incubated at room 1 hour, wherein antibody and breast
The volume ratio of micelle is 0.5:1；
Step 2: being added by carbodiimides in step 1 gained solution, incubated at room 2 hours, in every milliliter of latex particle
Add 5mg carbodiimides.
Step 3: after centrifugal for step 2 gained aaerosol solution, take precipitation 60mmol/L phosphate buffer (PH7.6) and wash
Wash and disperse and get final product.
Test kit concrete operation method is with embodiment 1.
Embodiment 5: the correlation test of detectable
Use this law invention test kit (concrete formula is with embodiment 1) and the BMG reagent of Roche Holding Ag of contrast agents Germany,
Use automatic 7170 automatic clinical chemistry analyzers that 50 parts of human serums (including normal and monstrosity) are entered by each autoregressive parameter simultaneously
Row measures, and measured value is carried out correlation analysis.Fig. 2, X is seen according to being measured measurement result with the parameter in above-mentioned " table 1 ",
Y-axis is measured value (the content mg/L of BMG),
Being found out by the result of Fig. 2, the phase relation of two kinds of reagent is R2=0.9984, regression equation is y=0.9876x+
0.0985.It is good that result shows that this reagent and import reagent measure patients serum's dependency, has good specificity with accurate
Property.Carry out additionally, above experiment is 7170 full automatic biochemical apparatus using Hitachi, Ltd to manufacture, but the reagent of the present invention does not limits
In above-mentioned instrument, apply also for other full-automatic or semi-automatic biochemical analyzers.
Test example 6: lowest detectable limit is tested
This experiment purpose is the detectable minimum check-up inducing degree when testing clinical sample.
Use experimental example 1 reagent, contrast agents (power of converging is biological), calibration object, blank solution (normal saline solution), normal person
Serum sample, low value sample.
Machine: Hitachi 7170 automatic biochemistry analyzer.
Operating procedure: use normal saline solution or deionized water dissolving low value sample, then 50% be diluted to 5 points,
Each test sample 5 times together with zero point, calculate meansigma methods, try to achieve SD numerical value.
Result resolves: according to detection data, calculates SD numerical value and CV numerical value, calculates 1SD, 2SD respectively, opens from minimum
Beginning, the numerical value of its meansigma methods-2SD is exactly the minimum check-up inducing degree of reagent at zero point more than meansigma methods+2SD.Table 2 shows, this
Invention kit reagent measure dilution 1/16,1/8,1/4,1/2 serum time, the numerical value of dilution meansigma methods-2SD is all higher than zero point
Meansigma methods+2SD, shows that kit reagent lowest detectable limit of the present invention at least can reach 0.05mg/L.Table 3 shows, power of converging is raw
Thing reagent measures dilution 1/16,1/8,1/4,1/2 serum, and compares serum meansigma methods-2SD and zero point meansigma methods+2SD size,
The numerical value of 1/8 and 1/16 dilute serum meansigma methods-2SD is respectively less than zero point meansigma methods+2SD, shows the remittance minimum inspection of power biological reagent
Survey is limited to about 0.2mg/L.
Table 2
Table 3
Test example 7: sensitivity experiment
This experiment purpose is the kit reagent absorbance when testing normal saline and certain density management serum
Changing value.
Use experimental example 1 reagent, contrast agents (power of converging is biological), calibration object, blank solution, the normal saline solution of 0.9%,
Concentration management serum time absorbance changing value.
Machine: Hitachi 7170 automatic biochemistry analyzer.
Operating procedure: use normal saline solution, low value sample, each test sample 5 times, calculate absorbance.
Table 4,5,6 shows, when reagent of the present invention measures normal saline, absorbance is changed to 13.4 (1/10000A), theoretical dense
Degree is changed to 289.2 (1/10000A) for absorbance during 1mg/L serum sample；Extinction when remittance power biological reagent measures normal saline
Degree is changed to 22.2 (1/10000A), and when theoretical concentration is 1mg/L serum sample, absorbance is changed to 130.2 (1/10000A)；
R1 does not adds albumen dissociate agent reagent measure normal saline time absorbance be changed to 10.8 (1/10000A), theoretical concentration is
During 1mg/L serum sample, absorbance is changed to 111.4 (1/10000A).Table 4,5,6 represents invention kit reagent, power of converging respectively
Biological reagent and R1 do not add the sensitivity of the reagent of the protein agent that dissociates, shows not add the dissociate reagent sensitivity of agent of albumen bright
Aobvious less than reagent of the present invention, and kit reagent sensitivity of the present invention is significantly better than remittance power biological reagent.
Table 4
Table 5
Table 6
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Claims (10)
1. the preparation method of a small fragment BMG antibody, it is characterised in that comprise the steps of
With protease, BMG antibody is hydrolyzed, obtains Fab fragment；
Open the disulfide bond of described Fab fragment with dithiothreitol, DTT, after exposing sulfydryl, carry out coupling with artificial polypeptide, obtain
Described small fragment BMG antibody.
2. the preparation method of small fragment BMG antibody as claimed in claim 1, it is characterised in that described protease is pepsin
One or more in enzyme, papain, subtilisin.
3. the preparation method of small fragment BMG antibody as claimed in claim 1, it is characterised in that described artificial polypeptide be tripeptides,
One or more in tetrapeptide, pentapeptide.
4. the beta 2-microglobulin detecting kit containing the small fragment BMG antibody described in claim 1, it is characterised in that
Including R1 reagent, R2 reagent and calibration object, described R1 reagent is the phosphate buffer containing the protein agent that dissociates, described reagent
R2 is the buffer system containing the latex particle being coated described small fragment BMG antibody, and described calibration object includes 5 different BMG
The Ox blood serum substrate of concentration.
5. beta 2-microglobulin detecting kit as claimed in claim 4, it is characterised in that described R1 reagent comprises following one-tenth
Point: pH is 7.0～7.6, concentration is 10～100mmol/L phosphate buffers, and concentration is 200～500mmol/L to dissociate albumen
Agent, concentration is 5～20mmol/L disodiumedetates, and concentration is 10～50mmol/L sodium chloride and volume fraction is
The preservative of 0.01%～0.05%.
The highly sensitive beta 2-microglobulin detecting kit of one the most according to claim 5, it is characterised in that described
The protein agent that dissociates is one or more in carbamide, triton x-100, guanidine hydrochloride；Described preservative is Proclin300.
The highly sensitive beta 2-microglobulin detecting kit of one the most according to claim 4, it is characterised in that described
R2 reagent comprise following composition: pH be 7.0～7.6, concentration be 10～100mmol/L phosphate buffers, small fragment BMG antibody
Coated sensitization latex particle.
The highly sensitive beta 2-microglobulin detecting kit of one the most according to claim 7, it is characterised in that described
Sensitization latex particle be poly (methyl methacrylate) micro-sphere.
The highly sensitive beta 2-microglobulin detecting kit of one the most according to claim 7, it is characterised in that described
The preparation method of small fragment BMG antibody coated sensitization latex particle comprises the steps:
S1: small fragment BMG antibody is added in the emulsion of sensitization latex particle that particle diameter is 100～200nm, controls small fragment
BMG antibodies Antibodies is 0.2～1:1 with the volume ratio of latex particle, and incubated at room 1 hour obtains solution；
S2: added by carbodiimides in step S1 gained solution, controls to add 5mg carbonization two in every milliliter of latex particle sub-
Amine, incubated at room 2 hours, obtain suspension；
S3: after step S2 gained suspension is carried out solid-liquid separation, takes the washing of solid portion phosphate buffer and disperses.
The highly sensitive beta 2-microglobulin detecting kit of one the most according to claim 4, it is characterised in that 5
The BMG concentration of described Ox blood serum substrate calibration object is respectively 1.25mg/L, 2.5mg/L, 5mg/L, 10mg/L and 20mg/L.
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| CN108445224A (en) * | 2018-02-26 | 2018-08-24 | 王贤俊 | A kind of cardic fatty acid binding protein antibody fragment compounded latex particle and preparation method thereof |
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| CN109942703A (en) * | 2019-03-25 | 2019-06-28 | 上海睿康生物科技有限公司 | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
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| CN109942703A (en) * | 2019-03-25 | 2019-06-28 | 上海睿康生物科技有限公司 | The preparation and α 1- microglobulin detection kit of α 1- microglobulin polyclonal antibody |
| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
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