CN106324251B - The preparation method and beta 2-microglobulin detecting kit of small fragment BMG antibody - Google Patents
The preparation method and beta 2-microglobulin detecting kit of small fragment BMG antibody Download PDFInfo
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- CN106324251B CN106324251B CN201610643873.3A CN201610643873A CN106324251B CN 106324251 B CN106324251 B CN 106324251B CN 201610643873 A CN201610643873 A CN 201610643873A CN 106324251 B CN106324251 B CN 106324251B
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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
Highly sensitive beta 2-microglobulin detecting kit the present invention provides a kind of preparation method of small fragment BMG antibody and containing the antibody.The kit is made of R1 reagent, R2 reagent and calibration object three parts, wherein R1 reagent is containing a kind of phosphate buffer for dissociating protein agent, R2 reagent is the buffer containing a certain amount of latex particle for being coated with small fragment BMG antibody, and calibration object is mainly cow's serum matrix.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 special small fragment BMG antibody is coated with using a kind of more stable, amplified signal effect more preferably latex particle, by the two means, to realize the high sensitivity of reagent of the present invention.Solves currently on the market the problems such as BMG reagent detection sensitivity is not high, expensive.
Description
Technical field
The present invention relates to a kind of preparation method of small fragment BMG antibody and the detection of the β2-microglobulin containing the antibody tries
Agent box, especially a kind of medical immunology in-vitro diagnosis field.
Background technique
β2-microglobulin is Sweden scholar Berggard in nineteen sixty-eight first from the Wilson ' s that tamm-Horsfall protein can be caused to urinate
Discovery is separated in the twenty-four-hour urine of patients such as disease and chronic cadium poisoning, is the serum proteins that molecular weight is 11815Da, molecule
A pair of of disulfide bond is included, without sugar, serum protein electrophoresis is located at 2 region β.
The synthesis of β2-microglobulin is more stable, its average generating rate is estimated as 2.4mg/kg/day under normal circumstances.
The metabolism of the albumen only relies upon kidney, has cell surface to fall off or be released into the β2-microglobulin of blood, from the free mistake of glomerulus
Filter, 99.9% in nearly kidney end tubule reabsorption and catabolism, and no longer reflux enters blood, thus serumβ 2- is micro- under normal circumstances
Globulin remains maintenance level.
β2-microglobulin molecular weight is small, it is thus possible to freely filtered from glomerulus, and β2-microglobulin in addition to by
The rarely catabolism outside kidney outside kidney excretion, generation constant rate in vivo, age, gender, body musculature
It will not influence its serum levels content.β2-microglobulin generates in not increased situation in vivo, serum beta-2-microglobulin water
Flat raising is the pole sensitive indexes for reflecting Glomerular filtration function injury, and than serum urea nitrogen, creatinine to glomerular filtration
Evaluation it is more sensitive.Serum beta-2-microglobulin level increases, however Urine β2- microglobulin level keeps normal, this is because kidney
Caused by the filtering function damage of dirty glomerulus, acute, chronic nephritis, renal failure etc. are usually seen.Proximal tubular is β 2-
Unique place that microglobulin is handled in vivo, Urine β2- microglobulin is the specific parameters for evaluating proximal tubular function, when close
When renal tubule being held slightly to be damaged, Urine β2- microglobulin can be obviously increased, and can accurately reflect the degree of proximal tubular damage.
Serum beta-2-microglobulin is normal and Urine β2- microglobulin raising is common in congenital proximal convoluted tubule functional defect, Fanconi synthesis
Sign, acute tubular necrosis, tubulointerstitial injury, Wilson ' s disease etc..
The detection method of β2-microglobulin 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-
The measurement of globulin immunoturbidimetry, will not cause radioactive pollution, stability, precision and accuracy performance are good to experimenter
It is good, in routine clinical measurement and clinical detection requirement can be able to satisfy.In the automatic clinical chemistry analyzer of clinical examination department indispensability
Upper use, it is easy to detect, examining report fast can be rapidly issued, and have to light Medium hemolysis and slight yellow subcutaneous ulcer, piarhemia
Certain anti-interference ability.The low problem of the generally existing sensitivity of β2-microglobulin detection reagent currently on the market, import examination
Agent price is again more expensive.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of preparation method of small fragment BMG antibody and
Beta 2-microglobulin detecting kit containing the antibody, the kit are used to detect the content of BMG in serum, to reach operation
Simplicity, high sensitivity, specificity are good, quickly, measurement result accurately and reliably purpose.
The present invention is achieved by the following technical solutions:
In a first aspect, it includes following steps the present invention provides a kind of preparation method of small fragment BMG antibody:
BMG antibody is hydrolyzed with protease, obtains Fab segment；
Dithiothreitol (DTT) is added under conditions of PH8.5, room temperature acts on the disulfide bond for opening the Fab segment for 5 hours, cruelly
After exposing sulfydryl, manually polypeptide is coupled, and obtains the small fragment BMG antibody.
The small fragment BMG antibody prepared in the present invention is anti-made of eliminating Fc segment in antibody, being coupled by polypeptide
Body.
Preferably, the protease be one of pepsin, papain, subtilopeptidase A or
It is several.
Preferably, the artificial polypeptide is one or more of tripeptides, tetrapeptide, pentapeptide.
Second aspect, the present invention also provides a kind of, and the β2-microglobulin containing small fragment BMG antibody above-mentioned detects examination
Agent box, which is characterized in that including R1 reagent, R2 reagent and calibration object, the R1 reagent is the phosphate containing dissociation protein agent
Buffer system, the reagent R2 are the buffer system containing the latex particle for being coated with the small fragment BMG antibody, the calibration
Product include the cow's serum matrix of 5 difference BMG concentration.
Preferably, the R1 reagent includes following ingredient: pH is 7.0~7.6, concentration is 10~100mmol/L
Phosphate buffer, concentration are that 200~500mmol/L dissociates protein agent, and concentration is 5~20mmol/L ethylenediamine tetra-acetic acid two
Sodium, the preservative that concentration is 10~50mmol/L sodium chloride and volume fraction is 0.01%~0.05%.
Preferably, the dissociation protein agent is one or more of urea, triton x-100, guanidine hydrochloride；Institute
Stating preservative is Proclin 300.
Preferably, the R2 reagent includes following ingredient: pH is 7.0~7.6, concentration is 10~100mmol/L
Phosphate buffer, the coated sensitization latex particle of small fragment BMG antibody.
Preferably, the sensitization latex particle is poly (methyl methacrylate) micro-sphere.
Preferably, the preparation method of the coated sensitization latex particle of the small fragment BMG antibody includes following step
It is rapid:
S1: small fragment BMG antibody is added in the lotion for the sensitization latex particle that partial size is 100~200nm, controls small pieces
The volume ratio of section BMG antibodies Antibodies and latex particle is 0.2~1:1, is incubated at room temperature 1 hour, obtains solution；
S2: carbodiimides is added in step S1 acquired solution, controls and 5mg carbonization two is added in every milliliter of latex particle
Imines is incubated at room temperature 2 hours, obtains suspension；
S3: after suspension obtained by step S2 is separated by solid-liquid separation, solid portion is taken to wash dispersion with phosphate buffer
?.
Preferably, the BMG concentration of 5 cow's serum matrix calibration objects be respectively 1.25mg/L, 2.5mg/L,
5mg/L, 10mg/L and 20mg/L.
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 small fragment coupling BMG antibody by a kind of preparation method of BMG antibody, should
Antibody stability is good, high specificity；
2, albumen is added in reagent R1 and dissociates agent, expose the BMG antigen site in sample sufficiently, improve antigen
Antibody Percentage bound.Meanwhile the poly- first more more stable than polystyrene microsphere is used during with small fragment coupling BMG antibody coating
Base benzene e pioic acid methyl ester microballoon, play the role of signal amplification, greatly improve reagent sensitivity, sample concentration down to
It is still can detect that when 0.05mg/L as a result, and the lowest detection of the reagent of the prior art is limited to 0.2mg/L；
3, sensitivity significantly improves kit of the present invention compared with prior art, when such as reagent of the present invention measures physiological saline
Absorbance change is 13.4 (1/10000A), and absorbance change is 289.2 (1/ when theoretical concentration is 1mg/L serum sample
10000A)；Absorbance change is generally 22.2 (1/10000A) when the reagent of the prior art measures physiological saline, and theoretical concentration is
Absorbance change is 130.2 (1/10000A) when 1mg/L serum sample；Meanwhile the reagent measurement of albumen dissociation agent is not added in R1
Absorbance change when physiological saline is 10.8 (1/10000A), and absorbance change is when theoretical concentration is 1mg/L serum sample
111.4(1/10000A).Show that albumen dissociation agent is added in reagent to be acted on obviously reagent sensitivity is improved.
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 BMG reference standard of 5 kinds of different contents；
Fig. 2 be respectively adopted the BMG reagent of reagent of the present invention and German Roche Holding Ag 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 preparation methods of small fragment BMG antibody, the specific steps are as follows:
BMG antibody is hydrolyzed with protease, obtains Fab segment；
Dithiothreitol (DTT) is added under conditions of PH8.5, room temperature acts on the disulfide bond for opening the Fab segment for 5 hours, cruelly
After exposing sulfydryl, manually polypeptide is coupled, and obtains the small fragment BMG antibody.
Embodiment 2
The present embodiment provides a kind of BMG detection kits of high sensitivity, form as follows:
1. reagent R1 are as follows:
2. reagent R2 preparation process are as follows:
Step 1: BMG antibody being added in the latex particle that partial size is 100nm, incubation at room temperature 1 hour, wherein antibody and cream
The volume ratio of micelle is 0.2:1；
Step 2: carbodiimides being added in step 1 acquired solution, is incubated at room temperature 2 hours, in every milliliter of latex particle
5mg carbodiimides is added；
Step 3: after the centrifugation of step 2 gained aaerosol solution, precipitating being taken to be washed with 10mmol/L phosphate buffer (PH7.0)
It washs and disperses to obtain the final product.
The BMG detection kit of the present embodiment description, is suitable for various types of full automatic biochemical apparatus, complete with Hitachi 7170
For automatic biochemical analyzer, operation such as table 1.Analysis method: Two point end assay, i.e. the dosage of reagent R1, R2 be respectively 240ul and
60ul, sample size 3ul (serum)/12ul (urine)；3ul serum sample or 12ul urine specimen is added in 37 in 240ul reagent R1
After DEG C being incubated for 5min, be added 60ul reagent R2,37 DEG C of incubations 30s, reading absorbance, be then incubated for again after 3min read it is another
Point；Detection dominant wavelength is 546nm.
Using this reagent and said determination method, using the BMG for 5 kinds of different contents that 7170 Biochemical Analyzer of Hitachi measures
The curve (as shown in Figure 1) of calibration object (self-control), each point represent the reference calibrations product an of content, and wherein X-axis indicates that BMG contains
It measures (mg/L)；Y-axis indicates absorbance.
Table 1
Embodiment 3
The present embodiment provides a kind of BMG detection kits of high sensitivity, form as follows:
1. reagent R1 are as follows:
2. reagent R2 preparation process are as follows:
Step 1: BMG antibody being added in the latex particle that partial size is 200nm, incubation at room temperature 1 hour, wherein antibody and cream
The volume ratio of micelle is 1:1；
Step 2: carbodiimides being added in step 1 acquired solution, is incubated at room temperature 2 hours, in every milliliter of latex particle
5mg carbodiimides is added；
Step 3: after the centrifugation of step 2 gained aaerosol solution, taking precipitating with 100mmol/L phosphate buffer (PH7..4)
Washing disperses to obtain the final product.
Kit concrete operation method is the same as embodiment 1.
Embodiment 4:
The present embodiment provides a kind of BMG detection kits of high sensitivity, form as follows:
1. reagent R1 are as follows:
2. reagent R2 preparation process are as follows:
Step 1: BMG antibody being added in the latex particle that partial size is 150nm, incubation at room temperature 1 hour, wherein antibody and cream
The volume ratio of micelle is 0.5:1；
Step 2: carbodiimides being added in step 1 acquired solution, is incubated at room temperature 2 hours, in every milliliter of latex particle
5mg carbodiimides is added.
Step 3: after the centrifugation of step 2 gained aaerosol solution, precipitating being taken to be washed with 60mmol/L phosphate buffer (PH7.6)
It washs and disperses to obtain the final product.
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.Fig. 2, X are seen according to measurement result is measured with the parameter in above-mentioned " table 1 ",
Y-axis is measured value (the content mg/L of BMG),
Found out by the result of Fig. 2, the phase relation of two kinds of reagents is R2=0.9984, regression equation y=0.9876x+
0.0985.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 (power of converging is biological), calibration object, blank solution (normal saline solution), normal person
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 0.05mg/L.Table 3 shows that power of converging is raw
Object 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 size,
The numerical value of 1/8 and 1/16 dilute serum average value -2SD is respectively less than zero point average value+2SD, shows the remittance minimum inspection of power biological reagent
Survey is limited to 0.2mg/L or so.
Table 2
Table 3
Test example 7: sensitivity experiment
This experiment purpose is absorbance of the kit reagent when testing physiological saline and certain density management serum
Changing value.
Using 1 reagent of experimental example, contrast agents (converge with power biology), calibration object, blank solution, 0.9% normal saline solution,
Absorbance change value when the management 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 4,5,6 shows that absorbance change is 13.4 (1/10000A) when reagent of the present invention measures physiological saline, theoretical dense
Absorbance change is 289.2 (1/10000A) when degree is 1mg/L serum sample；Extinction when the power biological reagent that converges measures physiological saline
Degree variation is 22.2 (1/10000A), and absorbance change is 130.2 (1/10000A) when theoretical concentration is 1mg/L serum sample；
The absorbance change when reagent of albumen dissociation agent not being added to measure physiological saline in R1 is 10.8 (1/10000A), and theoretical concentration is
Absorbance change is 111.4 (1/10000A) when 1mg/L serum sample.Table 4,5,6 respectively indicates invention kit reagent, remittance power
Do not add the sensitivity for dissociating the reagent of protein agent in biological reagent and R1, shows not plus the reagent sensitivity of albumen dissociation agent is bright
It is aobvious to be lower 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
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 (10)
1. a kind of preparation method of small fragment BMG antibody, which is characterized in that comprise the steps of:
BMG antibody is hydrolyzed with protease, obtains Fab segment；
The disulfide bond of the Fab segment is opened with dithiothreitol (DTT), after exposing sulfydryl, manually polypeptide is coupled, and is obtained
The small fragment BMG antibody.
2. the preparation method of small fragment BMG antibody as described in claim 1, which is characterized in that the protease is stomach cardia
One or more of enzyme, papain, subtilopeptidase A.
3. the preparation method of small fragment BMG antibody as described in claim 1, which is characterized in that the artificial polypeptide be tripeptides,
One or more of tetrapeptide, pentapeptide.
4. a kind of beta 2-microglobulin detecting kit containing small fragment BMG antibody described in claim 1, which is characterized in that
Including R1 reagent, R2 reagent and calibration object, the R1 reagent is the phosphate buffer containing dissociation protein agent, the reagent
R2 is the buffer system containing the latex particle for being coated with the small fragment BMG antibody, and the calibration object includes 5 difference BMG
The cow's serum matrix of concentration.
5. beta 2-microglobulin detecting kit as claimed in claim 4, which is characterized in that the R1 reagent include as follows at
Point: pH is 7.0~7.6, concentration is 10~100mmol/L phosphate buffer, and concentration is that 200~500mmol/L dissociates albumen
Agent, concentration are 5~20mmol/L disodium ethylene diamine tetraacetate, and concentration is 10~50mmol/L sodium chloride and volume fraction is
0.01%~0.05% preservative.
6. beta 2-microglobulin detecting kit according to claim 5, which is characterized in that the dissociation protein agent is urine
One or more of element, triton x-100, guanidine hydrochloride；The preservative is Proclin 300.
7. beta 2-microglobulin detecting kit according to claim 4, which is characterized in that the R2 reagent includes as follows
Ingredient: pH is 7.0~7.6, concentration is 10~100mmol/L phosphate buffer, the coated sensitization latex of small fragment BMG antibody
Particle.
8. beta 2-microglobulin detecting kit according to claim 7, which is characterized in that the sensitization latex particle
For poly (methyl methacrylate) micro-sphere.
9. beta 2-microglobulin detecting kit according to claim 7, which is characterized in that the small fragment BMG antibody packet
The preparation method of the sensitization latex particle of quilt includes the following steps:
S1: small fragment BMG antibody is added in the lotion for the sensitization latex particle that partial size is 100~200nm, controls small fragment
The volume ratio of BMG antibodies Antibodies and latex particle is 0.2~1:1, is incubated at room temperature 1 hour, obtains solution；
S2: carbodiimides is added in step S1 acquired solution, controls and two Asia of 5mg carbonization is added in every milliliter of latex particle
Amine is incubated at room temperature 2 hours, obtains suspension；
S3: after suspension obtained by step S2 is separated by solid-liquid separation, solid portion is taken to wash dispersion with phosphate buffer.
10. beta 2-microglobulin detecting kit according to claim 4, which is characterized in that 5 cow's serum matrix
The BMG concentration of 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 |
| CN109142749A (en) * | 2018-08-11 | 2019-01-04 | 金华市强盛生物科技有限公司 | A kind of cardic fatty acid binding protein detection kit |
| 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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