CN106093433A - Microalbumin test kit based on latex immunoturbidimetry and preparation method thereof - Google Patents
Microalbumin test kit based on latex immunoturbidimetry and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of microalbumin test kit based on latex immunoturbidimetry, comprising: reagent A, described reagent A is the glycine buffer system of pH=8.5～9.4, and the macromolecule containing 0.1～0.5g/L promotees poly-agent, 1～the surfactant of 10g/L and the electrolyte of 0.5～10g/L；And reagent B, described reagent B is the MES buffer system of pH=7.0～7.5, goat-anti people's coated latex of microalbumin polyclonal antibody, 1～the surfactant of 10g/L of goat-anti people's microalbumin polyclonal antibody, 5～the 10mL/L containing 2.5～3g/L and the electrolyte of 0.5～10g/L.The invention also discloses the method preparing reagent B.Described test kit can be prevented effectively from nonspecific reaction, and detection sensitivity is higher, and has relatively low detection limit, can be used for the mensuration of clinical microalbumin.
Description
Technical field
The invention belongs to technical field of immunoassay, be specifically related to a kind of microalbumin based on latex immunoturbidimetry
Test kit and preparation method thereof.
Background technology
Microalbumin refers to the microalbumin occurred in Human Urine.Albumin is the normal albumen in a kind of blood
Matter, but in physiological conditions only there is very small amount albumin in urine.Under normal circumstances, in urine, the concentration of microalbumin is not
More than 20mg/L, it is then microalbuminuria when the microalbumin concentration of urine reaches 20～200mg/L, reflects human body kidney
Dirty exceptional leakage protein.The detection of microalbumin is early discovery nephropathy diagnosis index most sensitive, most reliable.By urine
The testing result of liquid microalbumin, can relatively accurately diagnose the state of an illness in conjunction with the statement of incidence, symptom and medical history.And
Periodic detection Urinary microalbumin, can play positive role to the prevention of nephropathy and early treatment.
The method of the Clinical practice detecting microalbumin in prior art mainly has immunoturbidimetry and radioimmunity to expand
Arching pushings etc., wherein immunoturbidimetry is that antigen-antibody combines dynamic measurement method, operates relatively easy, low cost, divides at biochemistry
Use relatively broad in analyzer.Immunoturbidimetry can be divided into Immunity transmission turbidity and Immune scatter turbidimetry.Its ultimate principle
It is: when antigen and antibody reacts and during ratio properly (general antibody excess) in special dilution system, the solubility of formation
Immune complex in dilution system macromolecule promote poly-agent (Polyethylene Glycol etc.) effect under polymerization separate out a diameter of 340nm～
The granule of 700nm, makes reactant liquor turbidity occur.When antibody concentration is fixed, the amount of the immune complex of formation is along with in sample
The increase of amount of antigen and increase, the turbidity of reactant liquor is consequently increased.Can quilt when incident illumination irradiates the reactant liquor of different turbidity
Absorption in various degree, reflect and reflect, compareing with series of standards product by measuring the turbidity of reactant liquor, sample can be drawn
The content of middle antigen.In common Immunity transmission turbidity or Immune scatter turbidimetry, a small amount of little antigen-antibody is combined
Thing extremely difficult formation turbidity, unless placed the long period, but the sensitivity of detection can be relatively low, and the consumption strengthening antibody antigen can
Increase testing cost, and do not meet the requirement of milligram ammonia.Based on this, disclosed in prior art, there is latex enhancing immune turbidimetry
I.e. latex immunoturbidimetry, its principle is to be coated on the latex particle of certain diameter by the antibody corresponding with test substance,
The volume making antigen-antibody conjugate increases, and light is by afterwards, and the Strength Changes of transmission light and scattered light is the most notable, thus carries
The sensitivity of high test.
But in latex immunoturbidimetry, although the macromolecule of interpolation promotees poly-agent and easier combination of antibody can be made to resist
Former, but also can make other materials of antibodies simultaneously, cause non-specific enhancing；And activate latex and also make antibody itself
Reactivity dramatically increase, the macromolecule increasing excess promotees poly-agent, and more latex can be made to assemble, and further promotion is non-specific
Reaction, the latex particle particle diameter that result is easily caused gathering is uneven, and reaction repeatability is low, and characteristic wavelength during detection is not solid
Fixed.And during Clinical practice, parameter is all fixing, the variation of characteristic wavelength makes this technology in practical clinical
There is certain limitation.It addition, the latex used by existing latex immunoturbidimetry needs to separate excess in preparation process
The activating reagents such as EDC, NHS, and need to carry out sealing treatment, relatively complicated in operation.
Summary of the invention
Therefore, for when prior art measures microalbumin by latex immunoturbidimetry, non-specific responding easily occurs
Technical problem, it is an object of the invention to provide one and can be prevented effectively from nonspecific reaction, and detection sensitivity is high, detection
Limit low microalbumin test kit based on latex immunoturbidimetry.
The microalbumin test kit based on latex immunoturbidimetry of the present invention includes:
Reagent A, described reagent A is the glycine buffer system of pH=8.5～9.4, containing the macromolecule of 0.1～0.5g/L
Promote surfactant and the electrolyte of 0.5～10g/L of poly-agent, 1～10g/L；
And reagent B, described reagent B are the MES buffer system of pH=7.0～7.5, containing the goat-anti people of 2.5～3g/L
Goat-anti people's coated latex of microalbumin polyclonal antibody, 1～the 10g/L of microalbumin polyclonal antibody, 5～10mL/L
Surfactant and the electrolyte of 0.5～10g/L.
Described latex is the MES buffer system of pH=7.0～7.5, be particle diameter be 30～80nm preferably 40～the carboxylic of 50nm
Base microsphere is through NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate)
It is coated, by goat-anti people's microalbumin polyclonal antibody, the latex obtained again after activation；In described latex, the concentration of carboxyl microsphere
Being 1～2g/L preferred 1g/L, NHS is 0.05～0.1:1 with the mass ratio of carboxyl microsphere, and EDC with the mass ratio of carboxyl microsphere is
0.05～0.1:1, the goat-anti people's microalbumin polyclonal antibody being coated is 0.05～0.1 with the mass ratio of carboxyl microsphere:
1。
Preferably, described reagent A and described reagent B respectively possibly together with 0.5～1g/L preservative, described preservative is
Proclin300 and/or sodium azide.
Preferably, it is polyethylene glycol 6000 that the macromolecule in described reagent A promotees poly-agent, and polyethylene glycol 6000 is at described reagent
Concentration in A is 0.2～0.5g/L；Surfactant in described reagent A and described reagent B is triton X-100；Described
Electrolyte in reagent A and described reagent B is sodium chloride.
The 100mmol/L glycine buffer system that described reagent A is pH=9.0 of one preferred embodiment of the present invention, contains
There are the polyethylene glycol 6000 of 0.5g/L, the triton X-100 of 1g/L, the sodium chloride of 5g/L and the sodium azide of 1g/L.
The 50mmol/L MES buffer system that described reagent B is pH=7.0 of one preferred embodiment of the present invention, contains
Goat-anti people's microalbumin polyclonal antibody of 2.8g/L, goat-anti people's coated glue of microalbumin polyclonal antibody of 5mL/L
Breast, the triton X-100 of 1g/L, the sodium chloride of 5g/L and the sodium azide of 1g/L.
Another object of the present invention also resides in a kind of method preparing reagent B that discloses, and described reagent B is pH=7.0's～7.5
MES buffer system, the goat-anti people's trace containing goat-anti people's microalbumin polyclonal antibody, 5～the 10mL/L of 2.5～3g/L is white
The coated latex of protein polyclone antibody, 1～the surfactant of 10g/L and the electrolyte of 0.5～10g/L；
The method comprises the steps:
1) with the MES buffer of pH=7.0～7.5, carboxyl microsphere is diluted to 1～2g/L, adds NHS and EDC, 15
～reaction obtains activating latex for 20～30 minutes at 25 DEG C；
2) add, in activation latex, the goat-anti people's microalbumin polyclonal antibody being coated, shake at 25～37 DEG C
Hatch 2～4 hours, prepare goat-anti people's coated latex of microalbumin polyclonal antibody；
3) according to each concentration of component of described reagent B, in the MES buffer of pH=7.0～7.5, surface activity is added
Agent, electrolyte and goat-anti people's microalbumin polyclonal antibody, be subsequently adding step 2) prepare goat-anti people's microalbumin many
The coated latex of clonal antibody；
4) with 0.2 μ L nylon membrane filtration sterilization and bulky grain, reagent B is obtained.
Step 1) in carboxyl microspherulite diameter be 30～80nm preferably 40～50nm；NHS with the mass ratio of carboxyl microsphere is
0.05～0.1:1, EDC are 0.05～0.1:1 with the mass ratio of carboxyl microsphere；Step 2) in, the goat-anti people's trace being coated is white
Protein polyclone antibody is 0.05～0.1:1 with the mass ratio of carboxyl microsphere.
Step 2) and step 3) in goat-anti people's microalbumin polyclonal antibody of adding in advance with pH=7.0～7.5
MES buffer is diluted to 20～30g/L.
Step 3) in, it being also added with preservative proclin300 and/or sodium azide, it is 0.5 that described preservative adds concentration
～1g/L.
Key technology and the most progressive effect of the present invention are:
1, a key technology of the present invention is to control goat-anti people's microalbumin polyclonal antibody and activation latex in reagent B
In the mass ratio of carboxyl microsphere be 200～400:1, simultaneously the particle diameter of carboxyl microsphere be 30～80nm especially 40～
50nm, makes to be coated on a small quantity and uses antibody to be combined with carboxyl microsphere, the most just define common antibody and carry being coated of carboxyl microsphere
The mixture of antibody, and the microsphere assembled is removed by 0.2 μ L membrane filtration.When adding antigen, antigen will be proportionally
With both antibodies, assembling the particulate matter formed and will comprise 1 or several carboxyl microsphere, the performance of reaction increases
Add, avoid nonspecific reaction as far as possible.
2, another key technology of the present invention is the consumption that macromolecule promotees poly-agent, and concentration is 0.1～0.5g/L.Due to the fact that
Adding the activation latex containing carboxyl microsphere, the reactivity of antibody dramatically increases, but if exempts from for promotion by prior art
Epidemic disease complex is assembled and is added the macromolecule poly-agent of rush of excess, instead more carboxyl microsphere aggregation can be made, produces non-specific
Reaction.The present invention attempt adjust macromolecule promote poly-agent consumption to avoid nonspecific reaction as far as possible, due to containing activation
Carboxyl microsphere, reaction itself has certain sensitivity, it is contemplated that less macromolecule promote poly-agent the most just can have relatively
The poly-effect of good rush.The actual result of experiment also really so, when macromolecule promote the consumption of poly-agent be reduced to concentration be 0.1～
Preferably effect is can reach during 0.5g/L.
3, the present invention also has the preparation process that a key technology relates to described reagent B, first the carboxyl microsphere warp in latex
Mix with the goat-anti people's microalbumin polyclonal antibody being coated after NHS, EDC activation and hatch, then mix with remaining component,
It is configured to reagent B.Compared with traditional latex preparation process, control NHS, EDC and the goat-anti people's microalbumin being coated
The consumption of polyclonal antibody, can avoid NHS, EDC and activated carboxyl microsphere separation process, and activated carboxyl microsphere was closed
Journey, therefore more simplifies in operation.
To sum up, the described test kit of the present invention can be used for detecting microalbumin in urine on automatic clinical chemistry analyzer
Content, compared with existing immunoturbidimetry reagent, can be prevented effectively from nonspecific reaction, and characteristic absorption wavelength is stable, can improve
Reaction sensitivity, detection limit is relatively low, can be used for the detection of low concentration sample.And compared with traditional latex preparation process,
The preparation method of the present invention eliminates more solid-liquor separation and closed process, and operation more simplifies.
Accompanying drawing explanation
Fig. 1 is concentration and the corresponding scatterplot of Δ A value of microalbumin standard urine specimen.
Detailed description of the invention
Embodiment 1～3 test kit and preparation thereof
Test kit can be used for latex immunoturbidimetry and measures microalbumin, including reagent A and reagent B.
The component of reagent A is as shown in table 1.
The component of table 1 reagent A
The preparation steps of reagent A is (preparation 1L): respectively according to the concentration of component of embodiment in table 1 1～3, take glycine
(7.5g, 100mmol) is dissolved in purified water (1L), is subsequently adding the sodium chloride of corresponding amount, triton X-100, polyethylene glycol 6000
And sodium azide, mixing also obtains reagent A with sodium hydroxide regulation pH.
(2) component of reagent B is as shown in table 2.
The component of table 2 reagent B
Latex in table 2 is the MES buffer system of the carboxyl microsphere containing 1g/L, the carboxyl microsphere in latex through NHS and
EDC activation is also coated by goat-anti people's microalbumin polyclonal antibody, NHS, the EDC in the latex of embodiment 1～3 and goat-anti people
Microalbumin polyclonal antibody and carboxyl microspheres quality are than as shown in table 3.
Each component in table 3 latex and the mass ratio of carboxyl microsphere
The preparation steps of reagent B is (preparation 1L):
1) the carboxyl microsphere (being provided by Bangs laboratories, Inc) that 100 μ L solids contents are 10g/100mL is provided,
Be diluted to 10mL with MES buffer (50mmol/L, pH=7.0, lower with), be subsequently adding the NHS of ratio corresponding amount shown in table 3 and
EDC, room temperature reaction obtains activating latex for 20 minutes.
2) add in activation latex ratio corresponding volume shown in table 3 containing 30g/L goat-anti people's microalbumin polyclone
The MES buffer of antibody, then at 37 DEG C shaking to hatch 2 hours prepared goat-anti people's microalbumin polyclonal antibodies coated
Latex, i.e. latex described in table 2.
3) in the MES buffer of 500mL, the sodium chloride of ratio corresponding amount, triton X-100, sodium azide shown in table 2 is added
With the MES buffer containing 30g/L goat-anti people's microalbumin polyclonal antibody of ratio corresponding volume shown in table 2, it is subsequently adding
Step 2) goat-anti people's microalbumin polyclonal antibody coated 10mL latex of preparing, and complement to 1L with MES buffer.
4) filter with the nylon membrane of 0.22 μ L after mixing, degerming and remove bulky grain and obtain reagent B.
Effect example 1
Measure of merit test kit: the test kit of embodiment 1～3, comparative example microalbumin test kit (by Britain
RANDOX company produces).
Instrument: Hitachi 7170 biochemistry analyzer.
Test wavelength: dominant wavelength 340nm, commplementary wave length 700nm.
Method of testing: end-point method, incremental reacts.Under the conditions of 37 DEG C, first reagent A and the urine specimen of microalbumin
Mix 3～5 minutes, be subsequently adding reagent B and start reaction, wherein volume ratio reagent A: reagent B: sample=200:40:2；5 minutes
Time according to blank determination initial absorbance A1, then when 10 minutes according to blank determination absorbance A 2, calculate the difference of A2 Yu A1
Value Δ A.
1, the mensuration of standard curve
With embodiment 1～3 and the test kit of comparative example measure the micro-of series of standards concentration as shown in table 4 the most one by one
Measure the Δ A value of albuminous master sample, and the concentration and the Δ A value recorded according to a series of microalbumins can distinguish matching
Going out the standard curve of each test kit, standard curve is LOGIT-LOG4P function.
The concentration of the microalbumin scatterplot corresponding with the Δ A value recorded as it is shown in figure 1, as shown in Figure 1, embodiment
1～3 compared to comparative example, and the concentration of Complement C_3 is more preferable with the linear relationship of Δ A value, and the slope of the standard curve of matching will more
Greatly, accuracy and sensitivity have bigger lifting.
The Δ A value test result of table 4 standard urine specimen
2, the Detection results of standard curve
With a series of white containing concentration known trace shown in embodiment 1～3 and the table 5 that measures respectively of the test kit of comparative example
The Δ A value of the serum sample of albumen, then draws the mensuration concentration of correspondence, this mensuration concentration on the standard curve of matching respectively
Deviation is there will be, as shown in table 5 with actual concentrations.
Table 5 measurement result contrasts
Clinical accuracy requirement deviation is less than 10%.By table 4, the test kit of comparative example is at detection 1.9mg/L
Sample time deviation just exceeded the 10% of clinical requirement, its detection limit can only be 3.8mg/L.And the test kit of the present invention is in inspection
Survey 1.9mg/L sample acquired results within 10%, meets clinical accuracy requirement, therefore lowest detection with actual concentrations deviation
Limit can reach 1.9mg/L.
Claims (10)
1. a microalbumin test kit based on latex immunoturbidimetry, it is characterised in that described test kit includes:
Reagent A, described reagent A is the glycine buffer system of pH=8.5～9.4, and the macromolecule containing 0.1～0.5g/L promotees poly-
The surfactant of agent, 1～10g/L and the electrolyte of 0.5～10g/L；
And reagent B, described reagent B are the MES buffer system of pH=7.0～7.5, containing goat-anti people's trace of 2.5～3g/L
Goat-anti people's coated latex of microalbumin polyclonal antibody, 1～the table of 10g/L of albumin polyclonal antibody, 5～10mL/L
Face activating agent and the electrolyte of 0.5～10g/L.
2. test kit as claimed in claim 1, it is characterised in that described latex is the MES buffer system of pH=7.0～7.5,
It is that particle diameter is 30～80nm preferably 40～the carboxyl microsphere of 50nm is many by goat-anti people's microalbumin again after NHS and EDC activates
Clonal antibody is coated the latex obtained；In described latex, the concentration of carboxyl microsphere is 1～2g/L preferred 1g/L, and NHS is micro-with carboxyl
The mass ratio of ball is 0.05～0.1:1, and EDC is 0.05～0.1:1 with the mass ratio of carboxyl microsphere, the goat-anti people's trace being coated
Albumin polyclonal antibody is 0.05～0.1:1 with the mass ratio of carboxyl microsphere.
3. test kit as claimed in claim 1 or 2, it is characterised in that described reagent A and described reagent B are respectively possibly together with 0.5
～the preservative of 1g/L, described preservative is proclin300 and/or sodium azide.
4. test kit as claimed in claim 3, it is characterised in that it is Polyethylene Glycol that the macromolecule in described reagent A promotees poly-agent
6000, polyethylene glycol 6000 concentration in described reagent A is 0.2～0.5g/L；Table in described reagent A and described reagent B
Face activating agent is triton X-100；Electrolyte in described reagent A and described reagent B is sodium chloride.
5. test kit as claimed in claim 4, it is characterised in that described reagent A is the 100mmol/L glycine of pH=9.0
Buffer system, polyethylene glycol 6000, the triton X-100 of 1g/L, the sodium chloride of 5g/L and the nitrine of 1g/L containing 0.5g/L
Sodium.
6. test kit as claimed in claim 4, it is characterised in that described reagent B is the 50mmol/L MES buffering of pH=7.0
System, the goat-anti people's microalbumin polyclonal antibody containing 2.8g/L, goat-anti people's microalbumin polyclonal antibody of 5mL/L
Coated latex, the triton X-100 of 1g/L, the sodium chloride of 5g/L and the sodium azide of 1g/L.
7. the method preparing reagent B, it is characterised in that described reagent B is the MES buffer system of pH=7.0～7.5, contains
There is goat-anti people's microalbumin polyclonal antibody of goat-anti people's microalbumin polyclonal antibody, 5～the 10mL/L of 2.5～3g/L
The surfactant of coated latex, 1～10g/L and the electrolyte of 0.5～10g/L；
The method comprises the steps:
1) with the MES buffer of pH=7.0～7.5, carboxyl microsphere is diluted to 1～2g/L, adds NHS and EDC, 15～25
At DEG C, reaction obtains activating latex for 20～30 minutes；
2) add, in activation latex, the goat-anti people's microalbumin polyclonal antibody being coated, shake at 25～37 DEG C and hatch
2～4 hours, prepare goat-anti people's coated latex of microalbumin polyclonal antibody；
3) according to each concentration of component of described reagent B, in the MES buffer of pH=7.0～7.5, surfactant, electricity are added
Solve matter and goat-anti people's microalbumin polyclonal antibody, be subsequently adding step 2) goat-anti people's microalbumin Anti-TNF-α of preparing
The coated latex of body；
4) with 0.2 μ L nylon membrane filtration sterilization and bulky grain, reagent B is obtained.
8. method as claimed in claim 7, it is characterised in that step 1) in carboxyl microspherulite diameter be 30～80nm preferably 40
～50nm；NHS is 0.05～0.1:1 with the mass ratio of carboxyl microsphere, and EDC is 0.05～0.1:1 with the mass ratio of carboxyl microsphere；
Step 2) in, the goat-anti people's microalbumin polyclonal antibody being coated is 0.05～0.1:1 with the mass ratio of carboxyl microsphere.
9. method as claimed in claim 7, it is characterised in that step 2) and step 3) the middle goat-anti people's microalbumin added
Polyclonal antibody is diluted to 20～30g/L with the MES buffer of pH=7.0～7.5 in advance.
10. method as claimed in claim 6, it is characterised in that step 3) in, be also added with preservative proclin300 and/
Or sodium azide, it is 0.5～1g/L that described preservative adds concentration.
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Cited By (2)
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| CN109781990A (en) * | 2018-12-25 | 2019-05-21 | 无锡市人民医院 | A kind of β-trace protein detection kit and preparation method |
| CN112698040A (en) * | 2020-12-09 | 2021-04-23 | 宁波职业技术学院 | Urine microalbumin detection reagent and preparation method thereof |
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| CN102998468A (en) * | 2012-09-20 | 2013-03-27 | 武汉华美生物工程有限公司 | 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof |
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| CN109781990A (en) * | 2018-12-25 | 2019-05-21 | 无锡市人民医院 | A kind of β-trace protein detection kit and preparation method |
| CN112698040A (en) * | 2020-12-09 | 2021-04-23 | 宁波职业技术学院 | Urine microalbumin detection reagent and preparation method thereof |
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| CN106093433B (en) | 2018-05-08 |
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