CN106153885A - Complement C_3 test kit based on latex immunoturbidimetry and preparation method thereof - Google Patents
Complement C_3 test kit based on latex immunoturbidimetry and preparation method thereof Download PDFInfo
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- CN106153885A CN106153885A CN201610431532.XA CN201610431532A CN106153885A CN 106153885 A CN106153885 A CN 106153885A CN 201610431532 A CN201610431532 A CN 201610431532A CN 106153885 A CN106153885 A CN 106153885A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- G—PHYSICS
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a kind of Complement C_3 test kit based on latex immunoturbidimetry, comprising: reagent A, described reagent A is the glycine buffer system of pH=8.0～9.4, 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=6.5～7.3, the goat-anti coated latex of human complement c 3 polyclonal antibody, 1～the surfactant of 10g/L of goat-anti human complement c 3 polyclonal antibody, 2.5～the 5mL/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 used for the mensuration of human complement c 3, can be prevented effectively from nonspecific reaction, and detection sensitivity is higher, and has relatively low detection limit.
Description
Technical field
The invention belongs to technical field of immunoassay, be specifically related to a kind of Complement C_3 reagent based on latex immunoturbidimetry
Box and preparation method thereof.
Background technology
Complement C_3 is the complement component that in serum, content is the highest, and molecular weight is 195000, mainly by macrophage and liver
Synthesis, under the effect of C3 convertase, can be cleaved into two fragments of C3a and C3b, at complement Classical pathway and alternative activation
Approach all plays a significant role.
Complement dynamically changes and the most increasingly comes into one's own, in serum Complement C_3 concentration be raised and lowered often with
Human body situation is relevant, and such as, Gastritis total complement of serum and Complement C_3 that antigen-antibody complex causes all are decreased obviously,
And when infectious disease and tissue injury and acute inflammation, complement C2, C3, C4 all increase.For another example, tumor patient complement amount can rise
Height, particularly hepatocarcinoma, Complement C_3 raises the most obvious, and when diagnosing cancer of liver, the detection of Complement C_3 has certain meaning.
The method detecting Complement C_3 in prior art mainly has immunoturbidimetry and radioimmunodiffusion etc., wherein immunity
Turbidimetry is that antigen-antibody combines dynamic measurement method, operates relatively easy, low cost, uses the widest in biochemistry analyzer
General.Immunoturbidimetry can be divided into Immunity transmission turbidity and Immune scatter turbidimetry.Its ultimate principle is: when antigen and antibody exist
Reaction in special dilution system and during ratio suitable (general antibody excess), the soluble immune complex of formation is in dilution system
In system, under the effect that macromolecule promotees poly-agent (Polyethylene Glycol etc.), polymerization separates out the granule of a diameter of 340nm～700nm, makes reaction
There is turbidity in liquid.When antibody concentration is fixed, the amount of the immune complex of formation along with in sample the increase of amount of antigen and increase,
The turbidity of reactant liquor is consequently increased.Can be by absorption in various degree, reflection when incident illumination irradiates the reactant liquor of different turbidity
And refraction, compare with series of standards product by measuring the turbidity of reactant liquor, the content of antigen in sample can be drawn.Commonly
Immunity transmission turbidity or Immune scatter turbidimetry in, a small amount of little antigen antibody complex extremely difficult formation turbidity, unless
Place the long period, but the sensitivity of detection can be relatively low, and the consumption strengthening antibody antigen can increase testing cost, and 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, makes the body of antigen-antibody conjugate
Long-pending increase, light is by afterwards, and the Strength Changes of transmission light and scattered light is the most notable, thus improves the sensitivity of 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
Having certain limitation, the detection limit of the test kit of the latex immunoturbidimetry of existing mensuration Complement C_3 is higher, to low concentration
The Detection results of sample undesirable.It addition, the latex used by existing latex immunoturbidimetry needs point in preparation process
The activating reagents such as EDC, the NHS from excess, and need to carry out sealing treatment, relatively complicated in operation.
Summary of the invention
Therefore, for when prior art measures Complement C_3 by latex immunoturbidimetry, nonspecific reaction easily occurs,
The technical problem that detection limit is higher, it is an object of the invention to provide one and can be prevented effectively from nonspecific reaction, and detect
Complement C_3 test kit based on latex immunoturbidimetry highly sensitive, that detection limit is low.
The Complement C_3 test kit based on latex immunoturbidimetry of the present invention includes:
Reagent A, described reagent A is the glycine buffer system of pH=8.0～9.4, 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=6.5～7.3, containing the goat-anti people of 2.5～3g/L
The goat-anti coated latex of human complement c 3 polyclonal antibody, 1～the surface of 10g/L of Complement C_3 polyclonal antibody, 2.5～5mL/L
Activating agent and the electrolyte of 0.5～10g/L.
Described latex is the MES buffer system of pH=6.5～7.3, 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 human complement c 3 polyclonal antibody, the latex obtained again after activation；In described latex, the concentration of carboxyl microsphere be 1～
The mass ratio of the preferred 1g/L of 2g/L, NHS and carboxyl microsphere is 0.05～0.1:1, the mass ratio of EDC and carboxyl microsphere be 0.05～
0.1:1, the goat-anti human complement c 3 polyclonal antibody being coated is 0.05～0.1:1 with the mass ratio of carboxyl microsphere.
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 PVOH 6000 that the macromolecule in described reagent A promotees poly-agent, and PVOH 6000 is in described reagent A
Concentration be 0.2～0.5g/L；Surfactant in described reagent A and described reagent B is triton X-100；Described reagent
Electrolyte in A and described reagent B is sodium chloride.
The 100mmol/L glycine buffer system that described reagent A is pH=8.6 of one preferred embodiment of the present invention, contains
There are the PVOH 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=6.8 of one preferred embodiment of the present invention, contains
The goat-anti human complement c 3 polyclonal antibody of 2.8g/L, the goat-anti coated latex of human complement c 3 polyclonal antibody of 5mL/L, 1g/L
Triton X-100, 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=6.5's～7.3
MES buffer system, the goat-anti human complement c 3 containing goat-anti human complement c 3 polyclonal antibody, 2.5～the 5mL/L of 2.5～3g/L is many
The coated latex of clonal 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=6.5～7.3, 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 human complement c 3 polyclonal antibody being coated, shake at 25～37 DEG C and hatch
2～4 hours, prepare the goat-anti coated latex of human complement c 3 polyclonal antibody；
3) according to each concentration of component of described reagent B, in the MES buffer of pH=6.5～7.3, surface activity is added
Agent, electrolyte and goat-anti human complement c 3 polyclonal antibody, be subsequently adding step 2) prepare goat-anti human complement c 3 polyclonal antibody
Coated latex；
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 human complement c 3 being coated
Polyclonal antibody is 0.05～0.1:1 with the mass ratio of carboxyl microsphere.
Step 2) and step 3) in the goat-anti human complement c 3 polyclonal antibody that adds delay with the MES of pH=6.5～7.3 in advance
Rush liquid and be 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 human complement c 3 polyclonal antibody in reagent B and activate in latex
The mass ratio of carboxyl microsphere is 400～1000:1, and the particle diameter of carboxyl microsphere is 30～80nm especially 40～50nm simultaneously, makes
It is coated on a small quantity and is combined with carboxyl microsphere with antibody, the most just define common antibody and the mixing of the coated antibody carrying carboxyl microsphere
Zoarium, and the microsphere assembled is removed by 0.2 μ L membrane filtration.When add antigen time, antigen will proportionally with both
Antibodies, assembles the particulate matter formed and will comprise 1 or several carboxyl microsphere, and the performance of reaction is greatly increased, as far as possible
Avoid nonspecific reaction.
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 human complement c 3 polyclonal antibody being coated after NHS, EDC activation and hatch, then mix with remaining component, preparation
Become reagent B.Compared with traditional latex preparation process, control NHS, EDC and the goat-anti human complement c 3 Anti-TNF-α being coated
The consumption of body, can avoid NHS, EDC and activated carboxyl microsphere separation process, and activated carboxyl microsphere closed process, therefore grasp
More simplify on work.
To sum up, the described test kit of the present invention can be used for detecting on automatic clinical chemistry analyzer containing of complement C3
Amount, compared with existing immunoturbidimetry reagent, can be prevented effectively from nonspecific reaction, and characteristic absorption wavelength is stable, can improve anti-
Answering sensitivity, detection limit is relatively low, can be used for the detection of low concentration sample, and compared with traditional latex preparation process, this
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 Complement C_3 standard serum sample.
Detailed description of the invention
Embodiment 1～3 test kit and preparation thereof
Test kit can be used for latex immunoturbidimetry and measures Complement C_3, 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, PVOH 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 human complement c 3 polyclonal antibody, NHS, the EDC in the latex of embodiment 1～3 and goat-anti people's complement
C3 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=6.8, 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 human complement c 3 polyclonal antibody
MES buffer, then at 37 DEG C shaking hatch the 2 hours prepared goat-anti coated latex of human complement c 3 polyclonal antibody, 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 human complement c 3 polyclonal antibody of ratio corresponding volume shown in table 2, it is subsequently adding step
2) the goat-anti human complement c 3 polyclonal antibody coated 10mL latex prepared, 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, the Complement C_3 test kit of comparative example (are become by Sichuan newly health
Biological Co., Ltd. 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 mixes with the serum sample of Complement C_3
3～5 minutes, it is subsequently adding reagent B and starts reaction, wherein volume ratio reagent A: reagent B: sample=200:40:2；5 minute time ratio
According to blank determination initial absorbance A1, then when 10 minutes according to blank determination absorbance A 2, calculate the difference DELTA of A2 Yu A1
A。
1, the mensuration of standard curve
With embodiment 1～3 and the test kit of comparative example measure the benefit of series of standards concentration as shown in table 4 the most one by one
The Δ A value of the standard serum sample of body C3, and the concentration and the Δ A value recorded according to a series of Complement C_3 can simulate respectively respectively
The standard curve of test kit, standard curve is LOGIT-LOG4P function.
The scatterplot that the concentration of Complement C_3 is 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, 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 be bigger, accurate
Exactness and sensitivity have bigger lifting.
The Δ A value test result of table 4 standard serum sample
2, the Detection results of standard curve
With a series of containing concentration known Complement C_3 shown in embodiment 1～3 and the table 5 that measures respectively of the test kit of comparative example
The Δ A value of serum sample, then draw the mensuration concentration of correspondence, this mensuration concentration and reality respectively according to the standard curve of matching
Border concentration there will be deviation, as shown in table 5.
Table 5 measurement result contrasts
Clinical accuracy requirement deviation is less than 10%.By table 5, the test kit of comparative example is at detection 12.5mg/
During the sample of dL, deviation has just exceeded the 10% of clinical requirement, and its detection limit can only be 25.0mg/dL, with explanation 26.6mg/dL
Lowest detectable limit is basically identical.And the test kit of the present invention is in detection 12.5mg/dL sample acquired results and actual concentrations deviation
Within 10%, meeting clinical accuracy requirement, therefore lowest detectable limit can reach 12.5mg/dL.
Claims (10)
1. a Complement C_3 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=8.0～9.4, 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=6.5～7.3, containing goat-anti people's complement of 2.5～3g/L
The goat-anti coated latex of human complement c 3 polyclonal antibody, 1～the surface activity of 10g/L of C3 polyclonal antibody, 2.5～5mL/L
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=6.5～7.3,
Be particle diameter be 30～80nm preferably 40～the carboxyl microsphere of 50nm through NHS and EDC activate after again by goat-anti human complement c 3 polyclone
Antibody is coated the latex obtained；In described latex, the concentration of carboxyl microsphere is 1～2g/L preferred 1g/L, NHS and carboxyl microsphere
Mass ratio is 0.05～0.1:1, and EDC is 0.05～0.1:1 with the mass ratio of carboxyl microsphere, and the goat-anti human complement c 3 being coated is many
Clonal 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 PVOH that the macromolecule in described reagent A promotees poly-agent
6000, the PVOH 6000 concentration in described reagent A is 0.2～0.5g/L；Surface in described reagent A and described reagent B
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=8.6
Buffer system, PVOH 6000, the triton X-100 of 1g/L, the sodium chloride of 5g/L and the sodium azide of 1g/L containing 0.5g/L.
6. test kit as claimed in claim 4, it is characterised in that described reagent B is the 50mmol/L MES buffering of pH=6.8
System, the goat-anti human complement c 3 polyclonal antibody containing 2.8g/L, the goat-anti coated glue of human complement c 3 polyclonal antibody of 3mL/L
Breast, 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=6.5～7.3, contains
The goat-anti human complement c 3 polyclonal antibody having goat-anti human complement c 3 polyclonal antibody, 2.5～the 5mL/L of 2.5～3g/L is coated
The surfactant of latex, 1～10g/L and the electrolyte of 0.5～10g/L；
The method comprises the steps:
1) with the MES buffer of pH=6.5～7.3, 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) adding, in activation latex, the goat-anti human complement c 3 polyclonal antibody being coated, at 25～37 DEG C, shaking hatches 2～4
Hour, prepare the goat-anti coated latex of human complement c 3 polyclonal antibody；
3) according to each concentration of component of described reagent B, in the MES buffer of pH=6.5～7.3, surfactant, electricity are added
Solving matter and goat-anti human complement c 3 polyclonal antibody, be subsequently adding step 2) the goat-anti human complement c 3 polyclonal antibody for preparing is coated
Latex；
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 human complement c 3 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) middle many grams of the goat-anti human complement c 3 added
Grand antibody is diluted to 20～30g/L with the MES buffer of pH=6.5～7.3 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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| CN106932570A (en) * | 2017-03-07 | 2017-07-07 | 中国人民解放军第二军医大学 | A kind of kit for detecting PSA PSA and its application |
| CN107490676A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of Complement C_3 detection kit and detection method |
| CN107607724A (en) * | 2017-08-11 | 2018-01-19 | 中山市创艺生化工程有限公司 | A kind of compound stabilizer and its application for Complement C_3 measure kit |
| CN107607724B (en) * | 2017-08-11 | 2020-04-28 | 中山市创艺生化工程有限公司 | Composite stabilizer for complement C3 determination kit and application thereof |
| CN108982831A (en) * | 2018-07-27 | 2018-12-11 | 金华市强盛生物科技有限公司 | A kind of preparation method of new type latex enhancing immunoturbidimetry seminal plasma fructose detection kit 2 |
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