CN106124774B - Complement C4 kit based on latex immunoturbidimetry and preparation method thereof - Google Patents
Complement C4 kit based on latex immunoturbidimetry and preparation method thereof Download PDFInfo
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- CN106124774B CN106124774B CN201610431512.2A CN201610431512A CN106124774B CN 106124774 B CN106124774 B CN 106124774B CN 201610431512 A CN201610431512 A CN 201610431512A CN 106124774 B CN106124774 B CN 106124774B
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
Abstract
The invention discloses a kind of Complement C4 kit based on latex immunoturbidimetry, including：Reagent A, the reagent A are the glycine buffer system of pH=8.0~9.4, and the macromolecule containing 0.1~0.5g/L promotees poly- agent, the surfactant of 1~10g/L and the electrolyte of 0.5~10g/L；And reagent B, the reagent B be pH=6.5~7.3 MES buffer systems, the coated latex of goat-anti people's Complement C4 polyclonal antibody, the surfactant of 1~10g/L and the electrolyte of 0.5~10g/L of goat-anti people's Complement C4 polyclonal antibody, 5~10mL/L containing 1.5~2g/L.The invention also discloses the methods of reagent preparation B.The kit can be used for the measure of people's Complement C4, can effectively avoid nonspecific reaction, and detection sensitivity is higher, and be limited with relatively low detection.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of Complement C4 reagent based on latex immunoturbidimetry
Box and preparation method thereof.
Background technology
Complement C4 is a kind of multi-functional β 1- globulin being present in blood plasma.In the activation of complement classical pathway, C4 quilts
C1s is hydrolyzed to C4a, C4b, they complement activation, promote phagocytosis, prevent immune complex deposit and in and virus etc. hair
The effect of waving.C4 is an important component of complement Classical pathway, its measure contributes to the autoimmune diseases such as SLE to examine
Disconnected, treatment and CORD PARALYSIS.Such as the reduction of C4 be common in immune complex caused by ephritis, systemic loupus erythematosus, virus
Sexy dye, systemic lupus erythematosus syndrome, hepatic sclerosis, hepatitis etc., and rise is common in various infectious diseases, acute inflammation, tissue damage, more
Hair property myeloma etc..
The method for detecting Complement C4 in the prior art mainly has immunoturbidimetry and radioimmunodiffusion etc., wherein immune
Turbidimetry is antigen-antibody combination dynamic assay method, and operation is relatively easy, at low cost, using more wide on Biochemical Analyzer
It is general.Immunoturbidimetry can be divided into Immunity transmission turbidity and Immune scatter turbidimetry.Its basic principle is：When antigen and antibody exist
Reaction and during ratio suitable (general antibody excess) in special dilution system, the soluble immune complex of formation is in dilution
The particle of a diameter of 340nm~700nm is precipitated in polymerization under the action of macromolecule promotees poly- agent (polyethylene glycol etc.) in system, makes reaction
There is turbidity in liquid.When antibody concentration is fixed, the amount of the immune complex of formation increases with the increase of amount of antigen in sample,
The turbidity of reaction solution is consequently increased.It can be by different degrees of absorption, reflection when the reaction solution of the different turbidity of incident light irradiation
And refraction, it is compareed by the turbidity for measuring reaction solution with series of standards product, you can draw the content of antigen in sample.Common
Immunity transmission turbidity or Immune scatter turbidimetry in, a small amount of extremely difficult formation turbidity of small antigen antibody complex, unless
It places the long period, but the sensitivity detected can be relatively low, and the dosage for increasing antibody antigen can increase testing cost, and not
Meet the requirement of milligram ammonia.Based on this, latex enhancing immune turbidimetry i.e. latex immunoturbidimetry is disclosed in the prior art,
Principle is will to be coated on the corresponding antibody of test substance on the latex particle of certain diameter, make the body of antigen-antibody conjugate
Product increase, light is by the way that afterwards, the Strength Changes of transmitted light and scattering light are more notable, so as to improve the sensitivity of experiment.
But in latex immunoturbidimetry, resist although the poly- agent of macromolecule rush of addition can make antibody be easier combination
Original, but antibody can also be made to combine other substances simultaneously, cause non-specific enhancing；And activation latex also causes antibody in itself
Reactivity dramatically increase, the macromolecule for increasing excess promotees poly- agent, can assemble more latex, further promotes non-specific
Reaction, it is uneven to be as a result easy to cause the latex particle grain size of aggregation, and reaction reappearance is low, and characteristic wavelength during detection is not solid
It is fixed.And during Clinical practice, parameter is all fixed, and the variation of characteristic wavelength causes the technology in practical clinical
There is certain limitation, the detection limit of the kit of the existing latex immunoturbidimetry for measuring Complement C4 is higher, to low concentration
Sample detection result it is undesirable.In addition, the latex used in existing latex immunoturbidimetry needs point in preparation process
From activating reagents such as excessive EDC, NHS, and progress Seal treatment is needed, it is relatively complicated in operation.
The content of the invention
Therefore, be susceptible to nonspecific reaction for the kit for measuring Complement C4 in the prior art, detection limit compared with
The technical issues of high, it is an object of the invention to provide one kind can effectively avoid nonspecific reaction, detection sensitivity is high, detects
Limit the low Complement C4 kit based on latex immunoturbidimetry.
The Complement C4 kit based on latex immunoturbidimetry of the present invention includes：
Reagent A, the reagent A is the glycine buffer system of pH=8.0~9.4, the macromolecule containing 0.1~0.5g/L
Promote the electrolyte of poly- agent, the surfactant of 1~10g/L and 0.5~10g/L；
And the MES buffer systems that reagent B, the reagent B are pH=6.5~7.3, the goat-anti containing 1.5~2.0g/L
People's Complement C4 polyclonal antibody, the coated latex of goat-anti people's Complement C4 polyclonal antibody of 5~10mL/L, the surface of 1~10g/L
The electrolyte of activating agent and 0.5~10g/L.
The latex is the MES buffer systems of pH=6.5~7.3, is that grain size is 30~80nm preferably carboxylics of 40~50nm
Base microballoon is through NHS (N- hydroxysuccinimides) and EDC (1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate)
The latex being coated with again by goat-anti people's Complement C4 polyclonal antibody after activation；In the latex, the concentration of carboxyl microballoon for 1~
The mass ratio of 2g/L preferred 1g/L, NHS and carboxyl microballoon is 0.05~0.1:1, EDC with the mass ratio of carboxyl microballoon for 0.05~
0.1:1, goat-anti people's Complement C4 polyclonal antibody of coating is 0.05~0.1 with the mass ratio of carboxyl microballoon:1.
Preferably, also the preservative containing 0.5~1g/L, the preservative are respectively by the reagent A and the reagent B
Proclin300 and/or Sodium azide.
Preferably, the macromolecule in the reagent A promotees poly- agent as Macrogol 6000, and Macrogol 6000 is in the reagent
Concentration in A is 0.2~0.5g/L；Surfactant in the reagent A and the reagent B is triton X-100；It is described
Electrolyte in reagent A and the reagent B is sodium chloride.
The reagent A of the preferred embodiment of the present invention is the 100mmol/L glycine buffer systems of pH=8.6, is contained
There are the Macrogol 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 reagent B of the preferred embodiment of the present invention is the 50mmol/L MES buffer systems of pH=6.8, is contained
Goat-anti people's Complement C4 polyclonal antibody of 1.8g/L, the coated latex of goat-anti people's Complement C4 polyclonal antibody of 5mL/L, 1g/L
The Sodium azide of triton X-100, the sodium chloride of 5g/L and 1g/L.
Another object of the present invention also resides in a kind of method for disclosing reagent preparation B, and the reagent B is pH=6.5~7.3
MES buffer systems, goat-anti people's Complement C4 polyclonal antibody, goat-anti people's Complement C4 of 5~10mL/L containing 1.5~2.0g/L are more
The coated latex of clonal antibody, the surfactant of 1~10g/L and the electrolyte of 0.5~10g/L；
This method comprises the following steps：
1) carboxyl microballoon is diluted to 1~2g/L with the MES buffer solutions of pH=6.5~7.3, adds NHS and EDC, 15
It is reacted at~25 DEG C and obtains within 20~30 minutes activation latex；
2) goat-anti people's Complement C4 polyclonal antibody of coating is added in into activation latex, shakes and is incubated at 25~37 DEG C
2~4 it is small when, goat-anti people's Complement C4 polyclonal antibody coated latex is made；
3) according to each component concentration of the reagent B, surface-active is added in into the MES buffer solutions of pH=6.5~7.3
Then agent, electrolyte and goat-anti people's Complement C4 polyclonal antibody add in goat-anti people's Complement C4 polyclonal antibody made from step 2)
Coated latex；
4) with 0.2 μ L nylon membranes filtration sterilizations and bulky grain, reagent B is obtained.
Carboxyl microspherulite diameter in step 1) is 30~80nm preferably 40~50nm；The mass ratio of NHS and carboxyl microballoon is
0.05~0.1:1, EDC with the mass ratio of carboxyl microballoon is 0.05~0.1:1；In step 2), goat-anti people's Complement C4 of coating
The mass ratio of polyclonal antibody and carboxyl microballoon is 0.05~0.1:1.
The goat-anti people's Complement C4 polyclonal antibody added in step 2) and step 3) is delayed in advance with the MES of pH=6.5~7.3
Fliud flushing is diluted to 20g/L.
In step 3), also added with preservative proclin300 and/or Sodium azide, the preservative addition concentration is 0.5
~1g/L.
The key technology and positive effect of the present invention is：
1st, a key technology of the invention is that goat-anti people's Complement C4 polyclonal antibody is controlled in reagent B with activating in latex
The mass ratio of carboxyl microballoon is 100~200:1, while the grain size of carboxyl microballoon is 30~80nm especially 40~50nm, is made
A small amount of coating antibody is combined with carboxyl microballoon, is so just formd common antibody and is carried the mixed of the coated antibody of carboxyl microballoon
Zoarium, and pass through the microballoon of 0.2 μ L membrane filtrations removal aggregation.When adding in antigen, antigen will proportionally with both
Antibody combines, and assembles the particulate matter of formation and will be greatly increased comprising 1 or several carboxyl microballoons, the performance of reaction, as far as possible
Avoid nonspecific reaction.
2nd, another key technology of the present invention is the dosage that macromolecule promotees poly- agent, and concentration is 0.1~0.5g/L.The present invention due to
Add the activation latex containing carboxyl microballoon, the reactivity of antibody dramatically increases, if but by the prior art for promote exempt from
Epidemic disease compound is assembled and the macromolecule of excessive addition promotees poly- agent, instead can make more carboxyl microsphere aggregations, generates non-specific
Reaction.The present invention attempts to adjust the dosage of the poly- agent of macromolecule rush to avoid nonspecific reaction as far as possible, due to containing activation
Carboxyl microballoon, reaction has certain sensitivity in itself, it is contemplated that less macromolecule promote poly- agent whether just can with compared with
The good rush effect of gathering.The actual result of experiment also really in this way, when macromolecule promote poly- agent dosage be reduced to concentration for 0.1~
It can reach preferable effect during 0.5g/L.
3rd, the present invention also has the preparation process that a key technology is related to the reagent B, the carboxyl microballoon warp first in latex
Incubation is mixed with goat-anti people's Complement C4 polyclonal antibody of coating after NHS, EDC activation, is then mixed with remaining component, is prepared
Into reagent B.Compared with traditional latex preparation process, NHS, EDC and goat-anti people's Complement C4 Anti-TNF-α of coating are controlled
The dosage of body can avoid NHS, EDC and the separation process of activated carboxyl microballoon and activated carboxyl microballoon closed process, therefore grasp
More simplify on work.
To sum up, the kit of the invention can be used for detecting containing for complement C4 on automatic clinical chemistry analyzer
Amount compared with existing immunoturbidimetry reagent, can effectively avoid nonspecific reaction, and characteristic absorption wavelength is stablized, and can improve anti-
Sensitivity is answered, detection limit is relatively low, available for the detection of low concentration sample, and compared with traditional latex preparation process, this
Invention eliminates more solid-liquor separation and closed process, more simplifies in operation.
Description of the drawings
Fig. 1 is the concentration of Complement C4 standard serum sample and the correspondence scatter diagram of Δ A values.
Specific embodiment
Examples 1 to 3 kit and its preparation
Kit can be used for latex immunoturbidimetry to measure Complement C4, including reagent A and reagent B.
The component of reagent A is as shown in table 1.
The component of 1 reagent A of table
The preparation steps of reagent A are (preparing 1L)：Respectively according to the concentration of component of Examples 1 to 3 in table 1, glycine is taken
(7.5g, 100mmol) is dissolved in purified water (1L), then adds in sodium chloride, triton X-100, the Macrogol 6000 of corresponding amount
And Sodium azide, mixing simultaneously obtain reagent A with sodium hydroxide adjusting pH.
(2) component of reagent B is as shown in table 2.
The component of 2 reagent B of table
Latex in table 2 is the MES buffer systems of the carboxyl microballoon containing 1g/L, the carboxyl microballoon in latex through NHS and
EDC is activated and is coated with by goat-anti people's Complement C4 polyclonal antibody, NHS, EDC and goat-anti people's complement in the latex of Examples 1 to 3
C4 polyclonal antibodies are with carboxyl microspheres quality than as shown in table 3.
The mass ratio of each component and carboxyl microballoon in 3 latex of table
The preparation steps of reagent B are (preparing 1L)：
1) the carboxyl microballoon (being provided by Bangs laboratories, Inc) that 100 μ L solid contents are 10g/100mL is provided,
Be diluted to 10mL with MES buffer solutions (50mmol/L, pH=6.8, similarly hereinafter), then add in table 3 shown in ratio corresponding amount NHS and
EDC, room temperature reaction obtain activation latex for 20 minutes.
2) the people's Complement C4 polyclonal antibody of goat-anti containing 30g/L of ratio corresponding volume shown in table 3 is added in into activation latex
MES buffer solutions, then at 37 DEG C shaking be incubated 2 it is small when the coated latex of goat-anti people's Complement C4 polyclonal antibody is made, i.e.,
Latex described in table 2.
3) sodium chloride, triton X-100, the Sodium azide of ratio corresponding amount shown in table 2 are added in into the MES buffer solutions of 500mL
With the MES buffer solutions of the people's Complement C4 polyclonal antibody of goat-anti containing 30g/L of ratio corresponding volume shown in table 2, step is then added in
2) the coated 10mL latex of goat-anti people Complement C4 polyclonal antibody made from, and complement to 1L with MES buffer solutions.
4) with the nylon membrane filtration of 0.22 μ L, degerming and except bulky grain obtains reagent B after mixing.
Effect example 1
Measure of merit kit：The kit of Examples 1 to 3, the Complement C4 kit of comparative example (by the new health in Sichuan into
Biological Co., Ltd. produces).
Instrument：7170 Biochemical Analyzer of Hitachi.
Test wavelength：Dominant wavelength 340nm, commplementary wave length 700nm.
Test method：End-point method, incremental reaction.Under the conditions of 37 DEG C, the serum sample mixing of reagent A and Complement C4 first
It 3~5 minutes, then adds in reagent B and starts to react, wherein volume ratio reagent A:Reagent B:Sample=200:40:2；5 minute when ratio
According to blank determination initial absorbance A1, then at 10 minutes according to blank determination absorbance A 2, the difference DELTA of calculating A2 and A1
A。
1st, the measure of standard curve
Measure the benefit of series of standards concentration as shown in table 4 one by one respectively with the kit of Examples 1 to 3 and comparative example
The Δ A values of the standard serum sample of body C4, and can be fitted respectively respectively with the Δ A values measured according to a series of concentration of Complement C4s
The standard curve of kit, standard curve are LOGIT-LOG4P functions.
The concentration of Complement C4 is with measuring the corresponding scatter diagrams of Δ A values as shown in Figure 1, as shown in Figure 1, Examples 1 to 3 phase
Than in comparative example, the linear relationship of the concentration and Δ A values of Complement C4 is more preferable, the slope of the standard curve of fitting is by bigger, accurately
Degree and sensitivity have larger promotion.
The Δ A value test results of 4 standard serum sample of table
2nd, the detection result of standard curve
It is a series of containing known concentration Complement C4 shown in the table 5 measured respectively with the kit of Examples 1 to 3 and comparative example
Serum sample Δ A values, then draw corresponding measured concentration respectively on the standard curve of fitting, the measured concentration and reality
Border concentration is present with deviation, as shown in table 5.
5 measurement result of table compares
Clinical accuracy requirement deviation is no more than 10%.By table 5, the kit of comparative example is in detection 3.3mg/dL
Sample when deviation be more than just the 10% of clinical requirement, detection limit can only be 6.6mg/dL.And the kit of the present invention exists
3.3mg/dL samples acquired results and actual concentrations deviation are detected within 10%, meets clinical accuracy requirement, thus it is minimum
Detection limit can reach 3.3mg/dL.
Claims (11)
1. a kind of Complement C4 kit based on latex immunoturbidimetry, which is characterized in that the kit includes：
Reagent A, the reagent A are the glycine buffer system of pH=8.0~9.4, and the macromolecule containing 0.1~0.5g/L promotees poly-
The electrolyte of agent, the surfactant of 1~10g/L and 0.5~10g/L；
And the MES buffer systems that reagent B, the reagent B are pH=6.5~7.3, goat-anti people's complement containing 1.5~2g/L
The coated latex of goat-anti people's Complement C4 polyclonal antibody, the surfactant of 1~10g/L of C4 polyclonal antibodies, 5~10mL/L
With the electrolyte of 0.5~10g/L, wherein, the latex is the MES buffer systems of pH=6.5~7.3, be grain size for 30~
The latex that the carboxyl microballoon of 80nm is coated with after NHS and EDC activation by goat-anti people's Complement C4 polyclonal antibody again；The glue
In breast, the concentration of carboxyl microballoon is 1~2g/L.
2. kit as described in claim 1, which is characterized in that the latex is the MES buffer systems of pH=6.5~7.3,
It is that the carboxyl microballoon that grain size is 40~50nm is coated with to obtain by goat-anti people's Complement C4 polyclonal antibody again after NHS and EDC activation
Latex；In the latex, the concentration of carboxyl microballoon is 1g/L, and the mass ratio of NHS and carboxyl microballoon is 0.05~0.1:1, EDC
Mass ratio with carboxyl microballoon is 0.05~0.1:1, goat-anti people's Complement C4 polyclonal antibody of coating and the matter of carboxyl microballoon
Amount is than being 0.05~0.1:1.
3. kit as claimed in claim 1 or 2, which is characterized in that the reagent A and the reagent B also contain 0.5 respectively
The preservative of~1g/L, the preservative are proclin300 and/or Sodium azide.
4. kit as claimed in claim 3, which is characterized in that it is polyethylene glycol that the macromolecule in the reagent A, which promotees poly- agent,
6000, concentration of the Macrogol 6000 in the reagent A is 0.2~0.5g/L；Table in the reagent A and the reagent B
Face activating agent is triton X-100；Electrolyte in the reagent A and the reagent B is sodium chloride.
5. kit as claimed in claim 4, which is characterized in that the reagent A is the 100mmol/L glycine of pH=8.6
Buffer system, the nitrine of the triton X-100 of Macrogol 6000,1g/L, the sodium chloride of 5g/L and 1g/L containing 0.5g/L
Sodium.
6. kit as claimed in claim 4, which is characterized in that the 50mmol/L MES that the reagent B is pH=6.8 are buffered
System, the coated glue of goat-anti people's Complement C4 polyclonal antibody of goat-anti people's Complement C4 polyclonal antibody, 5mL/L containing 1.8g/L
The Sodium azide of breast, the triton X-100 of 1g/L, the sodium chloride of 5g/L and 1g/L.
7. a kind of prepare for the method for the reagent B in the Complement C4 kit based on latex immunoturbidimetry, which is characterized in that
The reagent B be pH=6.5~7.3 MES buffer systems, goat-anti people's Complement C4 polyclonal antibody containing 1.5~2g/L, 5
The coated latex of goat-anti people's Complement C4 polyclonal antibody of~10mL/L, the surfactant of 1~10g/L and 0.5~10g/L
Electrolyte；
This method comprises the following steps：
1) carboxyl microballoon is diluted to 1~2g/L with the MES buffer solutions of pH=6.5~7.3, adds NHS and EDC, 15~25
It is reacted at DEG C and obtains within 20~30 minutes activation latex；
2) goat-anti people's Complement C4 polyclonal antibody of coating is added in into activation latex, the shaking incubation 2~4 at 25~37 DEG C
Hour, the coated latex of goat-anti people's Complement C4 polyclonal antibody is made；
3) according to each component concentration of the reagent B, surfactant, electricity are added in into the MES buffer solutions of pH=6.5~7.3
Matter and goat-anti people's Complement C4 polyclonal antibody are solved, it is coated then to add in goat-anti people's Complement C4 polyclonal antibody made from step 2)
Latex；
4) with 0.2 μ L nylon membranes filtration sterilizations and bulky grain, reagent B is obtained.
8. the method for claim 7, which is characterized in that the carboxyl microspherulite diameter in step 1) is 30~80nm；NHS with
The mass ratio of carboxyl microballoon is 0.05~0.1:1, EDC with the mass ratio of carboxyl microballoon is 0.05~0.1:1；In step 2), bag
By the mass ratio of goat-anti people's Complement C4 polyclonal antibody and carboxyl microballoon be 0.05~0.1:1.
9. method as claimed in claim 8, which is characterized in that the carboxyl microspherulite diameter in step 1) is 40~50nm.
10. the method for claim 7, which is characterized in that the goat-anti people's Complement C4 added in step 2) and step 3) is more
Clonal antibody is diluted to 20~30g/L with the MES buffer solutions of pH=6.5~7.3 in advance.
11. the method for claim 7, which is characterized in that in step 3), also added with preservative proclin300 and/
Or Sodium azide, the preservative addition concentration is 0.5~1g/L.
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| CN109752332A (en) * | 2017-11-07 | 2019-05-14 | 重庆中元汇吉生物技术有限公司 | A kind of C1Q detection kit |
| CN111693709A (en) * | 2019-03-12 | 2020-09-22 | 程明 | Complement C1q determination kit and determination method thereof |
| CN111693699A (en) * | 2020-07-07 | 2020-09-22 | 上海怡珏生物科技有限公司 | Application of C4 antibody in preparation of detection kit |
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| CN102628867A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Double antibody latex enhanced retinol binding protein detection kit |
| 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 |
| CN102955033A (en) * | 2012-10-22 | 2013-03-06 | 金华市强盛生物科技有限公司 | Kit for determining glycocholic acid in human blood |
| CN103018464A (en) * | 2012-12-12 | 2013-04-03 | 元升生物科技（上海）有限公司 | Reagent for determining procalcitonin and preparation method of reagent |
| CN103604931A (en) * | 2013-11-15 | 2014-02-26 | 陆上苏 | Human S100 protein detection reagent and preparation method thereof |
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