CN109100515A - A kind of kit and preparation method thereof measuring cyclic citrullinated peptid concentration - Google Patents
A kind of kit and preparation method thereof measuring cyclic citrullinated peptid concentration Download PDFInfo
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Abstract
The present invention relates to field of biotechnology more particularly to a kind of kits for measuring cyclic citrullinated peptid concentration.The technical solution adopted by the present invention is that: a kind of kit measuring cyclic citrullinated peptid concentration, it is characterized by comprising reagent R1 and reagent R2, R1 reagent includes buffer, promotor, preservative, and R2 reagent includes that buffer, stabilizer, coupling have cyclic citrullinated peptide antigen-biotin-Streptavidin latex microsphere, preservative.The invention has the advantages that the kit of measurement cyclic citrullinated peptid concentration of the invention, Streptavidin 1:1 is connected using latex microsphere, cyclic citrullinated peptide antigen connects biotin 1:2, the two is again with 1:1 hybrid reaction, can obviously improve repeatability and sensitivity, the range of linearity can accomplish 5-150U/ml.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of kits and its system for measuring anti-cyclic citrulline peptide concentration
Preparation Method.
Background technique
Cyclic citrulline polypeptide antibody (anti-cyclic peptide containing citrulline, anti-
CCP): being more skin segments of cyclic annular polyguanidine albumen, the antibody based on IgG type.AntiCCP antibody is by RA patient's bone-marrow-derived lymphocyte
Autocrine, and other diseases patient and normal population bone-marrow-derived lymphocyte not Autocrine antiCCP antibody.Therefore, anti-CCP
Antibody is to RA specificity with higher.
Rheumatoid arthritis (RA) is a kind of common systemic autoimmune disease, with joint synovitis disease and
Symmetry, destructive arthropathy are main clinical characteristics, and the course of disease is long, easily repeatedly, very big pain is caused to patient.It should
Disease incidence of the disease in China is about 0.4%, and the whole world is 0.5-1%, 20-50 years old morbidity's age.
Studies have shown that can detecte APF ELISA (APF), anti-keratin antibody (AKA) in the serum of RA patient,
Its antigen site is cyclic citrulline polypeptide (CCP).And the detection of Anti-cyclic critrullinated polypeptide antibody has compared with APF and AKA detection
Certain superiority, therefore, the detection of Anti-cyclic critrullinated polypeptide antibody have great importance to the diagnosis of RA.In conventional method
AntiCCP antibody is directly coupled using latex microsphere, our company is poor using this method discovery sensitivity and linear measurement range.Therefore,
The new technical solution of one kind should be provided to solve the above problems.
Summary of the invention
The object of the present invention is to provide a kind of high sensitivity, a kind of reproducible measurement cyclic citrullinated peptid is dense
Kit of degree and preparation method thereof.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of kit measuring cyclic citrullinated peptid concentration, including reagent R1 and reagent R2, the reagent R1's
Each component and concentration include:
The reagent R2 includes the second buffer, save liquid, coupling has cyclic citrullinated peptide antigen-biotin-strepto- is affine
The latex microsphere of element, the concentration of second buffer are 8.05-27.30g/L, each component and concentration packet for saving liquid
It includes:
N, N- bicine N- 1.26-9.83g/L
Stabilizer 0.5-2.0g/L
Second preservative 0.8-2.0ml/L
The each component and concentration of latex microsphere conjugate include:
Further technical solution:
The first buffer solution A, the first buffer solution B in the reagent R1 and the second buffer in reagent R2 are PBS
Buffer, HEPES buffer solution, MES buffer, Tris buffer, glycine delay the combination of one or more of liquid.
Preferably, first buffer solution A is sodium dihydrogen phosphate dihydrate, the first buffer solution B is 12 hypophosphite monohydrate hydrogen
Disodium, the second buffer are 2- (N- morpholine) ethanesulfonic acid monohydrate
Preservative in the reagent R1 and R2 is Sodium azide, in Proclin-950, Proclin-300, thimerosal
One or more of combinations.
Stabilizer is the group of one or more of bovine serum albumin(BSA), casein, gelatin, mannitol in the reagent R2
It closes.
Preferably, stabilizer is bovine serum albumin(BSA) in the reagent R2.
Partial size selected by the latex microsphere is 50-300nm, and surface modification group is carboxyl, in hydroxyl, aldehyde radical, amino
One kind.
The pH of the reagent R1 and R2 is between 6.30-8.50.
Biotin combination cyclic citrullinated peptide antigen ratio is between 1:1-3:1, Streptavidin combination biotin-ring melon ammonia
Sour peptide antigen ratio is between 0.5:1-4:1.
Measure the preparation method of the kit of cyclic citrullinated peptid concentration, it is characterised in that: include the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept to put into the first buffer solution A while stirring with 0.8-3.2g/L standard, with the mark of 8.75-24.31g/L
Quasi- the first buffer solution B of investment is completely dissolved to material for stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating,
PH value is adjusted between 6.30-8.50, later with 5.8-20.0g/L standard, puts into sodium chloride, after material is completely dissolved again by
According to above-mentioned feeding mode respectively with the standard of 40-120 g/L, 0.8%-2.0%, PEG 6000, the first preservative are successively put into,
After the completion of feeding intake, continue stirring and be completely dissolved to whole materials for 5-30 minutes, solution is as clear as crystal, Agitation Tank bottom without precipitating,
It is settled to final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
B1, buffer are first added appropriate purified water on magnetic stirring apparatus in Agitation Tank, open magnetic stirring apparatus
Power switch adjusts stirrer to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 8.05-27.30g/L, side
It stirs side and puts into the second buffer, stirring is completely dissolved for 5-30 minutes to material, and the as clear as crystal Agitation Tank bottom of solution is without precipitating
Afterwards, it is as clear as crystal to solution between 6.30-8.50, continuing stirring 5-30 minutes to adjust pH value, Agitation Tank bottom is fixed without precipitating
Hold to final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B2, liquid preparation is saved, appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer is into
Shelves revolving speed, makes solution keep middling speed rotary state, puts into N while stirring with the standard of 1.26-9.83g/L, N- dihydroxy ethyl is sweet
Propylhomoserin is completely dissolved to material, adjusts pH value between 6.30-8.50, marked later with 0.5-2.0g/L for stirring 5-30 minutes
Standard puts into stabilizer, after material is completely dissolved, then with 0.8-2.0ml/L standard, puts into the second preservative, continues to stir 5-
It is completely dissolved to whole materials within 30 minutes, solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
It is spare, it is identified；
Latex microsphere is diluted to the concentration of 0.05-2.0% by the preparation of b3, latex microsphere antibody coupling matter with buffer,
Again with 1:1 ratio in conjunction with Streptavidin, cyclic citrullinated peptide antigen is diluted to the concentration of 0.05-3.0g/L with reaction solution,
It after in conjunction with ratio of the biotin in 3:1-1:1, is added in the microballoon containing Streptavidin, mixes, Streptavidin knot
Biotin-cyclic citrullinated peptide antigen ratio is closed between 0.5:1-4:1,1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Amine hydrochlorate is dissolved into the concentration of 0.005-0.3g/L with reaction solution, is added dropwise in above-mentioned mixed solution when shaking up, room temperature is anti-
Answer 150-200 minutes, be added bovine serum albumin(BSA) closing, oscillation mix, react at room temperature 40-80 minutes, by mixed liquor with
The revolving speed centrifugation of 14000rpm 40-80 minutes, sucks supernatant, is added and saves liquid, and ultrasound is resuspended, repeated centrifugation cleaning 3 times, most
It after supernatant is removed in primary centrifugation afterwards, is added and saves liquid, ultrasound is resuspended, the R2 prepared is fitted into finished pot, is identified.
Due to the application of above-mentioned technical proposal, the invention has the following advantages over the prior art:
The kit of measurement cyclic citrullinated peptid concentration of the invention, connects Streptavidin 1 using latex microsphere:
1, cyclic citrullinated peptide antigen connects biotin 1:2, and the two with 1:1 hybrid reaction, can obviously improve repeatability and sensitivity again,
The range of linearity can accomplish 5-150 U/ml.
Detailed description of the invention
Fig. 1 is the calibration curve figure that R2 reagent does not use Cascaded amplification to be coupled in the embodiment of the present invention 4.Wherein X-axis indicates
Standard concentration, Y-axis indicate absorbance.
Fig. 2 is the calibration curve figure that R2 reagent uses Cascaded amplification coupling in the embodiment of the present invention 4.Wherein X-axis indicates mark
Quasi- product concentration, Y-axis indicate absorbance.
Specific embodiment
The invention will be further described combined with specific embodiments below:
Embodiment 1:
A kind of kit measuring cyclic citrullinated peptid concentration, including reagent R1 and reagent R2, the reagent R1's
Each component and concentration include: the sodium dihydrogen phosphate dihydrate as the first buffer solution A, and concentration 0.8g/L is slow as first
The disodium hydrogen phosphate dodecahydrate of fliud flushing B, concentration 24.31g/L, as the sodium chloride of the first electrolyte, concentration is
5.8g/L, as the PEG 6000 of the first macromolecule promotor, concentration 40g/L, and as the first preservative
Proclin-950, concentration 2.0%.
The reagent R2 includes the second buffer, save liquid, coupling has cyclic citrullinated peptide antigen-biotin-strepto- is affine
The latex microsphere of element, second buffer are 2- (N- morpholine) ethanesulfonic acid monohydrate, concentration 8.05g/L；The guarantor
Liquid storage includes: the N that concentration is 9.83g/L, and N- bicine N-, the bovine serum albumin(BSA) as stabilizer, concentration is
0.5g/L's, as the Proclin-950 of the second preservative, concentration is 0.8 ml/L；The each component of latex microsphere conjugate
And concentration includes: the latex microsphere that concentration is 2%, concentration is the biotin of 5g/L, and concentration is the Streptavidin of 5g/L, concentration
1- (3- the dimethylamino-propyl) -3- ethyl carbon two that cyclic citrullinated peptide antigen and concentration for 0.05g/L are 0.005g/L
Inferior amine salt hydrochlorate.
The preparation method of the kit of the concentration of the kit of the above measurement cyclic citrullinated peptid concentration, including it is as follows
Step,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, investment concentration is 0.8 g/L sodium dihydrogen phosphate dihydrate while stirring and concentration is 24.31g/L's
Disodium hydrogen phosphate dodecahydrate is completely dissolved to material, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts for stirring 30 minutes
Saving pH value is 6.00, the sodium chloride that concentration is 5.8g/L is put into later, after material is completely dissolved, according still further to above-mentioned feeding mode
PEG 6000 and Proclin-950 is successively put into, the concentration of both of the above is respectively 40g/L and 2.0%, after the completion of feeding intake, after
Continuous stirring is completely dissolved for 5 minutes to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
B1, buffer are first added appropriate purified water on magnetic stirring apparatus in Agitation Tank, open magnetic stirring apparatus
Power switch adjusts stirrer to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 8.05g/L, while stirring
Put into 2- (N- morpholine) ethanesulfonic acid monohydrate, be completely dissolved to material within stirring 5 minutes, the as clear as crystal Agitation Tank bottom of solution without
After precipitating, adjusting pH value is 6.30, continue stirring 5 minutes it is as clear as crystal to solution, Agitation Tank bottom is settled to final without precipitating
Volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B2, liquid preparation is saved, appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer is into
Shelves revolving speed, makes solution keep middling speed rotary state, puts into N while stirring with the standard of 9.83 g/L, N- bicine N-,
It is completely dissolved to material within stirring 5 minutes, adjusting pH value is 6.30, later with 0.5g/L standard, bovine serum albumin(BSA) is put into, to object
After material is completely dissolved, then with 0.8ml/L standard, put into Proclin-950, continue stirring 30 minutes it is completely molten to whole materials
Solution, solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
It is spare, it is identified；
The preparation of b3, latex microsphere antibody coupling matter, partial size selected by latex microsphere are 50nm, and surface modification group is carboxylic
Latex microsphere is diluted to buffer 2.0% concentration by base, then with 1:1 ratio in conjunction with Streptavidin, by cyclic citrulline
Peptide antigen is diluted to the concentration of 0.05g/L with reaction solution, in conjunction with ratio of the biotin in 2:1 after, be added to containing strepto- parent
It in the microballoon of element, mixes, Streptavidin combination biotin-CCP cyclic citrullinated peptide antigen ratio is 1:1, by 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride is dissolved into the concentration of 0.005g/L with reaction solution, it is added dropwise to when shaking up above-mentioned
It in mixed solution, reacts at room temperature 150 minutes, bovine serum albumin(BSA) closing is added, oscillation mixes, and reacts at room temperature 40 minutes, will mix
It closes liquid to be centrifuged 40 minutes with the revolving speed of 14000rpm, sucks supernatant, be added and save liquid, ultrasound is resuspended, repeated centrifugation cleaning 3
It is secondary, it after supernatant is removed in last time centrifugation, is added and saves liquid, ultrasound is resuspended, the R2 prepared is fitted into finished pot, is marked
Know.
Embodiment 2
A kind of kit measuring cyclic citrullinated peptid concentration, including reagent R1 and reagent R2, the reagent R1's
Each component and concentration include the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 3.2g/L, as the first buffering
The disodium hydrogen phosphate dodecahydrate of liquid B, concentration 8.75g/L, as the sodium chloride of the first electrolyte, concentration 20.0g/
L, as the PEG 6000 of the first macromolecule promotor, concentration 80g/L, as the Proclin-300 of the first preservative,
Concentration is 0.8%, and the reagent R2 has cyclic citrullinated peptide antigen-biotin-strepto- including the second buffer, preservation liquid, coupling
The latex microsphere of Avidin, second buffer are 2- (N- morpholine) ethanesulfonic acid monohydrate, concentration 27.30g/L；Institute
Stating and saving liquid includes: the N that concentration is 5.0g/L, and N- bicine N-, the casein as stabilizer, concentration is
2.0g/L's, as the Proclin-300 of the second preservative, concentration 2.0ml/L；The each component of latex microsphere conjugate and
Concentration includes: the latex microsphere that concentration is 0.05%, and concentration is the biotin of 20g/L, and concentration is the Streptavidin of 20g/L,
1- (3- the dimethylamino-propyl) -3- ethyl carbon two that the cyclic citrullinated peptide antigen and concentration that concentration is 3.0g/L are 0.3g/L is sub-
Amine hydrochlorate.
The preparation method of the kit of the above measurement cyclic citrullinated peptid concentration, includes the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, investment concentration is 3.2 g/L sodium dihydrogen phosphate dihydrate while stirring and concentration is the ten of 8.75g/L
Two hypophosphite monohydrate disodium hydrogens are completely dissolved to material, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjust for stirring 5 minutes
PH value is 7.00, later put into concentration be 20g/L sodium chloride, after material is completely dissolved, according still further to above-mentioned feeding mode according to
Secondary investment PEG 6000 and Proclin-300, the concentration of both of the above is respectively 80g/L and 0.8%, after the completion of feeding intake, is continued
Stirring is completely dissolved for 30 minutes to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
B1, buffer are first added appropriate purified water on magnetic stirring apparatus in Agitation Tank, open magnetic stirring apparatus
Power switch adjusts stirrer to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 27.3g/L, while stirring
2- (N- morpholine) ethanesulfonic acid monohydrate is put into, is completely dissolved to material within stirring 30 minutes, the as clear as crystal Agitation Tank bottom of solution
After precipitating, adjust pH value to 7, continue stirring 18 minutes it is as clear as crystal to solution, Agitation Tank bottom is settled to final without precipitating
Volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B2, liquid preparation is saved, appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer is into
Shelves revolving speed, makes solution keep middling speed rotary state, puts into N while stirring with the standard of 5.0g/L, N- bicine N-,
It is completely dissolved to material within stirring 18 minutes, adjusting pH value is 7, later with 2.0g/L standard, puts into casein, completely molten to material
Xie Hou, then with 2.0ml/L standard, Proclin-300 is put into, continue stirring and be completely dissolved to whole materials for 18 minutes, solution is clear
Clear transparent, Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
It is spare, it is identified；
The preparation of b3, latex microsphere antibody coupling matter, partial size selected by latex microsphere are 180nm, and surface modification group is
Latex microsphere is diluted to buffer 0.05% concentration by hydroxyl, then with 1:1 ratio in conjunction with Streptavidin, by ring melon
Propylhomoserin peptide antigen is diluted to the concentration of 3.0g/L with reaction solution, in conjunction with ratio of the biotin in 3:1 after, be added to containing strepto-
It in the microballoon of Avidin, mixes, Streptavidin combination biotin-cyclic citrullinated peptide antigen ratio is 4:1, by 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride is dissolved into the concentration of 0.3g/L with reaction solution, it is added dropwise to when shaking up above-mentioned mixed
Close solution in, react at room temperature 200 minutes, be added casein closing, oscillation mix, react at room temperature 60 minutes, by mixed liquor with
The revolving speed centrifugation of 14000rpm 60 minutes, sucks supernatant, is added and saves liquid, and ultrasound is resuspended, repeated centrifugation cleaning 3 times, finally
It after supernatant is removed in primary centrifugation, is added and saves liquid, ultrasound is resuspended, the R2 prepared is fitted into finished pot, is identified.
Embodiment 3
A kind of kit measuring cyclic citrullinated peptid concentration, including reagent R1 and reagent R2, the reagent R1's
Each component and concentration include the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 2g/L, as the first buffering
The disodium hydrogen phosphate dodecahydrate of liquid B, concentration 16g/L, as the sodium chloride of the first electrolyte, concentration 12g/L makees
For the PEG 6000 of the first macromolecule promotor, concentration 120g/L, as the thimerosal of the first preservative, concentration is
1.0%, the reagent R2 have cyclic citrullinated peptide antigen-biotin-Streptavidin including the second buffer, preservation liquid, coupling
Latex microsphere, second buffer be 2- (N- morpholine) ethanesulfonic acid monohydrate, concentration 16.80g/L；The guarantor
Liquid storage includes: the N that concentration is 1.26g/L, N- bicine N-, the mannitol as stabilizer, concentration 1.2g/L
, as the Sodium azide of the second preservative, concentration 1.5ml/L；The each component and concentration of latex microsphere conjugate include: dense
The latex microsphere that degree is 1.0%, concentration are the biotin of 13g/L, and concentration is the Streptavidin of 13g/L, concentration 1.5g/L
Cyclic citrullinated peptide antigen and concentration be 0.15g/L 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride.
The preparation method of the kit of the concentration of the above measurement measurement cyclic citrullinated peptid concentration, including walk as follows
Suddenly,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, solution is made to keep middling speed rotary state, putting into concentration while stirring is bis- hypophosphite monohydrate dihydro of 2g/L
The disodium hydrogen phosphate dodecahydrate that sodium and concentration are 16g/L is completely dissolved for stirring 18 minutes to material, and solution is as clear as crystal to match liquid
After pot bottom is without precipitating, adjusting pH value is 8.5, puts into the sodium chloride that concentration is 12g/L later, after material is completely dissolved, then
PEG 6000 and thimerosal are successively put into according to above-mentioned feeding mode, the concentration of both of the above is respectively 120g/L and 1.0%,
After the completion of feeding intake, continue stirring and be completely dissolved to whole materials for 18 minutes, solution is as clear as crystal, and Agitation Tank bottom is fixed without precipitating
Hold to final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
B1, buffer are first added appropriate purified water on magnetic stirring apparatus in Agitation Tank, open magnetic stirring apparatus
Power switch adjusts stirrer to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 16.8g/L, while stirring
2- (N- morpholine) ethanesulfonic acid monohydrate is put into, is completely dissolved to material within stirring 18 minutes, the as clear as crystal Agitation Tank bottom of solution
After precipitating, adjust pH value to 7, continue stirring 30 minutes it is as clear as crystal to solution, Agitation Tank bottom is settled to final without precipitating
Volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B2, liquid preparation is saved, appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer is into
Shelves revolving speed, makes solution keep middling speed rotary state, puts into N while stirring with the standard of 1.26 g/L, N- bicine N-,
It is completely dissolved to material within stirring 30 minutes, adjusting pH value is 8.5, later with 1.2g/L standard, puts into mannitol, complete to material
After dissolution, then with 1.5ml/L standard, Sodium azide is put into, continues stirring and be completely dissolved to whole materials for 5 minutes, solution is limpid
Bright, Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
It is spare, it is identified；
The preparation of b3, latex microsphere antibody coupling matter, partial size selected by latex microsphere are 300nm, and surface modification group is
Latex microsphere is diluted to buffer 1.0% concentration by amino, then with 1:1 ratio in conjunction with Streptavidin, by ring melon ammonia
Sour peptide antigen is diluted to the concentration of 3g/L with reaction solution, in conjunction with ratio of the biotin in 1:1 after, be added to containing strepto- parent
It in the microballoon of element, mixes, Streptavidin combination biotin-cyclic citrullinated peptide antigen ratio is 0.5:1, by 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride is dissolved into the concentration of 0.15g/L with reaction solution, it is added dropwise to when shaking up above-mentioned
In mixed solution, react at room temperature 180 minutes, be added mannitol closing, oscillation mix, react at room temperature 80 minutes, by mixed liquor with
The revolving speed centrifugation of 14000rpm 80 minutes, sucks supernatant, is added and saves liquid, and ultrasound is resuspended, repeated centrifugation cleaning 3 times, finally
It after supernatant is removed in primary centrifugation, is added and saves liquid, ultrasound is resuspended, the R2 prepared is fitted into finished pot, is identified.
Embodiment 4
A kind of application for the kit for measuring cyclic citrullinated peptid concentration of the present invention, its working principle is that: a molecule
Streptavidin can be with high degree of specificity in conjunction with four molecular biosciences elements, and affinity between the two is extremely strong, form grade
Join iodine.Detect its corresponding Streptavidin of cyclic citrullinated peptid-Biotin-Antibody sensitization latex in sample
Particle meets in the solution, is reacted using Cascaded amplification, forms antigen-antibody complex, is aggregated latex particle, generates
Turbidity variation.Content of the height of turbidity variation in the presence of sufficient antibodies with cyclic citrullinated peptid in sample is at just
Than, under certain wavelength, by compared with the calibration object of known concentration, can quantitative detection to go out anti-cyclic citrullinated peptide in sample anti-
The content of body.
Specific measurement result is compared as follows:
R2 reagent is directly coupled CCP cyclic citrullinated peptide antigenic reagent box using latex microsphere, is calibrated using standard items,
The results are shown in Table 1, and calibration curve is as shown in Figure 1.
Table 1, R2 reagent do not use Cascaded amplification to be coupled, and latex microsphere is directly coupled cyclic citrullinated peptide antigen the calibration results:
As a result parse: as shown in figure 1 and table 1, there is hook effect when being greater than 100U/ml in concentration.
R2 reagent uses latex microsphere indirect conjugation cyclic citrullinated peptide antigenic reagent box, is calibrated using standard items, ties
Fruit is as shown in table 2, and calibration curve is as shown in Figure 2.
Table 2, R2 reagent are coupled using Cascaded amplification, latex microsphere indirect conjugation cyclic citrullinated peptide antigen the calibration results:
As a result parse: as shown in Fig. 2 and table 2, the range of linearity can accomplish 5-150U/ml.
Embodiment 5
Precision CV detection:
R2 reagent is directly coupled cyclic citrullinated peptide antigenic reagent box using latex microsphere, takes 3 parts of part clinical serum samples at random
This repeats detection 10 times to same sample using automatic clinical chemistry analyzer, and testing result is as shown in table 3.
Table 3: the cyclic citrullinated peptid content (10 times) and the coefficient of variation of sample are detected
As a result parse: precision CV is larger, basic > 5%.
R2 reagent uses the kit of latex microsphere indirect conjugation cyclic citrullinated peptide antigen, takes 3 parts of clinical serum samples at random
This repeats detection 10 times to same sample using automatic clinical chemistry analyzer, and testing result is as shown in table 4.
Table 4: the latex microsphere indirect conjugation cyclic citrullinated peptide antigenic content (10 times) and the coefficient of variation of sample are detected
As a result parse: precision CV is obviously improved, within 4%.
Conclusion: using the kit of latex microsphere indirect conjugation cyclic citrullinated peptide antigen, i.e. latex microsphere connection strepto- parent
Biotin 1:2 is connected with plain 1:1 cyclic citrullinated peptide antigen, the two with 1:1 hybrid reaction, can obviously improve repeated and sensitive again
Degree, the range of linearity can accomplish 5-150 U/ml.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements or replace, these are improved or replacement
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of kit for measuring cyclic citrullinated peptid concentration, it is characterised in that: described including reagent R1 and reagent R2
The each component and concentration of reagent R1 include:
The reagent R2 include the second buffer, save liquid, coupling have cyclic citrullinated peptide antigen-biotin-Streptavidin
Latex microsphere, the concentration of second buffer are 8.05-27.30g/L, and each component for saving liquid and concentration include:
N, N- bicine N- 1.26-9.83g/L
Stabilizer 0.5-2.0g/L
Second preservative 0.8-2.0ml/L
The each component and concentration of latex microsphere conjugate include:
Latex microsphere 0.05-2.0%
Biotin 5-20g/L
Streptavidin 5-20g/L
Cyclic citrullinated peptide antigen 0.05-3.0g/L
1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride 0.005-0.3g/L.
2. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: described
The first buffer solution A, the first buffer solution B in reagent R1 and the second buffer in reagent R2 are PBS buffer solution, HEPES
Buffer, MES buffer, Tris buffer, glycine delay the combination of one or more of liquid.
3. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: described
Preservative in reagent R1 and R2 is the group of one or more of Sodium azide, Proclin-950, Proclin-300, thimerosal
It closes.
4. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: described
Stabilizer is the combination of one or more of bovine serum albumin(BSA), casein, gelatin, mannitol in reagent R2.
5. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: described
Partial size selected by latex microsphere is 50-300nm, and surface modification group is one of carboxyl, hydroxyl, aldehyde radical, amino.
6. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: described
The pH of reagent R1 and R2 are between 6.30-8.50.
7. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 1, it is characterised in that: biology
Element combines cyclic citrullinated peptide antigen ratio between 1:1-3:1, Streptavidin combination biotin-cyclic citrullinated peptide antigen ratio
Between 0.5:1-4:1.
8. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 2, it is characterised in that: described
First buffer solution A is sodium dihydrogen phosphate dihydrate, the first buffer solution B is disodium hydrogen phosphate dodecahydrate, and the second buffer is 2-
(N- morpholine) ethanesulfonic acid monohydrate.
9. a kind of kit for measuring cyclic citrullinated peptid concentration according to claim 2, it is characterised in that: described
Stabilizer is bovine serum albumin(BSA) in reagent R2.
10. the preparation method of the kit such as any measurement cyclic citrullinated peptid concentration of claim 1-9, feature exist
In: include the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, keeps solution
Middling speed rotary state puts into the first buffer solution A with 0.8-3.2g/L standard while stirring, is thrown with the standard of 8.75-24.31g/L
Enter the first buffer solution B, is completely dissolved to material within stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts
PH value, later with 5.8-20.0g/L standard, puts into sodium chloride, according still further to upper after material is completely dissolved between 6.30-8.50
Feeding mode is stated respectively with the standard of 40-120g/L, 0.8%-2.0%, PEG 6000, the first preservative is successively put into, feeds intake
After the completion, continue stirring to be completely dissolved to whole materials for 5-30 minutes, solution is as clear as crystal, and Agitation Tank bottom is without precipitating, constant volume
To final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored in into
In product tank, it is identified；
B), prepared by reagent R2；
B1, buffer are first added appropriate purified water on magnetic stirring apparatus in Agitation Tank, open magnetic stirring apparatus power supply
Switch adjusts stirrer to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 8.05-27.30g/L, side stirring
Side puts into the second buffer, is completely dissolved to material within stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts
Section pH value is as clear as crystal to solution between 6.30-8.50, continuing stirring 5-30 minutes, and Agitation Tank bottom is settled to without precipitating
Final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored in into
In product tank, it is identified；
B2, liquid preparation is saved, appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade turns
Speed makes solution keep middling speed rotary state, puts into N while stirring with the standard of 1.26-9.83g/L, N- bicine N-,
It is completely dissolved to material within stirring 5-30 minutes, adjusts pH value between 6.30-8.50, later with 0.5-2.0g/L standard, put into
Stabilizer after material is completely dissolved, then with 0.8-2.0ml/L standard, puts into the second preservative, continues stirring 5-30 minutes extremely
Whole materials are completely dissolved, and solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution storage is spare,
It is identified；
Latex microsphere, is diluted to the concentration of 0.05-2.0% by the preparation of b3, latex microsphere antibody coupling matter with buffer, then with
Cyclic citrullinated peptide antigen is diluted to the concentration of 0.05-3.0g/L in conjunction with Streptavidin by 1:1 ratio with reaction solution, with life
It after object element is combined in the ratio of 3:1-1:1, is added in the microballoon containing Streptavidin, mixes, Streptavidin combines life
Object element-cyclic citrullinated peptide antigen ratio is between 0.5:1-4:1, by 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate is dissolved into the concentration of 0.005-0.3g/L with reaction solution, is added dropwise in above-mentioned mixed solution when shaking up, room temperature reaction
150-200 minutes, be added bovine serum albumin(BSA) closing, oscillation mix, react at room temperature 40-80 minutes, by mixed liquor with
The revolving speed centrifugation of 14000rpm 40-80 minutes, sucks supernatant, is added and saves liquid, and ultrasound is resuspended, repeated centrifugation cleaning 3 times, most
It after supernatant is removed in primary centrifugation afterwards, is added and saves liquid, ultrasound is resuspended, the R2 prepared is fitted into finished pot, is identified.
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| CN110763840A (en) * | 2019-11-01 | 2020-02-07 | 苏州普瑞斯生物科技有限公司 | Detection kit for plasminogen activator inhibitor and preparation method thereof |
| CN110760565A (en) * | 2019-11-07 | 2020-02-07 | 苏州普瑞斯生物科技有限公司 | Detection kit for paraoxonase 1 and preparation method thereof |
| CN110824160A (en) * | 2019-11-27 | 2020-02-21 | 迪亚莱博（张家港）生物科技有限公司 | Anti-cyclic citrullinated peptide antibody detection kit and preparation method thereof |
| CN113419069A (en) * | 2021-06-16 | 2021-09-21 | 东软威特曼生物科技(南京)有限公司 | Kit and method for detecting anti-cyclic citrullinated peptide antibody |
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Application publication date: 20181228 |