CN105652018A - C-peptide quantitative determination kit - Google Patents
C-peptide quantitative determination kit Download PDFInfo
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- CN105652018A CN105652018A CN201610081311.4A CN201610081311A CN105652018A CN 105652018 A CN105652018 A CN 105652018A CN 201610081311 A CN201610081311 A CN 201610081311A CN 105652018 A CN105652018 A CN 105652018A
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- 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
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Abstract
The invention relates to a C-peptide quantitative determination kit. The C-peptide quantitative determination kit comprises magnetic particles coated with anti-C-peptide antibodies, acridinium ester-marked anti-C-peptide antibodies, sample diluent, a standard C-peptide, a reaction cleaning solution, a preexciting solution and an exciting solution and is mainly used for diabetes detection. The C-peptide quantitative determination kit which is high in sensitivity and specificity, convenient to operate, wide in linearity range and quick in analysis is obtained by adopting a magnetic separation technology to be combined with an immune reaction and adopting acridinium ester or acridine sulfonamide or an acridine sulfonamide as a marker. Either serum or urine can be adopted as a detection sample, a detection result is accurate and reliable, and the C-peptide quantitative determination kit is long in preservation period and can be preserved for a year.
Description
Technical field
The invention belongs to the aided diagnosis technique field of diabetes, the test kit being specifically related in human serum or urine the quantitative assay of C-peptide.
Background technology
Diabetes are the metabolic diseases being feature with hyperglycemia caused by defect of insulin secretion or insulin action obstacle. C-peptide is the secretory product of beta Cell of islet, and it and islets of langerhans have a common precursor proinsulin. The proinsulin of one molecule, after enzyme action, is cracked into the insulin of a molecule and the C-peptide of a molecule. In the diabetic of use of exogenous insulin, the mensuration of insulin can be subject to exogenous insulin and the interference of peripheral circulation antibody. But carry out the insulin antibody detected by the patient of insulin treatment C-peptide is not interfered significantly with. C-peptide is not absorbed by hepatocyte after producing from human body, mainly excretes at renal metabolism and from kidney. C-peptide inactive, also the receptor on cell membrane is not combined, and the assay method of the C-peptide that is not degraded is to be inherently more, than insulin, the method indicating level of insulin secretion reliably. It addition, for the mensuration applying exogenous insulin, due to C-peptide and insulin antibody no cross reaction, not disturbed by insulin antibody, exogenous insulin is again without C-peptide, therefore C-peptide measures and seems even more important, thus understanding islet beta cell function situation, there is positive role for guiding treatment.
The method being used for measuring C-peptide at present clinically mainly has radioimmunoassay, RIA, enzyme immunoassay, time resolved fluoro-immunoassay and chemiluminescence immune assay etc. By the restriction of the operation principle of method own, its sensitivity and poor anti jamming capability, there is the problem such as radiation and pollution, substantially withdraw from the market in radioimmunoassay, RIA. And enzyme immunoassay is by methodological restriction, specificity and susceptiveness have much room for improvement, and generally as primary dcreening operation, present stage can't as qualitative, quantitative means. Time time resolved fluoro-immunoassay carries out ultramicroanalysis, the stray light of exciting light can be subject to, and then cause that sensitivity is restricted. Chemiluminescence immune assay analyzes method high sensitivity, high specific compared with other, easy to operate, the range of linearity wide, it is fast to analyze, and is that the main flow of present stage analyzes method. And find a kind of technical method new, that chemiluminescence immune assay can be substituted also it is critical that.
Summary of the invention
The present invention adopts magnetic separation technique to be combined with immunoreation, by adopting acridinium ester or acridine sulfonamide or sulfamide derivative as label, it is provided that a kind of high sensitivity, high specific, easy to operate, the range of linearity wide, analyzes quick C-peptide immue quantitative detection reagent box.Detection sample both can be in vitro serum, it is also possible to be in vitro urine, and accurately and reliably, and this C-peptide quantitative determination reagent kit is added with preservative to testing result in preparation process so that it is of a specified duration that this test kit preserves the time limit.
Provided by the invention utilize chemiluminescence magnetic microgranule immunoassay technology, by the test kit of C-peptide content in double antibody sandwich method detection by quantitative human serum or urine, can quickly detect C-peptide content in serum or urine, and testing result has significantly high sensitivity and specificity.
This invention address that the technical scheme that above-mentioned technical problem adopts is as follows:
A kind of C-peptide quantitative determination reagent kit, it is characterised in that: include the anti-C-peptide antibody of the anti-coated magnetic particle of C-peptide antibody, acridinium ester label;
The described anti-coated magnetic particle of C-peptide antibody includes the magnetic particle with carboxyl or p-toluenesulfonyl, anti-C-peptide antibody, is coated buffer, preserves liquid, confining liquid;
The anti-C-peptide antibody of described acridinium ester label is prepared from by label, anti-C-peptide antibody, labelling buffer, preservation liquid; Described label is any one in acridinium ester or acridine sulfonamide or acridine sulfamide derivative.
Preferably, the described anti-coated magnetic particle of C-peptide antibody with carboxyl also includes activator and coupling agent, and the described preparation method with the anti-coated magnetic particle of C-peptide antibody of carboxyl is as follows:
1) it is coated buffer, confining liquid, preservation liquid, activator, coupling agent described in preparation;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds in the antibody of concentration and is coated buffer, continue centrifugal, repeat 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) the anti-coated magnetic particle of C-peptide antibody is prepared: take the described magnetic particle with carboxyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add activator and coupling agent, fully mix suspended magnetic microgranule with eddy mixer, react 30-40min; Again with being coated buffer solution for cleaning 4-6 time, Magneto separate frame is removed supernatant, adds step 2) pretreated anti-C-peptide antibody, adds and is coated buffer, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour; With being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, adds confining liquid, at 25 DEG C, rotates with eddy mixer and is coated 4-5 hour, finally cleans 4-6 time with preservation liquid, after preserving liquid dilution, and 2-8 DEG C of preservation.
Preferably, the described anti-coated magnetic particle of C-peptide antibody with tosyl also includes catalyst, and the described preparation method with the anti-coated magnetic particle of C-peptide antibody of tosyl is as follows:
1) it is coated buffer, confining liquid, preservation liquid, catalyst described in preparation;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds in the antibody of concentration and is coated buffer, continue centrifugal, repeat 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) the anti-coated magnetic particle of C-peptide antibody is prepared: take the described magnetic particle with p-toluenesulfonyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add catalyst, fully mix suspended magnetic microgranule with eddy mixer, react 30-40min; Again with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, adds step 2) pretreated anti-C-peptide antibody, adds and is coated buffer, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour;With being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, adds confining liquid, at 25 DEG C, rotates with eddy mixer and is coated 4-5 hour, finally cleans 4-6 time with preservation liquid, after preserving liquid dilution, and 2-8 DEG C of preservation.
Preferably, the preparation method of the anti-C-peptide antibody of described acridinium ester label is as follows:
1) prepare described labelling buffer, preserve liquid;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds labelling buffer in the antibody of concentration, continues centrifugal, repeats 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) prepare the anti-C-peptide antibody of acridinium ester label: by described label add step 2) pretreatment antibody in, add labelling buffer, lucifuge 37 DEG C concussion reaction 16 hours, after having reacted, purify with rapidly and efficiently protein purification system, remove unconjugated label, collect the antibody of labelling, after preserving liquid dilution, 2-8 DEG C of preservation. Preferably, described C-peptide quantitative determination reagent kit also includes Sample dilution, reaction cleanout fluid, preexciting liquid, exciting liquid, C-poly saccharide peptide standard product.
Preferably, described Sample dilution is be added with the Tris buffer of surfactant and preservative or MES buffer or phosphate buffer.
Preferably, described reaction cleanout fluid is 0.01M phosphate buffer composition for being added with surfactant and preservative, concentration.
Preferably, described preexciting liquid to be concentration be 13-14g/L hydrogen peroxide and aqueous solution that concentration is 3-4g/L nitric acid composition.
Preferably, described exciting liquid is sodium hydrate aqueous solution.
Preferably, the preparation method of described C-poly saccharide peptide standard product is as follows:
(1) preparation standard dilutions;
(2) described C-poly saccharide peptide standard product is prepared: dissolve C-peptide antigen with standard dilutions, preparation aimed concn is the C-peptide antigen high concentration stock solution of 60ng/mL, C-poly saccharide peptide standard product is adopted to indicate, measure its actual concentration value, according to actual concentrations value, with standard dilutions dilution preparation variable concentrations C-poly saccharide peptide standard product, the C-poly saccharide peptide standard product prepared is dispensed in cillin bottle, every bottle of 1mL, after lyophilizing, 2-8 DEG C of preservation.
The Advantageous Effects of the present invention is as follows:
The present invention adopts magnetic separation technique to be combined with immunoreation, by adopting acridinium ester or acridine sulfonamide or acridine sulfamide derivative as label, it is provided that a kind of high sensitivity, high specific, easy to operate, the range of linearity wide, analyzes quick C-peptide quantitative determination reagent kit. Detection sample both can be serum, it is also possible to be urine, and accurately and reliably, and this C-peptide quantitative determination reagent kit adds preservative to testing result in preparation process so that this test kit preserves the time limit for a long time, can preserve 1 year as long as.
Detailed description of the invention
The invention will be further described below:
The C-peptide quantitative determination reagent kit of the present invention includes the anti-coated magnetic particle of C-peptide antibody (M reagent), the anti-C-peptide antibody (R reagent) of acridinium ester label, Sample dilution, C-poly saccharide peptide standard product, reaction cleanout fluid, preexciting liquid, exciting liquid.
Embodiment one
Described in this embodiment, the anti-coated magnetic particle of C-peptide antibody (M reagent) surface is with carboxyl, and its anti-C-peptide antibody is monoclonal antibody, and magnetic particle particle diameter is 1.5 ��m.
The preparation method of the described anti-coated magnetic particle of C-peptide antibody is as follows:
(1) preparation of solution:
It is coated buffer: 0.05M2-(N-morpholine) ethyl sulfonic acid (MES) buffer, wherein said is coated pH of buffer=5.5;
Activator: 100mL is coated buffer and adds 1g ethylene dichloride (EDC);
Coupling agent: 100mL is coated buffer and adds 2gN-hydroxysuccinimide (NHS);
Confining liquid: 100mL is coated buffer and adds 5g bovine serum albumin (BSA);
Preserve liquid: be 1% sodium azide, mass percent by concentration to be 25mMTris-HCl solution or MES solution or 3-(N-morpholine) propanesulfonic acid solutions (MOPS), concentration be 0.15MNaCl solution, mass percent to be 0.5%BSA, mass percent be 2% trehalose or sucrose, mass percent be that 0.05%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=7.3.
(2) the coated magnetic particle preparation process of anti-C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard the sodium azide or primary amine that contain in filtrate and filtrate, then the anti-C-peptide antibody of concentration is added and be coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1-2min antibody collecting ultra filtration standby, take the 40-100mg magnetic particle with carboxyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add 160 �� L activators and 250 �� L coupling agents, the magnetic particle of suspension is fully mixed with eddy mixer, reaction 30-40min, again with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add pretreated antibody, it is 1mL that interpolation is coated buffer to cumulative volume, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour, with being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, add 1mL confining liquid, at 25 DEG C, rotate with eddy mixer and be coated 4-5 hour, finally with preserving liquid cleaning 4-6 time, it is diluted to 300-1200 �� g/mL with preservation liquid, 2-8 DEG C of preservation.
Its anti-C-peptide antibody of anti-C-peptide antibody (R reagent) of described acridinium ester label is monoclonal antibody, and label is any one in acridinium ester or acridine sulfonamide or acridine sulfamide derivative.
The preparation method of the anti-C-peptide antibody of described acridinium ester label is as follows:
(1) preparation of solution:
Labelling buffer: preparation 0.05M phosphate buffer, pH=6.9
Preserve liquid: be 0.1% sodium azide, mass percent by concentration to be 25mMTris-HCl buffer or MES buffer or MOPS buffer, concentration be 0.15MNaCl solution, mass percent to be 0.5%BSA, mass percent be 0.5% trehalose or sucrose, mass percent be 0.05%Tween-20 or TritonX-100, wherein preserve liquid pH=7.3.
(2) the anti-C-peptide antibody preparation process of acridinium ester label:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add labelling buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is upside down in the centrifugal 1min of the 8000-10000r/min antibody collecting ultra filtration standby.
Take in the antibody of 0.01-0.1mg acridinium ester or acridine sulfonamide or acridine sulfamide derivative addition ultra filtration, wherein label needs to dissolve with DMSO solution or DMF solution before use, adding labelling buffer to cumulative volume is 0.5mL, 37 DEG C of lucifuge concussions are reacted 16 hours, after having reacted, purify with rapidly and efficiently protein purification system, remove unconjugated label, collect traget antibody, be diluted to 0.1-0.9 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
The preparation process of described C-poly saccharide peptide standard product is as follows:
(1) preparation of solution:
Standard dilutions: be that 0.05%Tween-20 forms by 25mMTris-HCl solution or phosphate buffer, 0.15MNaCl solution, mass percent to be 0.5%BSA, mass percent be 0.05% sodium azide, mass percent, wherein said standard dilutions pH=7.4.
(2) C-poly saccharide peptide standard product preparation process:
C-peptide antigen is dissolved with standard dilutions, preparation aimed concn is the C-peptide antigen high concentration stock solution of 60ng/mL, C-poly saccharide peptide standard product is adopted to indicate, measure its actual concentration value, according to actual concentrations value, with standard dilutions dilution preparation variable concentrations C-poly saccharide peptide standard product, the C-poly saccharide peptide standard product prepared is dispensed in cillin bottle, every bottle of 1mL, after lyophilizing, 2-8 DEG C of preservation.
Described Sample dilution is be added with the Tris buffer of surfactant and preservative or MES buffer or phosphate buffer. The formula of Sample dilution is as follows: be that 0.05% sodium azide, mass percent 0.05%Tween-20 form by concentration to be 25mMTris-HCl buffer or MES buffer or phosphate buffer, concentration be 0.15MNaCl solution, mass percent, wherein said Sample dilution pH=7.4.
Described reaction cleanout fluid is the 0.01M phosphate buffer being added with surfactant and preservative, and reaction cleanout fluid pH is 7.2-7.4.
Described preexciting liquid is 13g/L hydrogen peroxide by concentration and concentration is that the mixed liquor that 3g/L nitric acid forms is prepared from.
Described exciting liquid is sodium hydrate aqueous solution, and naoh concentration is 0.3mol/L.
Embodiment two
Described in this embodiment, the anti-coated magnetic particle of C-peptide antibody (M reagent) surface is with carboxyl, and its anti-C-peptide antibody is monoclonal antibody, and magnetic particle particle diameter is 3 ��m.
The preparation method of the described anti-coated magnetic particle of C-peptide antibody is as follows:
(1) preparation of solution:
It is coated buffer: 0.1M2-(N-morpholine) ethyl sulfonic acid (MES) buffer, wherein said is coated pH of buffer=4.5;
Activator: 100mL is coated buffer and adds 2g ethylene dichloride (EDC);
Coupling agent: 100mL is coated buffer and adds 1gN-hydroxysuccinimide (NHS);
Confining liquid: 100mL is coated buffer and adds 10g bovine serum albumin (BSA);
Preserve liquid: be 0.05% sodium azide, percent by volume by concentration to be 50mMTris-HCl solution or MES solution or 3-(N-morpholine) propanesulfonic acid solutions (MOPS), concentration be 0.2MNaCl solution, mass percent to be 5%BSA, mass percent be 0.5% trehalose or sucrose, mass percent be that 1%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=6.9.
(2) the coated magnetic particle preparation process of anti-C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard the sodium azide or primary amine that contain in filtrate and filtrate, then the anti-C-peptide antibody of concentration is added and be coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1-2min antibody collecting ultra filtration standby, take the 40-100mg magnetic particle with carboxyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add 400 �� L activators and 100 �� L coupling agents, the magnetic particle of suspension is fully mixed with eddy mixer, reaction 30-40min, again with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add pretreated antibody, it is 1mL that interpolation is coated buffer to cumulative volume, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour, with being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, add 3mL confining liquid, at 25 DEG C, rotate with eddy mixer and be coated 4-5 hour, finally with preserving liquid cleaning 4-6 time, it is diluted to 300-1200 �� g/mL with preservation liquid, 2-8 DEG C of preservation.
Its anti-C-peptide antibody of anti-C-peptide antibody (R reagent) of described acridinium ester label is monoclonal antibody, and label is any one in acridinium ester or acridine sulfonamide or acridine sulfamide derivative.
The preparation method of the anti-C-peptide antibody of described acridinium ester label is as follows:
(1) preparation of solution:
Labelling buffer: preparation 0.2M phosphate buffer, pH=5.9
Preserve liquid: be 0.05% sodium azide, percent by volume by concentration to be 50mMTris-HCl buffer or MES buffer or MOPS buffer, concentration be 0.2MNaCl solution, mass percent to be 5%BSA, mass percent be 2% trehalose or sucrose, mass percent be 1%Tween-20 or TritonX-100, wherein preserve liquid pH=6.9.
(2) the anti-C-peptide antibody preparation process of acridinium ester label:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add labelling buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is upside down in the centrifugal 1min of the 8000-10000r/min antibody collecting ultra filtration standby.
Take in the antibody of 0.01-0.1mg acridinium ester or acridine sulfonamide or acridine sulfamide derivative addition ultra filtration, wherein label needs to dissolve with DMSO solution or DMF solution before use, adding labelling buffer to cumulative volume is 0.5mL, 37 DEG C of lucifuge concussions are reacted 16 hours, after having reacted, purify with rapidly and efficiently protein purification system, remove unconjugated acridinium ester or acridine sulfonamide and derivant thereof, collect traget antibody, be diluted to 0.1-0.9 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
The preparation process of described C-poly saccharide peptide standard product is as follows:
(1) preparation of solution:
Standard dilutions: be that 1%Tween-20 forms by 50mMTris-HCl solution or phosphate buffer, 0.2MNaCl solution, mass percent to be 5%BSA, mass percent be 1% sodium azide, percent by volume, wherein said standard dilutions pH=7.1.
(2) C-poly saccharide peptide standard product preparation process:
C-peptide antigen is dissolved with standard dilutions, preparation aimed concn is the C-peptide antigen high concentration stock solution of 60ng/mL, C-poly saccharide peptide standard product is adopted to indicate, measure its actual concentration value, according to actual concentrations value, with standard dilutions dilution preparation variable concentrations C-poly saccharide peptide standard product, the C-poly saccharide peptide standard product prepared is dispensed in cillin bottle, every bottle of 1mL, after lyophilizing, 2-8 DEG C of preservation.
Described Sample dilution is be added with the Tris buffer of surfactant and preservative or MES buffer or phosphate buffer. The formula of Sample dilution is as follows: be that 1% sodium azide, percent by volume 1%Tween-20 form by concentration to be 50mMTris-HCl buffer or MES buffer or phosphate buffer, concentration be 0.2MNaCl solution, mass percent, wherein said Sample dilution pH=7.1.
Described reaction cleanout fluid is the 0.01M phosphate buffer being added with surfactant and preservative, and reaction cleanout fluid pH is 7.2-7.4.
Described preexciting liquid is 14g/L hydrogen peroxide by concentration and concentration is that the mixed liquor that 4g/L nitric acid forms is prepared from.
Described exciting liquid is sodium hydrate aqueous solution, and naoh concentration is 0.5mol/L.
Embodiment three
Described in this embodiment, the anti-coated magnetic particle of C-peptide antibody (M reagent) surface is with carboxyl, and its anti-C-peptide antibody is monoclonal antibody, and magnetic particle particle diameter is 2 ��m.
The preparation method of the described anti-coated magnetic particle of C-peptide antibody is as follows:
(1) preparation of solution:
It is coated buffer: 0.75M2-(N-morpholine) ethyl sulfonic acid (MES) buffer, wherein said is coated pH of buffer=4.5;
Activator: 100mL is coated buffer and adds 1.5g ethylene dichloride (EDC);
Coupling agent: 100mL is coated buffer and adds 1.5gN-hydroxysuccinimide (NHS);
Confining liquid: 100mL is coated buffer and adds 8g bovine serum albumin (BSA);
Preserve liquid: be 0.5% sodium azide, percent by volume by concentration to be 35mMTris-HCl solution or MES solution or 3-(N-morpholine) propanesulfonic acid solutions (MOPS), concentration be 0.18MNaCl solution, mass percent to be 3%BSA, mass percent be 1% trehalose or sucrose, mass percent be that 0.2%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=7.1.
(2) the coated magnetic particle preparation process of anti-C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard the sodium azide or primary amine that contain in filtrate and filtrate, the anti-C-peptide antibody of concentration is added and is coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1-2min antibody collecting ultra filtration standby, take the 40-100mg magnetic particle with carboxyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add 250 �� L activators and 200 �� L coupling agents, the magnetic particle of suspension is fully mixed with eddy mixer, reaction 30-40min, again with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add pretreated antibody, it is 1mL that interpolation is coated buffer to cumulative volume, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour, with being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, add 2mL confining liquid, at 25 DEG C, rotate with eddy mixer and be coated 4-5 hour, finally with preserving liquid cleaning 4-6 time, it is diluted to 300-1200 �� g/mL with preservation liquid, 2-8 DEG C of preservation.
Its anti-C-peptide antibody of anti-C-peptide antibody (R reagent) of described acridinium ester label is monoclonal antibody, and label is any one in acridinium ester or acridine sulfonamide or acridine sulfamide derivative.
The preparation method of the anti-C-peptide antibody of described acridinium ester label is as follows:
(1) preparation of solution:
Labelling buffer: preparation 0.1M phosphate buffer, pH=6.4;
Preserve liquid: be 0.5% sodium azide, percent by volume by concentration to be 35mMTris-HCl buffer or MES buffer or MOPS buffer, concentration be 0.18MNaCl solution, mass percent to be 3%BSA, mass percent be 1% trehalose or sucrose, mass percent be 0.2%Tween-20 or TritonX-100, wherein preserve liquid pH=7.1.
(2) the anti-C-peptide antibody preparation process of acridinium ester label:
Take the anti-C-peptide antibody of 1mg, with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add labelling buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is upside down in the centrifugal 1min of the 8000-10000r/min antibody collecting ultra filtration standby.
Take in the antibody of 0.01-0.1mg acridinium ester or acridine sulfonamide or acridine sulfamide derivative addition ultra filtration, wherein label needs to dissolve with DMSO solution or DMF solution before use, adding labelling buffer to cumulative volume is 0.5mL, 37 DEG C of lucifuge concussions are reacted 16 hours, after having reacted, purify with rapidly and efficiently protein purification system, remove unconjugated acridinium ester or acridine sulfonamide and derivant thereof, collect traget antibody, be diluted to 0.1-0.9 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
The preparation process of described C-poly saccharide peptide standard product is as follows:
(1) preparation of solution:
Standard dilutions: be that 0.2%Tween-20 forms by 30mMTris-HCl solution or phosphate buffer, 0.18MNaCl solution, mass percent to be 3%BSA, mass percent be 0.5% sodium azide, mass percent, wherein said standard dilutions pH=7.2.
(2) C-poly saccharide peptide standard product preparation process:
C-peptide antigen is dissolved with standard dilutions, preparation aimed concn is the C-peptide antigen high concentration stock solution of 60ng/mL, C-poly saccharide peptide standard product is adopted to indicate, measure its actual concentration value, according to actual concentrations value, with standard dilutions dilution preparation variable concentrations C-poly saccharide peptide standard product, the C-poly saccharide peptide standard product prepared is dispensed in cillin bottle, every bottle of 1mL, after lyophilizing, 2-8 DEG C of preservation.
Described Sample dilution is be added with the Tris buffer of surfactant and preservative or MES buffer or phosphate buffer. The formula of Sample dilution is as follows: be made up of concentration to be 30mMTris-HCl buffer or MES buffer or phosphate buffer, concentration be 0.18MNaCl solution, mass percent 0.5% sodium azide, volume basis 0.2%Tween-20, wherein said Sample dilution pH=7.3.
Described reaction cleanout fluid is the 0.01M phosphate buffer being added with surfactant and preservative, and reaction cleanout fluid pH is 7.2-7.4.
Described preexciting liquid is 13.5g/L hydrogen peroxide by concentration and concentration is that the mixed liquor that 3.5g/L nitric acid forms is prepared from.
Described exciting liquid is sodium hydrate aqueous solution, and naoh concentration is 0.4mol/L.
Embodiment four:
The preparation method that the present embodiment and the difference of embodiment one are the described coated magnetic particles of C-peptide antibody is different.
The coated magnetic particle surface of C-peptide antibody described in this embodiment is with p-toluenesulfonyl, and wherein magnetic particle particle diameter is 1.5 ��m, and its preparation method is as follows:
(1) preparation of solution:
It is coated buffer: 0.05M borate buffer, wherein said is coated pH of buffer=9.5;
Catalyst: 1L is coated buffer and adds 2M ammonium sulfate
Confining liquid: 100mL is coated buffer and adds 5gBSA;
Preserve liquid: be 1% sodium azide, percent by volume by concentration to be 25mMTris-HCl buffer or MES buffer or MOPS, concentration be 0.15MNaCl solution, mass percent to be 0.5%BSA, mass percent be 2% trehalose or sucrose, mass percent be that 0.05%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=7.3.
(2) the coated magnetic particle preparation process of C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, concentrate with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add and be coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1min antibody collecting ultra filtration standby.
Take the 40-100mg magnetic particle with p-toluenesulfonyl with being coated buffer solution for cleaning 4-6 time; Magneto separate removes supernatant; add pretreated antibody; it is 2mL that interpolation is coated buffer to cumulative volume; add 2mL catalyst; at 37 DEG C; rotate with eddy mixer and be coated 18-20 hour; 1mL confining liquid is added after having reacted; at 37 DEG C, continuation eddy mixer rotates and is coated 5-7 hour, finally with preserving liquid cleaning 4-6 time; it is diluted to 300-1200 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
Embodiment five
The preparation method that the present embodiment and the difference of embodiment two are the described coated magnetic particles of C-peptide antibody is different.
The coated magnetic particle surface of C-peptide antibody described in this embodiment is with p-toluenesulfonyl, and wherein magnetic particle particle diameter is 3 ��m, and its preparation method is as follows:
(1) preparation of solution:
It is coated buffer: 0.1M borate buffer, wherein said is coated pH of buffer=8.5;
Catalyst: 1L is coated buffer and adds 4M ammonium sulfate
Confining liquid: 100mL is coated buffer and adds 10gBSA;
Preserve liquid: be 0.05% sodium azide, percent by volume by concentration to be 50mMTris-HCl buffer or MES buffer or MOPS, concentration be 0.2MNaCl solution, mass percent to be 5%BSA, mass percent be 0.5% trehalose or sucrose, mass percent be that 1%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=6.9.
(2) the coated magnetic particle preparation process of C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, concentrate with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add and be coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1min antibody collecting ultra filtration standby.
Take the 40-100mg magnetic particle with p-toluenesulfonyl with being coated buffer solution for cleaning 4-6 time; Magneto separate removes supernatant; add pretreated antibody; it is 5mL that interpolation is coated buffer to cumulative volume; add 5mL catalyst; at 37 DEG C; rotate with eddy mixer and be coated 18-20 hour; 1mL confining liquid is added after having reacted; at 37 DEG C, continuation eddy mixer rotates and is coated 5-7 hour, finally with preserving liquid cleaning 4-6 time; it is diluted to 300-1200 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
Embodiment six
The preparation method that the present embodiment and the difference of embodiment three are the described coated magnetic particles of C-peptide antibody is different.
The coated magnetic particle surface of C-peptide antibody described in this embodiment is with p-toluenesulfonyl, and wherein magnetic particle particle diameter is 2 ��m, and its preparation method is as follows:
(1) preparation of solution:
It is coated buffer: 0.075M borate buffer, wherein said is coated pH of buffer=9.2;
Catalyst: 1L is coated buffer and adds 3M ammonium sulfate
Confining liquid: 100mL is coated buffer and adds 8gBSA;
Preserve liquid: be 0.5% sodium azide, percent by volume by concentration to be 35mMTris-HCl buffer or MES buffer or MOPS, concentration be 0.18MNaCl solution, mass percent to be 2%BSA, mass percent be 1% trehalose or sucrose, mass percent be that 0.5%Tween-20 or TritonX-100 forms, wherein said preservation liquid pH=7.1.
(2) the coated magnetic particle preparation process of C-peptide antibody:
Take the anti-C-peptide antibody of 1mg, concentrate with the ultra-filtration centrifuge tube of 30K or 50K centrifugal 3-5min under 8000-10000r/min, discard filtrate, add and be coated buffer, continue centrifugal, repeat 4-6 time, centrifuge tube is inverted the centrifugal 1min antibody collecting ultra filtration standby.
Take the 40-100mg magnetic particle with p-toluenesulfonyl with being coated buffer solution for cleaning 4-6 time; Magneto separate removes supernatant; add pretreated antibody; it is 3mL that interpolation is coated buffer to cumulative volume; add 4mL catalyst; at 37 DEG C; rotate with eddy mixer and be coated 18-20 hour; 1mL confining liquid is added after having reacted; at 37 DEG C, continuation eddy mixer rotates and is coated 5-7 hour, finally with preserving liquid cleaning 4-6 time; it is diluted to 300-1200 �� g/mL, 2-8 DEG C of preservation with preservation liquid.
Application Example
The in vitro serum sample picking up from the on an empty stomach health check-ups of 237 example healthy populations is detected, testing result carried out statistical analysis, it is determined that in vitro serum sample C-peptide content range of normal value is: 0.79-5.10ng/mL.
In vitro urine specimen picks up from 89 example healthy population twenty-four-hour urine liquid, and testing result is carried out statistical analysis, it is determined that in vitro urine specimen, C-peptide content range of normal value is: 24.0-208.2ng/mL.
The concrete detecting step of in vitro sample is as follows:
1, add 25 �� LC-peptide calibration objects or in vitro sample to reaction cup, need before detecting in vitro sample to dilute 10 times with Sample dilution;
2, adding 50 �� LM reagent to reaction cup, mixing concussion 37 DEG C is reacted 15 minutes;
3, after having reacted, use Magneto separate frame, separate magnetic particle, remove supernatant, add 200 �� L and react cleanout fluid, clean magnetic particle, repeatedly clean 5 times;
4, after cleaning, removing supernatant, add 100 �� LR reagent, mixing concussion 37 DEG C is reacted 15 minutes;
5, after having reacted, use Magneto separate frame, separate magnetic particle, remove supernatant, add 200 �� L and react cleanout fluid, clean magnetic particle, repeatedly clean 5 times;
6, after cleaning, remove supernatant, add 100 �� L preexciting liquid, mix magnetic particle, prepare detection luminous value;
7, adding 100 �� L exciting liquids to reaction cup, detect with ready luminometer rapidly, wherein C-peptide calibration object need to prepare the sample of 5 variable concentrations, and is detected respectively twice by every part of standard substance, according to corresponding luminous value, drawing standard curve.
Comprehensive above in vitro sample C-peptide concentration distribution situation, this test kit C peptide normal reference value is as follows:
Comparative example
C-peptide quantitative determination reagent kit is as follows with the contrast test of Roche C peptide quantitative determination reagent kit:
Detecting the 146 in vitro samples of example, sample type is in vitro serum. Wherein, male 71 example (48.6%), women 75 example (51.4%); Age distribution was at 2 months-84 years old; Diabetics 49 example (33.6%), healthy sample 34 example (23.3%), other cases 63 example (43.1%). The testing result adopting national standard is as follows, Low value anomaly sample 25 example (17.1%), normal sample 102 example (69.9%), high-value sector sample 19 example (13.0%).
Through C-peptide quantitative determination reagent kit and the detection of Roche C peptide quantitative determination reagent kit, 146 example in vitro sample Roche C peptide quantitative determination reagent kit (Electrochemiluminescince) detects exceptional sample 44 example, normal sample 102 example; Detecting exceptional sample 41 example with test kit of the present invention, normal sample 105 example, both have 5 examples by inconsistent sample. Test kit of the present invention is compared with the testing result of national standard, treat that the abnormal coincidence rate of examination reagent reaches 90.9%, normal coincidence rate reaches 99.0%, total coincidence rate reaches 96.6%, Kappa value reaches 0.917 (P < 0.001), visible, the test kit accuracy of the present invention is high, highly sensitive, specificity is good. From quantitative measure analysis, compared with contrast agent measured value, correlation coefficient is 0.993 (P < 0.01), and equation of linear regression is Y=0.75+0.974X (P < 0.001), and dependency is good.
The C-peptide quantitative determination reagent kit of the present invention be by magnetic separation technique be with nanometer or the magnetic particle of microsize grade for carrier, in conjunction with the special affinity characteristic that the protein in magnetic particle surface provides, under the oriented control adding magnetic field, by affine absorption, cleaning, desorption operations, a step can be separated to target molecule from complicated living things system, there is the many merits such as Magnetic Isolation absorption high specific simple and convenient, affine and hypersensitivity. ��?�� 9. pay kitchen Singapore is controlled Xian?Pan garden, Zhi Yuan border, Yuan Minxipan garden and is thrown that 15. imperial decree �� manger's helmet mould ? all of a sudden is in a difficult position admittedly lifts up Min play from the �� brown tool well-behaved ? of order and blame the uncommon ? cloth of the fragrance lush cruel Jing of promise beans to pour from �� mace crafty glue Fan Xi ? ash ? ? to throw the octogenarian ? egg of a louse Men Tong��Kui moving to and fro Herba Alii fistulosi institute boundless and indistinct ? moisture in the soil enlightening remote ? ? that swells and control an ancient wine vessel made of horn S and send cheat to sentence dizzy 17. 5 noisy originally to convert words Kang ? .1-10 ��m, in a liquid in suspended state, approximate liquid phase reactor, the surface of solid phase carriers of the surface area ratio equivalent of several ten million microgranules amasss much bigger, adsorbable more antibody, immunoreation speed can be accelerated, shorten the response time.
This C-peptide quantitative determination reagent kit is mainly used for the analysis of diabetes and understands the function of insulin B cell. No matter it is 1 type or type 2 diabetes mellitus patient, just all can pass through this test kit detection insulin level during disease to judge the function of insulin B cell, guiding treatment is had good effect.
The above; it is only the specific embodiment of the present invention; but protection scope of the present invention is not limited thereto; any those of ordinary skill in the art are in the technical scope that disclosed herein; the change can expected without creative work or replacement, all should be encompassed within protection scope of the present invention. Therefore, protection scope of the present invention should be as the criterion with claims protection defined.
Claims (10)
1. a C-peptide quantitative determination reagent kit, it is characterised in that: include the anti-C-peptide antibody of the anti-coated magnetic particle of C-peptide antibody, acridinium ester label;
The described anti-coated magnetic particle of C-peptide antibody includes the magnetic particle with carboxyl or p-toluenesulfonyl, anti-C-peptide antibody, is coated buffer, preserves liquid, confining liquid;
The anti-C-peptide antibody of described acridinium ester label is prepared from by label, anti-C-peptide antibody, labelling buffer, preservation liquid; Described label is any one in acridinium ester or acridine sulfonamide or acridine sulfamide derivative.
2. the C-peptide quantitative determination reagent kit piece described in Ju claim 1; it is characterized in that: the described anti-coated magnetic particle of C-peptide antibody with carboxyl also includes activator and coupling agent, and the described preparation method with the anti-coated magnetic particle of C-peptide antibody of carboxyl is as follows:
1) it is coated buffer, confining liquid, preservation liquid, activator, coupling agent described in preparation;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds in the antibody of concentration and is coated buffer, continue centrifugal, repeat 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) the anti-coated magnetic particle of C-peptide antibody is prepared: take the described magnetic particle with carboxyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add activator and coupling agent, fully mix suspended magnetic microgranule with eddy mixer, react 30-40min; Again with being coated buffer solution for cleaning 4-6 time, Magneto separate frame is removed supernatant, adds step 2) pretreated anti-C-peptide antibody, adds and is coated buffer, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour; With being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, adds confining liquid, at 25 DEG C, rotates with eddy mixer and is coated 4-5 hour, finally cleans 4-6 time with preservation liquid, after preserving liquid dilution, and 2-8 DEG C of preservation.
3. the C-peptide quantitative determination reagent kit piece described in Ju claim 1; it is characterized in that: the described anti-coated magnetic particle of C-peptide antibody with tosyl also includes catalyst, and the described preparation method with the anti-coated magnetic particle of C-peptide antibody of tosyl is as follows:
1) it is coated buffer, confining liquid, preservation liquid, catalyst described in preparation;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds in the antibody of concentration and is coated buffer, continue centrifugal, repeat 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) the anti-coated magnetic particle of C-peptide antibody is prepared: take the described magnetic particle with p-toluenesulfonyl with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, add catalyst, fully mix suspended magnetic microgranule with eddy mixer, react 30-40min; Again with being coated buffer solution for cleaning 4-6 time, Magneto separate removes supernatant, adds step 2) pretreated anti-C-peptide antibody, adds and is coated buffer, at 25 DEG C, rotate with eddy mixer and be coated 15-20 hour; With being coated buffer solution for cleaning 4-6 time after having reacted, Magneto separate removes supernatant, adds confining liquid, at 25 DEG C, rotates with eddy mixer and is coated 4-5 hour, finally cleans 4-6 time with preservation liquid, after preserving liquid dilution, and 2-8 DEG C of preservation.
4. the C-peptide quantitative determination reagent kit piece described in Ju claim 1, it is characterised in that: the preparation method of the anti-C-peptide antibody of described acridinium ester label is as follows:
1) prepare described labelling buffer, preserve liquid;
2) anti-C-peptide antibody pretreatment: take described anti-C-peptide antibody ultra-filtration centrifuge tube and be centrifuged, discard filtrate, adds labelling buffer in the antibody of concentration, continues centrifugal, repeats 4-6 time, is then inverted by centrifuge tube centrifugal, and it is standby that antibody is collected in concentration;
3) prepare the anti-C-peptide antibody of acridinium ester label: by described label add step 2) pretreatment antibody in, add labelling buffer, lucifuge 37 DEG C concussion reaction 16 hours, after having reacted, purify with rapidly and efficiently protein purification system, remove unconjugated label, collect the antibody of labelling, after preserving liquid dilution, 2-8 DEG C of preservation.
5. the C-peptide quantitative determination reagent kit piece described in Ju claim 1, it is characterised in that: described C-peptide quantitative determination reagent kit also includes Sample dilution, reaction cleanout fluid, preexciting liquid, exciting liquid, C-poly saccharide peptide standard product.
6. the C-peptide quantitative determination reagent kit piece described in Ju claim 5, it is characterised in that: described Sample dilution is be added with the Tris buffer of surfactant and preservative or MES buffer or phosphate buffer.
7. the C-peptide quantitative determination reagent kit piece described in Ju claim 5, it is characterised in that: described reaction cleanout fluid is 0.01M phosphate buffer composition for being added with surfactant and preservative, concentration.
8. the C-peptide quantitative determination reagent kit piece described in Ju claim 5, it is characterised in that: described preexciting liquid is concentration is 13-14g/L hydrogen peroxide and aqueous solution that concentration is 3-4g/L nitric acid composition.
9. the C-peptide quantitative determination reagent kit piece described in Ju claim 5, it is characterised in that: described exciting liquid is sodium hydrate aqueous solution.
10. the C-peptide quantitative determination reagent kit described in root Ju claim 5, it is characterised in that: the preparation method of described C-poly saccharide peptide standard product is as follows:
(1) preparation standard dilutions;
(2) C-poly saccharide peptide standard product is prepared: dissolve C-peptide antigen with standard dilutions, preparation aimed concn is the C-peptide antigen high concentration stock solution of 60ng/mL, C-poly saccharide peptide standard product is adopted to indicate, measure its actual concentration value, according to actual concentrations value, with standard dilutions dilution preparation variable concentrations C-poly saccharide peptide standard product, the C-poly saccharide peptide standard product prepared is dispensed in cillin bottle, every bottle of 1mL, after lyophilizing, 2-8 DEG C of preservation.
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| CN201610081311.4A CN105652018A (en) | 2016-02-04 | 2016-02-04 | C-peptide quantitative determination kit |
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| CN113960306A (en) * | 2020-07-20 | 2022-01-21 | 菲鹏生物股份有限公司 | Reagent for stabilizing acridinium ester marker protein and application thereof |
| CN113960306B (en) * | 2020-07-20 | 2023-04-28 | 菲鹏生物股份有限公司 | Reagent for stabilizing acridinium ester marker protein and application thereof |
| CN113588949A (en) * | 2021-08-17 | 2021-11-02 | 广州艺得诺生物科技有限公司 | Preparation and use method of canine coronavirus chemiluminescence detection reagent |
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Application publication date: 20160608 |