CN102680713A - Competitive latex-particle-enhanced immunoturbidimetric assay kit for C-peptide and preparation method thereof - Google Patents
Competitive latex-particle-enhanced immunoturbidimetric assay kit for C-peptide and preparation method thereof Download PDFInfo
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- CN102680713A CN102680713A CN2012102003471A CN201210200347A CN102680713A CN 102680713 A CN102680713 A CN 102680713A CN 2012102003471 A CN2012102003471 A CN 2012102003471A CN 201210200347 A CN201210200347 A CN 201210200347A CN 102680713 A CN102680713 A CN 102680713A
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Abstract
The invention discloses a competitive latex-particle-enhanced immunoturbidimetric assay kit for C-peptide and a preparation method thereof, and solves the problems of high difficulty in C-peptide assay, long time for assay and the like in the current sandwich method. The kit disclosed by the invention is composed of latex particles and reaction buffer, and is characterized in that the latex particles are coated with the following artificially-synthesized amino acid sequences: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X, and X consists of 2 to 4 Ks. The invention further provides a preparation method of the kit. The kit disclosed by the invention has the advantages of high accuracy, short time for assay, high reliability, etc.
Description
Technical field
What the present invention relates to is biological technical field, be specifically related to be competition law latex particle enhance immunity than turbid C peptide detection kit, the invention still further relates to the preparation method of this kit.
Background technology
Measure the C Toplink and learn the function of islet cells, diagnosis of diabetes and treatment are had very big meaning, the C peptide does not have the function of insulin; And the insulin of B cell secretion and C peptide are the equimolecular relation; This that is to say, secretes several insulin molecules, must secrete several C peptide molecules simultaneously.But serum C peptide concentration indirect reaction insulin concentration.The C peptide does not receive the liver enzyme deactivation, and the half life period is longer than insulin, directly in urine, drains through kidney, so the concentration of C peptide can reflect the function of insulin better in the blood.
Under the arm's length basis state C peptide level be 0.4 ± 0.2 receive rub/liter.In oral glucose tolerance test (test of standard bread meal), can draw blood and measure the serum C peptide level that fasting blood-glucose was loaded back 1 hour, 2 hours, 3 hours, normal person's C peptide level after obeying sugared 60 minutes is increased to more than 3 times of foundation level.Type 1 diabetes C level extremely low (＜0.2 receive rub/liter), hypoinsulinism person C peptide after the meal rises main amplitude and often is lower than 3 times.For the patient who accepts insulinize, when measuring in the blood insulin level and can not estimate self islet function, can measure C peptide level and estimate self pancreas B cell function.
Present existing C peptide assay method has the detection of chemical/electrochemical electrochemiluminescent immunoassay, radio-immunity detection, enzyme linked immunosorbent detection, and its shortcoming is generally to grow (about 30min) detection time, and testing cost is expensive.
What traditional latex particle enhance immunity turbidimetry adopted when detecting antigenic substance is that sandwich method detects principle; Be detected material antigen and hatch certain hour after sample dilution (reagent 1) mixes; With the nano particle that is coated with corresponding antibody (reagent 2) antigen-antibody reaction takes place again; Form the not immune complex of capacitive, on full automatic biochemical apparatus, show as absorbance and rise (forming certain absorbance changing value) changing value of this absorbance and the positive correlation of measured matter antigenic content; Use the standard items drawing standard curve of concentration known, then can go out its content according to the reaction absorbance change calculations of tested sample.Its requirement is that antigenic substance will have plural no sterically hindered epitope.But the C peptide is owing to have only 31 amino acid, and it is less not have sterically hindered epitope, adopts the sandwich method detection difficulty higher.
Summary of the invention
The objective of the invention is to solve present sandwich method, to detect C peptide difficulty higher, the problem that detection time is long, provide a kind of speed fast, detect easy competition law latex particle enhance immunity than turbid C peptide detection kit and preparation method.
To achieve these goals, the technical scheme of the present invention's employing is following:
Competition law latex particle enhance immunity is made up of latex particle and reaction buffer than turbid C peptide detection kit, and said latex particle is coated with the amino acid sequence of following synthetic:
EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X; X is made up of 2～4 K.
Further, the monoclonal antibody that contains anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope in the said reaction buffer.
Further, said competition law latex particle enhance immunity is based on immune competition law than turbid C peptide detection kit the C peptide that detects in the sample is carried out quantitative measurement.
Competition law latex particle enhance immunity comprises the preparation of latex particle and the preparation of reaction buffer than the preparation method of turbid C peptide detection kit, and the preparation method of said latex particle is made up of following steps:
(a1) the activation latex particle is centrifugal, removes supernatant, uses the HEPES damping fluid to redissolve;
(a2) the amino acid sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X of adding synthetic, reaction is 2 hours under the room temperature;
(a3) the 1M glycocoll of the above-mentioned reaction volume 1/100 of adding, and 10%BSA solution, reaction 30min;
(a4) centrifugal, remove supernatant, use the HEPES damping fluid to redissolve and promptly process finished product.
Obtain required latex particle for more effective; Said centrifugal condition is 22000rpm, and the centrifugal time is 10min.
As a kind of preferred, the reagent that the activation latex particle is adopted in said (a1) is EDC and S-NHS.
Further, to prepare process following for said reaction buffer:
In the MES of 100mM damping fluid, adding final concentration is the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope of 0.001～0.1mg/ml.
The principle of the invention is: what nano particle encapsulated is and the same or analogous antigen of tested antigenic substance; With a certain amount of corresponding antibody generation antigen-antibody reaction; Form the not immune complex of capacitive, on full automatic biochemical apparatus, show as and form certain absorbance changing value.When containing this kind antigen in the measured object; Because competition principle; Formed absorbance changing value is compared decline during with no measured matter; The changing value of absorbance and measured matter antigenic content negative correlation in competition law, the standard items drawing standard curve of use concentration known then can go out its content according to the reaction absorbance change calculations of tested sample.
The present invention has the following advantages and beneficial effect:
1, in the antigen measuring of C peptide, because its antigen is too little, the difficult a plurality of epitopes that form the sandwich immunoassay compound that provide; Use the present invention to need not to form a plurality of epitopes, can effectively detect the C peptide, make detection method easier, testing result is more accurate.
2, adopt kit of the present invention to detect the C peptide fast, only need 10min detection time.
3, the present invention's reaction buffer of adopting the latex particle of the amino acid EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X be coated with synthetic and containing the monoclonal antibody of anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope detects the C peptide; Therefore, the present invention has that specificity is good, advantage of high accuracy.
4, detect through kit of the present invention, it is cheap relatively that it detects cost, is fit to apply.
5, immune competition law of the present invention is applicable to the mensuration of other micromolecule antigens simultaneously.
Description of drawings
Fig. 1 is the typical curve of the C reference peptide standard of different content of the present invention.
Embodiment
Below in conjunction with embodiment the present invention is described further, but embodiment of the present invention is not limited to the following example.
Embodiment 1
The present invention is made up of latex particle and reaction buffer.
Said latex particle is coated with the amino acid sequence of following synthetic: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X; X is made up of 2～4 K.The monoclonal antibody that contains anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope in the said reaction buffer.This EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ is existing amino acid sequence.
The amino acid sequence of said synthetic: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X, synthetic by Sangon Biotech (Shanghai) Co., Ltd..The monoclonal antibody of anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope is provided by Abcam company.
(a) preparation method of said latex particle is made up of following steps:
(a1) adopt EDC and S-NHS activation latex particle, soak time is 15min, and the material after the activation is centrifugal 10min under the condition of 22000rpm, removes supernatant, uses the HEPES damping fluid to redissolve;
(a2) the amino acid sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKK of adding synthetic at room temperature reacted 2 hours;
(a3) the 1M glycocoll of the above-mentioned reaction volume 1/100 of adding, and 10%BSA solution, reaction 30min;
(a4) centrifugal 10min under the condition of 22000rpm removes supernatant, uses the HEPES damping fluid to redissolve.
Can prepare latex particle required for the present invention according to above-mentioned steps.EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in the above-mentioned steps; Above-mentioned S-NHS is the N-hydroxy thiosuccinimide.
(b) to prepare process following for said reaction buffer:
In the MES of 100mM damping fluid, adding final concentration is the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope of 0.001～0.1mg/ml.Said MES is a MES; The pH of MES damping fluid is 5.0～7.5.The final concentration of the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope that is adopted in the present embodiment is 0.001 mg/ml.
The latex particle and the reaction buffer that adopt above-mentioned steps to prepare detect the absorbance of the C poly saccharide peptide standard product under the variable concentrations, process typical curve of the present invention through this concentration and absorbance; Its testing process is following:
In Hitachi's 7060 automatic clinical chemistry analyzer devices, add latex particle of the present invention and reaction buffer, latex particle 200ul, reaction buffer 50ul.The sample that in this full automatic biochemical apparatus, adds 30ul again.Detect detected parameters: reaction time 10min, 18～31 read a little, the single wavelength of 570nm.
The manufacturing process of typical curve is following: adopt following master sample calibration point concentration 0.00nmol/l, 0.5nmol/l, 1.0nmol/l, 2.0nmol/l, 4.0nmol/l; With 5 Spline or Log4p model calibration, produce typical curve of the present invention through testing result.The typical curve of present embodiment is as shown in Figure 1, and wherein the X axle is represented the C peptide content, and the Y axle is represented the absorbance changing value.
After accomplishing typical curve, can carry out sample and measure, calculate C peptide content in the sample automatically by instrument.
Present embodiment originally detects 9 increments; Simultaneously, adopt the automatic cold light immunity analysis instrument of CENTAUR and the C peptide detectable of Bayer A.G sample to be detected as control experiment testing result such as table 1.
Embodiment 2
Present embodiment is that with the difference of embodiment 1 amino acid sequence of the synthetic that latex particle encapsulates is different; The final concentration of the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope that is adopted in the present embodiment simultaneously, is 0.05 mg/ml.The amino acid sequence of the synthetic that present embodiment adopts is: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKKK.Testing result such as table 1.
Embodiment 3
Present embodiment is that with the difference of embodiment 1 amino acid sequence of the synthetic that latex particle encapsulates is different; The final concentration of the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope that is adopted in the present embodiment simultaneously, is 0.1 mg/ml.The amino acid sequence of the synthetic that present embodiment adopts is: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKKKK.Testing result such as table 1.
Table 1
| Sample | Embodiment 1 | Embodiment 2 | Embodiment 3 | Control experiment |
| 1 | 0.121 | 0.128 | 0.129 | 0.132 |
| 2 | 0.580 | 0.591 | 0.592 | 0.610 |
| 3 | 0.383 | 0.393 | 0.391 | 0.383 |
| 4 | 0.628 | 0.639 | 0.634 | 0.667 |
| 5 | 1.075 | 1.099 | 1.089 | 1.113 |
| 6 | 0.945 | 0.968 | 0.973 | 0.983 |
| 7 | 0.239 | 0.245 | 0.248 | 0.251 |
| 8 | 0.101 | 0.111 | 0.114 | 0.114 |
| 9 | 0.745 | 0.762 | 0.777 | 0.790 |
Through last table 1, can effectively show: can effectively detect the content (nmol/l) of C peptide in the sample through the present invention, and its accuracy is high, detection time is short, reliability is high, method is easy, is fit to apply.
According to the foregoing description, just can realize the present invention well.
Claims (7)
1. competition law latex particle enhance immunity is made up of latex particle and reaction buffer than turbid C peptide detection kit, and it is characterized in that: said latex particle is coated with the amino acid sequence of following synthetic:
EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X; X is made up of 2～4 K.
2. competition law latex particle enhance immunity according to claim 1 is characterized in that than turbid C peptide detection kit, contains the monoclonal antibody of anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope in the said reaction buffer.
3. competition law latex particle enhance immunity according to claim 1 and 2 is characterized in that than turbid C peptide detection kit, is based on immune competition law the C peptide that detects in the sample is carried out quantitative measurement.
4. according to the preparation method of each described competition law latex particle enhance immunity of claim 1～3 than turbid C peptide detection kit; Comprise the preparation of latex particle and the preparation of reaction buffer; It is characterized in that the preparation method of said latex particle is made up of following steps:
(a1) the activation latex particle is centrifugal, removes supernatant, uses the HEPES damping fluid to redissolve;
(a2) the amino acid sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-X of adding synthetic, reaction is 2 hours under the room temperature;
(a3) the 1M glycocoll of the above-mentioned reaction volume 1/100 of adding, and 10%BSA solution, reaction 30min;
(a4) centrifugal, remove supernatant, use the HEPES damping fluid to redissolve and promptly process finished product.
5. competition law latex particle enhance immunity according to claim 4 is characterized in that than the preparation method of turbid C peptide detection kit said centrifugal condition is 22000rpm, and the centrifugal time is 10min.
6. competition law latex particle enhance immunity according to claim 5 is characterized in that than the preparation method of turbid C peptide detection kit the reagent that the activation latex particle is adopted in said (a1) is EDC and S-NHS.
7. according to the preparation method of each described competition law latex particle enhance immunity of claim 4～6, it is characterized in that it is following that said reaction buffer prepares process than turbid C peptide detection kit:
In the MES of 100mM damping fluid, adding final concentration is the monoclonal antibody of the anti-EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ epitope of 0.001～0.1mg/ml.
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Cited By (4)
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| CN103852584A (en) * | 2014-03-28 | 2014-06-11 | 重庆中元生物技术有限公司 | Latex immune enhancement turbidimetric kit for detecting peptide C quantitatively |
| CN104991077A (en) * | 2015-07-29 | 2015-10-21 | 安徽省煦棠医疗科技有限公司 | Troponin I competition turbidimetry detecting kit |
| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
| CN114878825A (en) * | 2022-03-29 | 2022-08-09 | 北京世纪沃德生物科技有限公司 | C peptide determination kit and method for detecting content of C peptide in human serum |
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| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
| CN114878825A (en) * | 2022-03-29 | 2022-08-09 | 北京世纪沃德生物科技有限公司 | C peptide determination kit and method for detecting content of C peptide in human serum |
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