CN103926405A - Creatine kinase isozyme detection kit and preparation thereof - Google Patents
Creatine kinase isozyme detection kit and preparation thereof Download PDFInfo
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Abstract
The invention provides a creatine kinase isozyme detection kit. The kit is based on colloidal gold immunoturbidimetry, and comprises reagents R2, and the reagents R2 are solutions of gold nanoparticles marked with creatine kinase isozyme antibodies. The kit is characterized in that the particle diameter of the gold nanoparticles is 62.2 nm to 79.1 nm, and the mass ratio of the gold nanoparticles and the antibodies is 50:20-60. The invention further provides a preparation method of the kit. The kit has the advantages of being high in sensitivity, strong in specificity, fast in response and good in stability, no sediment is generated after a reaction, a biochemical analyzer is convenient to clean, and the service life of the biochemical analyzer is prolonged.
Description
Technical field
The application relates to a kind of creatine kinase isozyme detection kit and preparation thereof based on colloid gold immune turbidimetry.
Background technology
Creatine kinase (CK) has three kinds of main isodynamic enzymes, i.e. CK-MM, CK-MB, CK-BB.CK-MB (CK-MB), mainly be present in cardiac muscle, in myocardial enzymes, to have specific enzyme most, from the eighties, be known as " goldstandard " of Myocardial Enzymologic diagnosis, being applied to the early diagnosis of acute myocardial infarction (AMI), is to diagnose and monitor patient's AMI state of an illness sensitivity and special index.
The most generally adopt clinically immunodepression, its principle: the anti-CK-M antibody suppression CK-M subunit in immunodepression reagent, the activity that suppresses the CK-M subunit of CK-MM activity and CK-MB, by the vigor (being equivalent to half CK-MB vigor) of not repressed CK-B monomer in working sample, so result is multiplied by 2, calculate the vigor of CK-MB, the detection sensitivity of CK-MB reagent is corresponding also than the CK left and right that is at half, due to CK-MB reference value <25U/L, the per minute absorbance changing value of its upper limit 25U/L only has 0.003 left and right, the temperature of detecting instrument is waftd and the interference of the turbid sample of fat, capital produces larger measuring error, make measurement result unreliable.The patent of application number 200610114997.9 is active by reaction Hydrogen Peroxide+luminol+peroxidase chemiluminescence determination CK-MB, although there is higher detection sensitivity, but because creatine kinase is easy to inactivation in serum, conventionally to add sulfhydryl reagent to keep its vigor, sulfhydryl reagent meeting severe inhibition hydrogen peroxide+peroxidase reaction, makes its measurement result unreliability.
Other conventional detections also have immunoturbidimetry and enzyme linked immunosorbent assay etc.Because enzyme linked immunosorbent assay detects consuming time and complicated operation, can not meet the requirement of large hospital Quantitative detection, and universal along with full automatic biochemical apparatus of immunoturbidimetry develops out multiple tachysynthesis than turbid detection technique.
The ultimate principle that latex strengthens immune turbidimetry is, first antibody is adsorbed on a kind of latex particle, when running into corresponding antigen, antigen-antibody in conjunction with and there is latex agglutination.The size of single latex particle is within incident wavelength, and light can see through, and when plural latex particle agglutination, can hinder light and see through, and transmitted light is reduced, and its minimizing degree is directly proportional to the amount of antigen.This method has improved the sensitivity and the accuracy that detect, is widely used.Yet, after Immunoturbidimetry reaction, can produce precipitation, be unfavorable for the cleaning of biochemical instruments, interference test result, and latex manufacturing cost is higher, causes immunoturbidimetry kit price and testing cost higher.Therefore there is again the immunoturbidimetry of nano-particle reinforcement, as patent CN101680890 just discloses, a kind ofly by turbidimetry, measure TIA method and the agent combination that people presses down half dirty propylhomoserin protease protein C, and in Chinese patent CN101819208, disclose a kind of microballoon immunoturbidimetry that utilizes and detect brain natriuretic peptide kit.In patent CN101680890, specifically disclose, finishing the particle diameter of being made by polystyrene, Polyvinylchloride, epoxy resin, poly-inclined to one side vinylidene chloride, poly--Alpha-Naphthyl methyl-prop diluted acid ester, polyethylene naphthalenedicarboxylate and their corresponding multipolymers of antibody at the organic polymer colloidal nano particle of 80-105nm.
The present invention's research shows, does not meet the requirement of test within this scope of many nano particles, such as: when 40nm is above, due to factors such as paramagnetism and proportions, easy coagulation cannot reach due stability to the magnetic nanoparticles such as iron oxide; Gold nano grain is too poor and improper etc. at the scope internal stability of particle diameter 80-150nm.According to the instruction of Chinese patent CN102749454, the present invention be take the colloid gold particle that diameter is 35nm-60nm and is tested, find, sensitivity is poor, cannot meet in clinical sensitivity is less than to the requirement of 1ng/mL, with the requirement of lowest detectable limit 5ng/mL, and the range of linearity not cover the concentration of clinical middle normal physiological and pathologic condition interval, therefore usually cause false-negative result; Also there is the reaction time long (equilibration time 5-10min) simultaneously, cannot reach the requirement of quick test.
Summary of the invention
The mistake instruction existing in order to overcome prior art, solves the above-mentioned defect existing in prior art, the invention provides a kind of creatine kinase isozyme detection kit based on colloid gold immune turbidimetry.This kit range of linearity is wide, highly sensitive, and low cost of manufacture does not produce precipitation after reaction, facilitates biochemical instruments to clean.
According to an aspect of the present invention, a kind of creatine kinase isozyme detection kit based on colloid gold immune turbidimetry is provided, this kit comprises reagent R2, described reagent R2 is the solution of gold nano grain of creatine kinase isozyme antibody that contained mark, the particle diameter of described gold nano grain is 62.2nm-79.1nm, preferably 67.2nm-72.4nm; Described gold nano grain and antibody mass ratio be at 50:20 – 60, preferably 50:40-60.
Specifically, described reagent R2 is pH value 5.0-9.5, preferably the solution of 6.0-8.5.
Further, described reagent R2 also contains set accelerator, stabilizing agent, sucrose and glycerine, wherein, set accelerator is that w/v 0.4% is with interior PEG20000, stabilizing agent is that w/v 2% is with interior BSA, the w/v of sucrose is below 20%, and the w/v of glycerine is below 20%.
As preferably, above-mentioned R2 is to contain particle diameter be 72.4 ± 1.5nm and the mark gold nano grain of creatine kinase isozyme, w/v 20% sucrose, w/v 0.2%PEG20000, w/v 0.5%BSA and w/v 5% glycerine, the phosphate buffer of pH7.2, wherein, the weight ratio of gold nano grain and antibody is 50:40-60.
Further, the described creatine kinase isozyme detection kit based on colloid gold immune turbidimetry has also comprised the reagent R1 of buffering and stabilization, described reagent R1 is the NaN3 that contains w/v 0.2%BSA, w/v 0.1% and w/v 0.05%Tween20's, the phosphate buffer of pH7.2.In kit of the present invention, inventive point is mainly at reagent R2, and reagent R1 also can be used the reagent R1 of detection kit in existing immunoturbidimetry.
Mentioned reagent R2 is prepared by the following method:
Prepare gold nano grain;
Antibody coupling, to gold nano grain, is obtained to the nanogold particle solution of coupling antibody;
In the nanogold particle solution of coupling antibody, add stabilizing agent and set accelerator;
Centrifugal 2 hours of 2800rpm;
Centrifugation is with containing w/v 20% sucrose, w/v 0.2%PEG20000, w/v 0.5%BSA and w/v 5% glycerine, and the phosphate buffer of pH value 7.2 is resuspended, adjusts concentration to OD
540nmbe 0.2.
The activity of antibody is easily subject to the impacts such as the decomposition of proteinase, high-temperature denatured, osmotic pressure, can add stabilizing agent to carry out the structure and activity of stabilization of antibodies.Described stabilizing agent can be one or more in the inorganic salts of alkali and alkaline earth metal ions of protein, PEG, sugar and solubility; Wherein, the preferred bovine serum albumin(BSA) of described protein (BSA) or gelatin; Described sugar can be monose, disaccharides or polysaccharide, wherein, and the preferred mannose of monose, galactose and/or glucose; The preferred lactose of disaccharides and/or sucrose; One or more in the preferred trehalose of polysaccharide, glucosan, mannose and glucose; The inorganic salts of the preferred halogen alkali and alkaline earth metal ions of described inorganic salts, more preferably one or more in sodium chloride, potassium chloride, lime chloride.
In kit of the present invention, reagent R2 can also add set accelerator, to increase antigen-antibody reaction speed.
In kit provided by the present invention, R2 can be one or more buffer solution systems in phosphate buffer, borate buffer solution, glycine buffer, citric acid-phosphate buffer, Tris damping fluid and HEPES damping fluid; Preferably phosphate buffer solution system.
The present invention has at least realized following beneficial effect:
Kit of the present invention has highly sensitive, high specificity, the feature of good stability, reaction time is short, and the chromogenic reaction time is less than 30s, and equilibration time is less than 2min, can be used for detecting creatine kinase isozyme content in serum or blood plasma, be applicable to clinical automatic clinical chemistry analyzer.The sensitivity that kit of the present invention detects creatine kinase isozyme can reach 0.01ng/mL, detects the range of linearity and reaches 0～600.0ng/mL.
After kit reaction of the present invention, do not produce precipitation, be convenient to the cleaning of biochemical instruments, extended the serviceable life of biochemical instruments.
Kit of the present invention adopts colloidal gold labeled monoclonal antibody, has reduced the manufacturing cost of the creatine kinase isozyme detection kit of colloid gold immune turbidimetry, has the larger market competitiveness.
Accompanying drawing explanation
The typical curve that Fig. 1: embodiment of the present invention 1-7 detects.
The typical curve that Fig. 2: embodiment of the present invention 9-15 detects.
Embodiment
Those skilled in the art below, will describe specific embodiments of the invention in detail, so that can understand the present invention in more detail.
Gold nano grain exists with similar colloidal state, claims again collaurum.
In creatine kinase isozyme detection kit based on colloid gold immune turbidimetry provided by the present invention, reagent R1 can be used the reagent R1 in immunoturbidimetry detection kit in prior art, i.e. existing commercial product.Specifically, reagent R1 can contain 0.2%BSA (w/v) as stabilizing agent, 0.1%NaN3 (w/v) as antiseptic and 0.05%Tween20 (w/v) as set accelerator, the phosphate buffer of 50mM (pH7.2) is that a kind of antigen site that impels exposes the buffer memory liquid that promotes antigen-antibody reaction.
Following embodiment only prepares reagent R2.
Embodiment 1
The preparation of the present embodiment reagent R2 is divided into three steps, first prepares colloidal gold solution, then by antibody coupling to gold nano grain, finally become rounded system with buffer salt, set accelerator and antiseptic etc. are miscible.
Detailed process is as follows:
1) preparation of the collaurum of 50nm is as follows:
In the 1000ml round-bottomed flask cleaning up, put into 1 piece of magnetic stir bar, add 500ml ultrapure water;
Adding 5ml concentration is 1% (w/v) gold chloride, 600rpm left and right high-speed stirred, and be heated to solution boiling, then by 6ml concentration, be rapidly that 1% (w/v) sodium citrate solution joins in flask fast, keep heating and vigorous stirring 10min, solution colour first turns black, and then purpling is red gradually;
Close heater switch and continue and stir 10min, then stop stirring and be cooled to room temperature, with ultrapure water, return to original volume 500ml;
Use the size of the colloid gold particle that transmission electron microscope observing makes, add up rear diameter be about 50.4 ± 1.2nm to 1000, ultrafiltration is removed after little molecule standby.
2) by antibody coupling to gold nano grain, detailed process is as follows:
Antibody to be marked is moved in bag filter, and dialysis in the phosphate buffer (pH7.2) of 10mM, to remove other small-molecule substances in antibody-solutions;
Antibody after dialysis is transferred in centrifuge tube, and the centrifugal 60min of 15000rpm collects supernatant;
On ultraviolet-visible spectrophotometer, measure solution O D
260nmabsorption value, calculates the concentration of antibody in solution, and with 10mM phosphate buffer by antibody dilution to 1mg/ml (w/v);
Get the centrifuge tube of 10 1.5 milliliters, add 1 milliliter of colloidal gold solution preparing;
Solution of potassium carbonate with 10% is adjusted to pH9 by the colloidal gold solution in centrifuge tube;
By 1,2,4,8,10,20,40,60,80, the antibody of 100ul is added to respectively in above-mentioned 10 centrifuge tubes, shake 30 minutes, now in every test tube, the mass ratio of gold nano grain and antibody is respectively 50:1,50:2,50:4,50:8,50:10,50:20,50:40,50:60,50:80,50:100;
To adding 100ul concentration in above-mentioned solution, be 10% NaCl solution, mix, after standing 2h, observe each pipe, discovery is when the mass ratio of gold nano grain and antibody reaches 50:100, have color change and a small amount of Precipitation, this shows when ratio reaches 50:100, is a maximum demand of labelled protein, the mass ratio of gold nano grain and antibody is less than 50:100 without color change with without Precipitation;
According to above result, 500ml colloidal gold solution is added in triangular flask, with 10% sal tartari, adjust while stirring the pH value to 9 of solution;
The antibody that is less than 50ml is joined in above-mentioned solution, continue to stir 30 minutes, in this process antibody with nano particle under the effect of Coulomb force and Van der Waals force, break through the hydrated sheath of colloidal grain surface and realize contact, in the interaction of electrostatic force and Robert Van de Walle moral power, colloidal solid is realized coupling with protein molecule;
3) preparation of reagent R2 is carried out as follows:
In colloidal gold solution after coupling, add a certain amount of BSA as stabilizing agent, final concentration is 1% (w/v), stirs 5min and makes it to dissolve completely;
In above-mentioned solution, add a certain amount of PEG20000, final concentration is 0.2% (w/v), stirs 10 minutes;
Above-mentioned solution is moved in centrifuge tube, with the centrifugal 2h of 2800rpm, abandon supernatant, retain precipitation;
By concentration, be 20% sucrose as stabilizing agent, 0.2%PEG20000 as set accelerator, 0.5%BSA as stabilizing agent and 5% glycerine as the resuspended sediment of phosphate buffer (pH7.2) of the 10mmol of stabilizing agent, adjust concentration to OD
540nmbe 0.2;
Obtain R2 solution, packing is standby.
Embodiment 2
Embodiment 2 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 2 preparations is 55nm.
It is 5.6ml that preparation 55nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 56.2 ± 1.5nm.
Embodiment 3
Embodiment 3 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 3 preparations is 62.2nm.
It is 5.4ml that preparation 62.2nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 62.2 ± 1.1nm.
Embodiment 4
Embodiment 4 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 4 preparations is 67nm.
It is 5.2ml that preparation 67nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 67.2 ± 1.5nm.
Embodiment 5
Embodiment 5 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 5 preparations is 72nm.
It is 5.1ml that preparation 72nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 72.4 ± 1.5nm.
Embodiment 6
Embodiment 6 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 6 preparations is 79nm.
It is 4.9ml that preparation 79nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 79.1 ± 1.6nm.
Embodiment 7
Embodiment 7 is only with the difference of embodiment 1, adds the amount of reductive agent by adjusting, and making the colloid gold particle particle diameter of embodiment 7 preparations is 83nm.
It is 4.7ml that preparation 83nm particle adds reductive agent 1% sodium citrate.The colloid gold particle that transmission electron microscope observing makes, diameter is about 83.5 ± 1.6nm.
Embodiment 8
Based on colloid gold immune, than the clinical result of use of turbid creatine kinase isozyme detection kit, measure:
(1) test condition of kit of the present invention and parameter are as follows
A) determination step: first add 240ul reagent R1, then add 3ul sample, 37 ℃ add 60ul reagent R2 after hatching 5min, in 540nm wavelength measurement absorbance, after three seconds, read first data, react after 5 minutes and read another point, obtain the difference of absorbance.
B) computing method: 6 scaling methods, take splines as computation schema.According to the value of absorbance and the reference serum curve of working, sample size can be calculated according to its absorbance on working curve.
(2) examination criteria curve
By method of the present invention, adopt Hitachi's 7180 type automatic analyzers to detect the creatine kinase isozyme reference material of 6 kinds of different contents, according to result, do typical curve, X-axis represents the content of creatine kinase isozyme, Y-axis represents absorbance difference.
(3) by above-mentioned method to prepare in above-described embodiment 7 kit measurement typical curves, analyze optimum particle diameter.Result is as follows:
When gold nano grain size is 50.4nm, y=-0.0004x+1.5896, R
2=0.2993;
When gold nano grain size is 56.2nm, y=-0.0029x+1.5936, R
2=0.5812;
When gold nano grain size is 62.2nm, y=-0.0676x+1.5744, R
2=0.9309;
When gold nano grain size is 67.2nm, y=-0.0646x+1.5668, R
2=0.9729;
When gold nano grain size is 72.4nm, y=-0.0698x+1.5942, R
2=0.9889;
When gold nano grain size is 79.1nm, y=-0.066x+1.4889, R
2=0.9119;
When gold nano grain size is 83.5nm, y=-0.0519x+0.9958, R
2=0.3743.
(4), to the above results, carry out sensitivity, linearly dependent coefficient, the range of linearity and stability analysis.
Sensitivity definition is the difference of test system to the standard model test value of variable concentrations, and difference shows more greatly approximately sensitive, does not have difference to show that system is too stable, can not cause than turbid change.Sensitivity can be by the typical curve after matching the absolute value of slope characterize.
Linearly dependent coefficient is the R value of correlativity equation, shows the registration of experimental test result and matched curve, and R value is higher, shows that linearity is better.
The range of linearity tests interval linear in corresponding result, the wide sample that is conducive to test wider concentration of the range of linearity.
Stability refers in this system collaurum stable existence, do not occur to reunite or the degree of precipitation, and the easier more greatly coagulation of particle, causes the change of turbidity in general, and particle is more little more stable, insensitive to test agent concentration change, i.e. poor sensitivity.Test agent is placed in the environment of 40 ℃, represents the quality of stability with the time length of stability time, the test duration is Dec.
By the contrast of above-mentioned four parameters, we can therefrom select optimal particle size range, by the table 1 that the results are summarized under various gold grain size conditions.
Table 1
Comprehensively relatively above result, can find, particle diameter is more than or equal to the nano particle of 62.2nm, and sensitivity just can reach requirement, can select 62.2nm-79.1nm Au colloidal nanoparticles, and the colloid gold particle that is greater than 83.5nm is due to too large, less stable.
Embodiment 9
The preparation process of reagent R2 is with reference to embodiment 1.According to the test result of embodiment 8, select to have compared with the Au colloidal nanoparticles of the 72.4nm of dominator as body series, change the antibody amount of colloid surface coupling.Antibody coupling is about on 72.4 ± 1.5nm gold nano grain to diameter, and the concentration of gold nano grain and antibody (w/v) is than being 50:100.
Embodiment 10
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:80.
Embodiment 11
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:60.
Embodiment 12
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:40.
Embodiment 13
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:20.
Embodiment 14
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:10.
Embodiment 15
Be with the difference of embodiment 9, the concentration of gold nano grain and antibody (w/v) is than being 50:1.
Embodiment 16
Adopt the method in embodiment 8, the kit of preparation in embodiment 9 to 15 is analyzed relatively.Test result is as follows.
The amount of gold grain: during the amount=50:100 of antibody, y=-0.00001x+1.58704, R
2=0.74585
The amount of gold grain: during the amount=50:80 of antibody, y=-0.00009x+1.5954, R
2=0.7564
The amount of gold grain: during the amount=50:60 of antibody, y=-0.0007x+1.5921, R
2=0.9965
The amount of gold grain: during the amount=50:40 of antibody, y=-0.0017x+1.5911, R
2=0.9929
The amount of gold grain: during the amount=50:20 of antibody, y=-0.0017x+1.5088, R
2=0.9795
The amount of gold grain: during the amount=50:10 of antibody, y=-0.0016x+1.1717, R
2=0.6112
The amount of gold grain: during the amount=50:1 of antibody, y=-0.0013x+0.9575, R
2=0.3753
Above-mentioned seven kinds of kit results are gathered and relatively arrive table 2:
Table 2
Based on the above results, gold nano grain and antibody concentration highly sensitive than between 50:20-60 time, linear relationship good, the range of linearity is wide and stability is better simultaneously, can select, and is more preferably 50:40-60.
Embodiment 17
By embodiment 8 and embodiment 16, we optimization the top condition of kit R2, now the condition of stabilizing agent, set accelerator in R2 is optimized.
Selecting gold nano grain is 72.4 ± 1.5nm, and the ratio of gold nano grain and antibody is at 50:60, and reagent R2 is chosen as phosphate-buffered liquor, selects respectively pH4.0,5.0,6.0,7.2,8.5,9.5,10.0 seven points.
Table 3 is sensitivity under three conditions, linearly dependent coefficient, the range of linearity and stability test result.
Table 3
From upper table, obtaining data can find out, at pH, be 4.0 and 10.0 o'clock, this kit does not meet the requirement of testing laboratory in sensitivity, linearly dependent coefficient, the range of linearity or stability, therefore the bounds of pH is 5.0-9.5, wherein sensitivity within the scope of 6.0-8.5, linearly dependent coefficient, the range of linearity and stability test result are best, and numerical value is close, show that within the scope of this, pH impact is not remarkable.
The same, selection gold nano grain is 72.4 ± 1.5nm, and the ratio of gold nano grain and antibody is at 50:60, and set accelerator is selected PEG20000, and the scope of concentration is 0～0.4% (w/v), selects 0,0.1%, 0.2% and 0.4%4 level, in Table 4.
Table 4
From upper table data can be found out, in solidifying agent, select PEG20000 sensitivity in 0～0.4% (w/v) scope, linearly dependent coefficient, the range of linearity close with stability test result, show and at 0.2% optimum.
The same, selection gold nano grain is 72.4 ± 1.5nm, and the ratio of gold nano grain and antibody is at 50:60, and stabilizing agent is selected BSA, sucrose and glycerine, and wherein BSA concentration is selected respectively 0,0.5% and 1% and 2%4 level; Sucrose is selected 0,5%, 10% and 20%4 level; Glycerine is selected 0,5%, 10% and 20%4 level; To carrying out above the orthogonal test of 3 factors and 4 levels, set up L16 (45) orthogonal arrage, carry out 16 tests, therefrom select optimal conditions, in Table 5 and table 6.
Table 5: 3 factors of stabilizing agent and 4 water-glasses
Table 6: orthogonal test scheme and orthogonal experiments
By above orthogonal test, best sensitivity and precision and the range of linearity appear at, gold nano grain is 72.4 ± 1.5nm, the ratio of gold nano grain and antibody is at 50:40-60, in the phosphate buffer of 10mmol (pH7.2), contain 20% sucrose as stabilizing agent, 0.2%PEG20000 as set accelerator, 0.5%BSA as stabilizing agent and 5% glycerine as stabilizing agent.
Embodiment 18
Reagent R2 gold nano grain is 72.4 ± 1.5nm, the ratio of gold nano grain and antibody is at 50:60, in the phosphate buffer of 10mmol (pH7.2), contain 20% sucrose as stabilizing agent, 0.2%PEG20000 as set accelerator, 0.5%BSA as stabilizing agent and 5% glycerine as stabilizing agent.
(1) sensitivity test
The optimum sensitivity of mentioned reagent box reaches 0.2ng/mL.
(2) linear determination
Get abnormal high value serum 1 portion of (1.) and 1 part of normal human serum (5.) of patient, get 1. isopyknic and 5. mix and form 3., get 1. isopyknic and 3. mix and form 2., get 3. isopyknic and 5. mix and form 4..Measure 1.～the 5. content of creatine kinase isozyme in serum.Measurement result shows, mentioned reagent box serum dilution effect is not obvious, has good linearity.
(3) precision test
With detecting, the Quality Control of albumen definite value, normal person's pooled serum and the abnormal patient's pooled serum of kidney merit are a, and METHOD FOR CONTINUOUS DETERMINATION 20 days, measures three every day, calculates in a few days and day to day precision.Measurement result shows that the mean value of definite value Quality Control is 12.2ng/mL, its in a few days imprecision be 2.1%, imprecision is 1.18% in the daytime.The mean value of normal person's pooled serum is 4.2ng/mL, and in a few days imprecision is 3.7%, and imprecision is 2.9% in the daytime.The abnormal patients serum's of kidney merit mean value is 9.7ng/mL, and in a few days imprecision is 1.18%, and imprecision is 0.66% in the daytime.These measurement results show that the precision of this kit can meet clinical requirement.
(4) interference test
In normal human serum, add separately a certain amount of cholerythrin, bovine hemoglobin, fat emulsion and rheumatoid factor, make it reach the concentration shown in table 7, add the deionized water of equal volume as noiseless thing serum simultaneously, measure the concentration of these samples simultaneously, the results are shown in Table 7.The result of table 7 show to using annoyance level as 10% as this mensuration system the highest limit of standing to chaff interference, Ascorbic Acid in Blood Serum is at 4.5g/L, cholerythrin is below 500M, haemoglobin is below 5g/L, fat emulsion is below 0.3%, and liquaemin is below 62.5U/ml, and rheumatoid factor is below 100IU/mL, do not affect mensuration, the results are shown in Table 7.
Table 7
Although represent with reference to preferred embodiment and described the present invention, it should be appreciated by those skilled in the art that in the situation that do not depart from the spirit and scope of the present invention that are defined by the claims, can carry out various modifications and conversion to these embodiment.
Claims (10)
1. a creatine kinase isozyme detection kit, described kit is based on colloid gold immune turbidimetry, comprise reagent R2, described reagent R2 is the solution of gold nano grain of creatine kinase isozyme antibody that contained mark, it is characterized in that, the particle diameter of described gold nano grain is 62.2nm-79.1nm, and described gold nano grain and antibody mass ratio are 50:20 – 60.
2. kit described in claim 1, is characterized in that, the particle diameter of described gold nano grain is 67.2nm-72.4nm.
3. kit described in claim 1, is characterized in that, described gold nano grain and antibody mass ratio are 50:40-60.
4. kit described in claim 2 or 3 any one, is characterized in that, described reagent R2 is the solution of pH value 5.0-9.5.
5. kit described in claim 4, is characterized in that, described reagent R2 is the solution of pH value 6.0-8.5.
6. kit described in claim 5, is characterized in that, described reagent R2 also contains set accelerator, stabilizing agent, sucrose and glycerine, and wherein, described stabilizing agent is one or more in the inorganic salts of alkali and alkaline earth metal ions of protein, PEG, sugar and solubility; Described protein is bovine serum albumin(BSA) or gelatin; Described sugar is monose, disaccharides or polysaccharide; Described inorganic salts are the inorganic salts of halogen alkali and alkaline earth metal ions; Reagent R2 is to be one or more the combination in phosphate buffer, borate buffer solution, glycine buffer, citric acid-phosphate buffer, Tris damping fluid and HEPES damping fluid.
7. kit described in claim 6, it is characterized in that, described set accelerator is that w/v 0.4% is with interior PEG20000, described stabilizing agent is that w/v 2% is with interior bovine serum albumin(BSA), the w/v of described sucrose is below 20%, and the w/v of described glycerine is below 20%.
8. kit described in claim 7, it is characterized in that, described reagent R2 is to contain particle diameter be 72.4 ± 1.5nm and the mark gold nano grain of creatine kinase isozyme antibody, w/v 20% sucrose, w/v 0.2%PEG20000, w/v 0.5%BSA and w/v 5% glycerine, the phosphate buffer of pH7.2, wherein, the weight ratio of gold nano grain and antibody is 50:40-60.
9. kit described in claim 7, it is characterized in that, described kit also comprises reagent R1, and described reagent R1 is the NaN3 that contains w/v 0.2%BSA, w/v 0.1% and w/v 0.05%Tween20, the phosphate buffer of pH7.2.
10. the method for preparing kit described in claim 1-9 any one, is characterized in that, described method comprises:
Prepare gold nano grain;
Antibody coupling, to gold nano grain, is obtained to the nanogold particle solution of coupling antibody;
In the nanogold particle solution of coupling antibody, add stabilizing agent and set accelerator;
Centrifugal 2 hours of 2800rpm;
Centrifugation is with containing w/v 20% sucrose, w/v 0.2%PEG20000, w/v 0.5%BSA and w/v 5% glycerine, and the phosphate buffer of pH value 7.2 is resuspended, adjusts concentration to OD
540nmbe 0.2.
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