CN104020295B - Calcitonin detection kit and preparation thereof - Google Patents
Calcitonin detection kit and preparation thereof Download PDFInfo
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Abstract
The invention provides a kind of calcitonin detection kit, described kit is based on colloid gold immune turbidimetry, comprise reagent R2, described reagent R2 is the solution containing the gold nano grain that marked calcitonin antibody, 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 are than being 50:20 – 60.Also the present invention also provides the preparation method of this kit.Kit of the present invention has highly sensitive, and high specificity is swift in response, and the feature of good stability, does not produce precipitation after reaction, is convenient to the cleaning of biochemical instruments, extends the serviceable life of biochemical instruments.
Description
Technical field
The application relates to a kind of calcitonin detection kit based on colloid gold immune turbidimetry and preparation thereof.
Background technology
Procalcitonin (procalcitonin, PCT) is the calcitonin precursor substance without hormonal activity, is made up of 116 amino acid, and molecular weight is the glycoprotein of 13KD.PCT secretes (comprising thyroid gland, lung and pancreatic tissue cells) by neural inner cell and expresses, and cuts be decomposed into calcitonin (CT), c-terminal peptides and amino terminal peptide through enzyme.The half life period of CT in human body is 25-30h, and good stability, in normal human serum, content is extremely low.
In the many situations causing SIRS (SIRS), such as bacterial infection, pancreatitis, burn to death, multiple injury, in blood, the content of CT is abnormal raises, and CT is without significant change.In blood, the order of severity of CT level and infection and damage is proportionate, and the efficiency index of the diagnosis become for pyemia, severe sepsis, septic shock and curative effect monitoring.
The conventional detection of CT has immunoturbidimetry and enzyme linked immunosorbent assay etc.Because enzyme linked immunosorbent assay detects consuming time and complicated operation, the requirement of large hospital Quantitative detection can not be met, and universal along with full automatic biochemical apparatus of immunoturbidimetry, develop out multiple tachysynthesis than turbid detection technique.
The ultimate principle that latex strengthens Immunity transmission turbidity is first adsorbed on by antibody on a kind of latex particle, and when running into corresponding antigen, antigen-antibody combines and occurs latex agglutination.The size of single latex particle is within incident wavelength, and light-transmissive, when plural latex particle agglutination, can hinder light therethrough, transmitted light is reduced, and its minimizing degree is directly proportional to the amount of antigen.This method improves sensitivity and the accuracy of detection, is widely used.But, Immunoturbidimetry reaction after can produce precipitation, be unfavorable for the cleaning of biochemical instruments, interference test result, and latex manufacturing cost is higher, cause Immunoturbidimetric kit price and testing cost higher.Therefore there is again the immunoturbidimetry of nano-particle reinforcement, a kind ofly measure people by turbidimetry as patent CN101680890 just discloses and press down the TIA method of half dirty serine protease PROTEIN C and agent combination, and disclose one in Chinese patent CN101819208 and utilize microballoon immunoturbidimetry to detect brain natriuretic peptide kit.Specifically disclose in patent CN101680890, the particle diameter be made up of polystyrene, Polyvinylchloride, epoxy resin, poly-inclined vinylidene chloride, poly-Alpha-Naphthyl methacrylate ester, polyethylene naphthalenedicarboxylate and their corresponding multipolymers of finishing antibody is at the organic polymer colloidal nanoparticles of 80-105nm.
The present invention's research shows, does not meet the requirement of test, such as: easy coagulation cannot reach due stability to the magnetic nanoparticles such as iron oxide due to the factor such as paramagnetism and proportion when more than 40nm within this scope of many nano particles; 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 is that the colloid gold particle of 35nm-60nm is tested with diameter, find, sensitivity is poor, cannot meet clinical in sensitivity is less than to the requirement of 1ng/mL, with the requirement of lowest detectable limit 5ng/mL, and the range of linearity does not cover the concentration ranges of clinical middle normal physiological and pathologic condition, therefore usually causes false-negative result; Also there is the reaction time long (equilibration time 5-10min) simultaneously, the requirement of quick test cannot be reached.
Summary of the invention
In order to overcome the mistake instruction that prior art exists, solving the above-mentioned defect existed in prior art, the invention provides a kind of calcitonin 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 calcitonin detection kit based on colloid gold immune turbidimetry is provided, this kit comprises reagent R2, described reagent R2 is the solution containing the gold nano grain that marked calcitonin antibody, the particle diameter of described gold nano grain is 62.2nm-79.1nm, preferred 67.2nm-72.4nm; Described gold nano grain and antibody mass compare at 50:20 – 60, preferred 50:40-60.
Specifically, described reagent R2 is the solution of pH value 5.0-9.5, preferred 6.0-8.5.
Further, described reagent R2 is also containing set accelerator, stabilizing agent, sucrose and glycerine, wherein, set accelerator is the PEG20000 within w/v 0.4%, stabilizing agent is the BSA within w/v 2%, the w/v of sucrose is less than 20%, and the w/v of glycerine is less than 20%.
As preferably, above-mentioned R2 is 72.4 ± 1.5nm containing particle diameter and marked the gold nano grain of calcitonin, 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 calcitonin detection kit based on colloid gold immune turbidimetry has also comprised the reagent R1 of buffering and stabilization, described reagent R1 is NaN3's containing w/v 0.2%BSA, w/v 0.1% and w/v 0.05%Tween20, the phosphate buffer of pH7.2.In kit of the present invention, inventive point is mainly at reagent R2, and reagent R1 also can use the reagent R1 of detection kit in existing immunoturbidimetry.
Mentioned reagent R2 is prepared by the following method:
Prepare gold nano grain;
By antibody coupling to gold nano grain, obtain the nanogold particle solution of coupled antibody;
Stabilizing agent and set accelerator is added in the nanogold particle solution of coupled antibody;
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, and adjustment concentration is 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 the structure and activity that stabilizing agent carrys out stabilization of antibodies.Described stabilizing agent can be one or more in the inorganic salts of the 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, one or more more preferably 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, citrate-phosphate salt buffer, Tris damping fluid and HEPES damping fluid; Preferably phosphate buffer solution system.
The present invention at least achieves following beneficial effect:
Kit of the present invention has highly sensitive, high specificity, the feature of good stability, and the 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 calcitonin content in serum or blood plasma, is applicable to clinical automatic clinical chemistry analyzer.The sensitivity that kit of the present invention detects calcitonin can reach 0.1ng/mL, and detecting the range of linearity is at 0-160ng/mL.
Do not produce precipitation after kit reaction of the present invention, be convenient to the cleaning of biochemical instruments, extend the serviceable life of biochemical instruments.
Kit of the present invention adopts colloidal gold labeled monoclonal antibody, reduces the manufacturing cost of the calcitonin detection kit of colloid gold immune turbidimetry, has the larger market competitiveness.
Accompanying drawing explanation
Fig. 1: the embodiment of the present invention 1-7 typical curve detected.
Fig. 2: the embodiment of the present invention 9-15 typical curve detected.
Embodiment
Those skilled in the art below in detail, specific embodiments of the invention will be described, so that can understand the present invention in more detail.
Gold nano grain exists, also known as collaurum with similar colloidal state.
Provided by the present invention based in the calcitonin detection kit of colloid gold immune turbidimetry, reagent R1 can use the reagent R1 in prior art in immunoturbidimetry detection kit, i.e. existing commercial product.Specifically, reagent R1 can be containing 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 (pH7.2) of 50mM is that a kind of antigen site that impels exposes the buffer memory liquid promoting 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 on gold nano grain, finally miscible with buffer salt, set accelerator and antiseptic etc. become rounded system.
Detailed process is as follows:
1) preparation of the collaurum of 50nm is as follows:
In the 1000ml round-bottomed flask cleaned up, put into magnetic stir bar 1 piece, add 500ml ultrapure water;
Adding 5ml concentration is 1% (w/v) gold chloride, about 600rpm high-speed stirred, and be heated to solution boiling, then rapid is that 1% (w/v) sodium citrate solution joins in flask fast by 6ml concentration, keep heating and vigorous stirring 10min, solution colour first turns black, and then purpling is red gradually;
Close heater switch and continue to stir 10min, then stopping stirring being cooled to room temperature, return to original volume 500ml with ultrapure water;
The size of the colloid gold particle using transmission electron microscope observing to obtain, add up rear diameter to 1000 and be about 50.4 ± 1.2nm, ultrafiltration is for subsequent use after removing Small molecular.
2) by antibody coupling on gold nano grain, detailed process is as follows:
Moved into by antibody to be marked in bag filter, 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, the centrifugal 60min of 15000rpm, collects supernatant;
Solution O D measured by UV-vis spectroscopy degree meter
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 prepared;
Colloidal gold solution in centrifuge tube is adjusted to pH9 by the solution of potassium carbonate with 10%;
By 1,2,4,8,10,20,40,60,80, the antibody of 100ul is added in above-mentioned 10 centrifuge tubes respectively, shake 30 minutes, the mass ratio now often propping up gold nano grain and antibody in test tube is respectively 50:1,50:2,50:4,50:8,50:10,50:20,50:40,50:60,50:80,50:100;
The NaCl solution that 100ul concentration is 10% is added in above-mentioned solution, mixing, each pipe is observed after leaving standstill 2h, find 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, while stirring with 10% sal tartari adjustment solution pH value to 9;
The antibody being less than 50ml is joined in above-mentioned solution, continue stirring 30 minutes, in the process antibody with nano particle under the effect of Coulomb force and Van der Waals force, the hydrated sheath breaking through colloidal grain surface realizes contact, in the interaction of electrostatic force and Robert Van de Walle moral power, colloidal solid realizes coupling with protein molecule;
3) preparation of reagent R2 is carried out as follows:
Add a certain amount of BSA in colloidal gold solution after coupling 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, abandons supernatant, retain precipitation;
Be 20% sucrose as stabilizing agent, 0.2%PEG20000 as set accelerator, 0.5%BSA as stabilizing agent and 5% glycerine phosphate buffer (pH7.2) the resuspended sediment as the 10mmol of stabilizing agent by concentration, adjustment concentration is to OD
540nmbe 0.2;
Obtain R2 solution, packing is for subsequent use.
Embodiment 2
Embodiment 2 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 2 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 is obtained, diameter is about 56.2 ± 1.5nm.
Embodiment 3
Embodiment 3 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 3 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 is obtained, diameter is about 62.2 ± 1.1nm.
Embodiment 4
Embodiment 4 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 4 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 is obtained, diameter is about 67.2 ± 1.5nm.
Embodiment 5
Embodiment 5 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 5 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 is obtained, diameter is about 72.4 ± 1.5nm.
Embodiment 6
Embodiment 6 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 6 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 is obtained, diameter is about 79.1 ± 1.6nm.
Embodiment 7
Embodiment 7 is only with the difference of embodiment 1, by adjustment add the amount of reductive agent, colloid gold particle particle diameter prepared by embodiment 7 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 is obtained, diameter is about 83.5 ± 1.6nm.
Embodiment 8
Based on the Clinical practice effect measuring of colloid gold immune than turbid calcitonin detection kit:
(1) test condition of kit of the present invention and parameter as follows
A) determination step: first add 240ul reagent R1, then add 3ul sample, 37 DEG C hatch 5min after add 60ul reagent R2, in 540nm wavelength measurement absorbance, read the first point data after three seconds, react and read another point after 5 minutes, obtain the difference of absorbance.
B) computing method: 6 scaling methods take splines as computation schema.To work curve according to the value of absorbance and reference serum, sample size can calculate on working curve according to its absorbance.
(2) examination criteria curve
By method of the present invention, adopt Hitachi 7180 type automatic analyzer to detect the calcitonin reference material of 6 kinds of different contents, do typical curve according to result, X-axis represents the content of calcitonin, and Y-axis represents absorbance difference.
(3) by above-mentioned method to prepare in above-described embodiment 7 kit measurement typical curves, analyze optimized particle size.Result is as follows:
When gold nano grain size is 50.4nm, y=-0.0000007x+1.5871, R
2=0.3216;
When gold nano grain size is 56.2nm, y=-0.0003x+1.6009, R
2=0.9266;
When gold nano grain size is 62.2nm, y=-0.0066x+1.5993, R
2=0.9866;
When gold nano grain size is 67.2nm, y=-0.0068x+1.6015, R
2=0.9879;
When gold nano grain size is 72.4nm, y=-0.007x+1.6105, R
2=0.9919;
When gold nano grain size is 79.1nm, y=-0.0068x+1.5101, R
2=0.9473;
When gold nano grain size is 83.5nm, y=-0.0054x+1.0172, R
2=0.3652.
(4) to the above results, sensitivity, linearly dependent coefficient, the range of linearity and stability analysis is carried out.
Sensitivity definition is the difference of test system to the standard model test value of variable concentrations, and difference shows more greatly about sensitive, and system is too stable not have difference then to show, can not cause than turbid change.Sensitivity can be characterized by the absolute value of the slope of the typical curve after matching.
The R value of linearly dependent coefficient and relationship equation, shows the registration of experimental test result and matched curve, and R value is higher, and it is linear better to show.
Namely the range of linearity tests interval linear in corresponding result, and the range of linearity is wide is conducive to the sample testing wider concentration.
Stability refers in this system collaurum stable existence, do not occur to reunite or the degree of precipitation, and the more large easier coagulation of particle in general, cause the change of turbidity, particle is more little more stable, insensitive to test agent concentration change, i.e. poor sensitivity.Test agent be placed in the environment of 40 DEG C, represent 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, will the results are summarized in table 1 under various gold grain size condition.
Table 1
The above result of Integrated comparative, can find, particle diameter is more than or equal to the nano particle of 62.2nm, sensitivity just can reach requirement, can select 62.2nm-79.1nm Au colloidal nanoparticles, and the colloid gold particle being 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 Au colloidal nanoparticles compared with the 72.4nm of dominator as body series, change the antibody amount of colloid surface coupling.Be about on 72.4 ± 1.5nm gold nano grain by antibody coupling to diameter, the concentration (w/v) of gold nano grain and antibody is than being 50:100.
Embodiment 10
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:80.
Embodiment 11
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:60.
Embodiment 12
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:40.
Embodiment 13
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:20.
Embodiment 14
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:10.
Embodiment 15
Be with the difference of embodiment 9, the concentration (w/v) of gold nano grain and antibody is than being 50:1.
Embodiment 16
Adopt the method in embodiment 8, com-parison and analysis is carried out to the kit of preparation in embodiment 9 to 15.Test result is as follows.
The amount of gold grain the: during amount=50:1 of antibody, y=-0.0000019x+1.58706, R
2=0.81919
The amount of gold grain the: during amount=50:10 of antibody, y=-0.0004x+1.6019, R
2=0.8239
The amount of gold grain the: during amount=50:20 of antibody, y=-0.0027x+1.6336, R
2=0.9764
The amount of gold grain the: during amount=50:40 of antibody, y=-0.0067x+1.6948, R
2=0.9662
The amount of gold grain the: during amount=50:60 of antibody, y=-0.0067x+1.6209, R
2=0.9955
The amount of gold grain the: during amount=50:80 of antibody, y=-0.0072x+1.3317, R
2=0.7765
The amount of gold grain the: during amount=50:100 of antibody, y=-0.0061x+1.1312, R
2=0.5509
Above-mentioned seven kinds of kit results are gathered and compare table 2:
Table 2
Based on the above results, gold nano grain and antibody concentration than between 50:20-60 time highly sensitive, linear relationship good, the range of linearity is wide and stability better simultaneously, can select, be more preferably 50:40-60.
Embodiment 17
By embodiment 8 and embodiment 16, we are the optimization top condition of kit R2, is optimized the condition of stabilizing agent, set accelerator in R2 now.
Gold nano grain is selected to be 72.4 ± 1.5nm, the ratio of gold nano grain and antibody is at 50:60, reagent R2 is chosen as phosphate buffer, select pH4.0,5.0,6.0,7.2,8.5,9.5,10.0 seven points respectively, table 3 is sensitivity under three conditions, linearly dependent coefficient, the range of linearity and stability test result.
Table 3
Data are obtained as can be seen from upper table, when pH is 4.0 and 10.0, 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 PEG20000 selected by set accelerator, 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
As can be seen from upper table data, select PEG20000 sensitivity in 0 ~ 0.4% (w/v) scope, linearly dependent coefficient, the range of linearity close with stability test result in solidifying agent, table and optimum 0.2%.
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 selects BSA, sucrose and glycerine, and wherein BSA concentration selects 0,0.5% and 1% and 2%4 levels respectively; 0,5%, 10% and 20%4 levels selected by sucrose; 0,5%, 10% and 20%4 levels selected by glycerine; To the orthogonal test of carrying out 3 factors and 4 levels above, 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 in the phosphate buffer (pH7.2) of 50:40-60,10mmol containing 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 in the phosphate buffer (pH7.2) of 50:60,10mmol containing 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 level serum 1 part (1.) and the normal human serum 1 part (5.) of patient, get 1. isopyknic and 5. mix formation 3., get 1. isopyknic and 3. mix formation 2., get 3. isopyknic and 5. mix formation 4..Measure 1. ~ 5. content of calcitonin in serum.Measurement result shows, mentioned reagent box serum-dilution effect is not obvious, has good linear.
(3) precision test
A with the Quality Control of detection albumen definite value, normal person's pooled serum and the abnormal patient's pooled serum of kidney merit, 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 mean value of the abnormal patients serum of kidney merit is 99.7ng/mL, and in a few days imprecision is 1.8%, and imprecision is 0.95% in the daytime.These measurement results show that the precision of this kit can meet clinical requirement.
(4) interference test
A certain amount of cholerythrin, bovine hemoglobin, fat emulsion and rheumatoid factor is added separately in normal human serum, it is made to reach the concentration shown in table 7, add the deionized water of equal volume as noiseless thing serum, the concentration of these samples of Simultaneously test, the results are shown in Table 7 simultaneously.The result of table 7 show using annoyance level be 10% as the highest the stand limit of this Analytical system to chaff interference, Ascorbic Acid in Blood Serum is at 4.5g/L, cholerythrin is at below 500M, haemoglobin is at below 5g/L, fat emulsion is below 0.3%, and liquaemin is at below 62.5U/ml, and rheumatoid factor is at below 100IU/mL, do not affect mensuration, the results are shown in Table 7.
Table 7
Although show and describe the present invention with reference to preferred embodiment, it should be appreciated by those skilled in the art that when not departing from the spirit and scope of the present invention be defined by the claims, various amendment and conversion can be carried out to these embodiments.
Claims (9)
1. a calcitonin detection kit, described kit is based on colloid gold immune turbidimetry, comprise reagent R2, described reagent R2 is the solution containing the gold nano grain that marked calcitonin antibody, it is characterized in that, the particle diameter of described gold nano grain is 72.4 ± 1.5nm, and described gold nano grain and antibody mass are than being 50:20 – 60.
2. kit described in claim 1, is characterized in that, described gold nano grain and antibody mass are than being 50:40 – 60.
3. kit described in claim 1 or 2 any one, it is characterized in that, described reagent R2 is the solution of pH value 5.0-9.5.
4. kit described in claim 3, is characterized in that, described reagent R2 is the solution of pH value 6.0-8.5.
5. kit described in claim 4, is characterized in that, described reagent R2 is also containing set accelerator, stabilizing agent, sucrose and glycerine, and wherein, described stabilizing agent is one or more in the inorganic salts of the 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 contains one or more the combination in phosphate buffer, borate buffer solution, glycine buffer, citrate-phosphate salt buffer, Tris damping fluid and HEPES damping fluid.
6. kit described in claim 5, it is characterized in that, described set accelerator is the PEG20000 within w/v 0.4%, and described stabilizing agent is the bovine serum albumin(BSA) within w/v 2%, the w/v of described sucrose is less than 20%, and the w/v of described glycerine is less than 20%.
7. kit described in claim 6, it is characterized in that, described reagent R2 is 72.4 ± 1.5nm containing particle diameter and marked the gold nano grain of calcitonin 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.
8. kit described in claim 6, is characterized in that, described kit also comprises reagent R1, and described reagent R1 is NaN3 containing w/v 0.2%BSA, w/v 0.1% and w/v 0.05%Tween20, the phosphate buffer of pH7.2.
9. prepare the method for kit described in claim 7, it is characterized in that, described method comprises:
Prepare gold nano grain;
By antibody coupling to gold nano grain, obtain the nanogold particle solution of coupled antibody;
Stabilizing agent and set accelerator is added in the nanogold particle solution of coupled antibody;
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, and adjustment concentration is to OD
540nmbe 0.2.
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| CN104391118B (en) * | 2014-11-12 | 2017-03-15 | 深圳市锦瑞生物科技有限公司 | The detection means of Procalcitonin. and the preparation method of detection material |
| CN105891497A (en) * | 2014-12-19 | 2016-08-24 | 北京科美生物技术有限公司 | Procalcitonin collaurum immune colorimetric determination detection kit and preparation method thereof |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
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