CN102628872A - Reagent box for detecting 25 hydroxyl vitamin D and manufacturing method thereof - Google Patents
Reagent box for detecting 25 hydroxyl vitamin D and manufacturing method thereof Download PDFInfo
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- CN102628872A CN102628872A CN2012100942583A CN201210094258A CN102628872A CN 102628872 A CN102628872 A CN 102628872A CN 2012100942583 A CN2012100942583 A CN 2012100942583A CN 201210094258 A CN201210094258 A CN 201210094258A CN 102628872 A CN102628872 A CN 102628872A
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Abstract
The invention discloses a reagent box for detecting 25 hydroxyl vitamin D and a manufacturing method thereof. The reagent box for detecting the 25 hydroxyl vitamin D mainly comprises a microporous plate wrapping high-specificity rat 25 hydroxyl vitamin D monoclonal antibodies, wrapping amount of the high-specificity rat 25 hydroxyl vitamin D monoclonal antibodies in each micropore of the microporous plate is 0.1-0.5mug, concentration of the 25 hydroxyl vitamin D labeled by biotin is 0.1mug/ml, and dosage of each micropore is 25mul. Sensitivity of the reagent box for detecting the 25 hydroxyl vitamin D is about 5nmol/L, linear range of the reagent box is larger than that of a similar reagent box, precision degree and accuracy degree of the reagent box are both in requirement range of reagent boxes, a blood serum sample can be directly added without dilution, an operation method of the reagent box is simpler than the similar reagent box, and the reagent box can be operated by common technical personnel. Compared with the similar reagent box, operation time of the reagent box is reduced by half.
Description
Technical field
The present invention relates to detection kit, particularly relate to 25 hydroxy-vitamine D detection kit and preparation method thereof.
Background technology
Human body and animal must absorb an amount of vitamin D every day from food, just can keep normal growth and health.Adult's suggestion daily intake is 5 μ g, and the gestational period and lactation, the women should increase the intake about 1 times, and trick every day is revealed 30 centimetres, shines in the sun 30 minutes, effectively prevents the shortage of vitamin D.But the vitamin D hyperphagia can cause poisoning, and some scholars think that taking in 25 μ g vitamin Ds long-term every day can cause poisoning, and this wherein possibly comprise the people that some are responsive to vitamin D, then causes poisoning certainly but take in 125 μ g vitamin Ds long-term every day.It is 20 μ g that China's formulation vitamin D can tolerate the highest intake.
25 (OH) VD is the existence form of topmost serum VD, and its serum-concentration has reacted the state of available VD in the body, and its result's reliability is better than serum 1,25 (OH) the VD level that detects.Serum 25 (OH) VD concentration reduced with the age.The elderly reduces sunshine relatively, and aging skin produces VD quantity and descends, and the absorption of inadequate diet, causes the elderly generally to lack VD, has increased the risk of suffering from osteoporosis.Therefore, detect 25 (OH) VD serum-concentration and can assist clinical assessment, also can be used for detecting the reason of the improper serum calcium level of patient the VD deficiency state.
Present 25 hydroxy-vitamine D detection kit mainly are to be main with imported product, and stable better products mainly is ELISA and radioimmunoassay method series products.The radioimmunoassay method detects the checker that 25 hydroxy-vitamine D kits exist needs operating experience abundant, has alpha-contamination problem, and usable range is restricted.But the at present normally used serum sample of the kit that ELISA detects 25 hydroxy-vitamine Ds that utilizes need dilute just application of sample, and the running time is long, and the method for operating step is more loaded down with trivial details.
Summary of the invention
Based on this, be necessary to provide a kind of susceptibility height, high specificity, easy and simple to handle, good stability, 25 hydroxy-vitamine D detection kit of pollution-free, good reproducibility.
A kind of 25 hydroxy-vitamine D detection kit mainly include:
(A) be coated with the microwell plate of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies, the package amount of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies is 0.1～0.5 μ g in each micropore of said microwell plate;
(B) concentration is biotin labeled 25 hydroxy-vitamine Ds of 0.1 μ g/ml, and consumption is every micropore 25 μ l.
Among embodiment, said 25 hydroxy-vitamine D detection kit comprise that also working concentration is the avidin of the HRPO mark of 0.5～2.0 μ g/ml therein;
Therein among embodiment, the package amount of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies is 0.2 μ g/ml in said each micropore that encapsulates plate.
Among embodiment, the working concentration of the avidin of said HRPO mark is 1.0 μ g/ml therein.
The present invention also provides the preparation method of above-mentioned 25 hydroxy-vitamine D detection kit.
A kind of preparation method of 25 hydroxy-vitamine D detection kit may further comprise the steps:
(1) preparation encapsulates the microwell plate of high specific mouse 25 (OH) D monoclonal antibody
The concentration that encapsulates by 0.1～0.5 μ g encapsulates high specific mouse 25 (OH) D monoclonal antibody to micropore, and 4 ℃ are spent the night, washing, and sealing seals, and obtains encapsulating the microwell plate of high specific mouse 25 (OH) D monoclonal antibody;
(2) prepare biotin labeled 25 hydroxy-vitamine Ds by conventional method, the concentration of said biotin labeled 25 hydroxy-vitamine Ds is 0.1 μ g/ml.
25 hydroxy-vitamine D detection kit of the present invention are 25 hydroxy-vitamine Ds and other hydroxylation metabolism products that adopt in competition law principle quantitative measurement human serum or the blood plasma.Standard items, quality-control product and sample are added in the micropore of the microwell plate that encapsulates high specific mouse 25 (OH) D monoclonal antibody in advance, add again under biotin labeled 25 (OH) D antigen room temperature and hatched 30 minutes, afterwards flushing.Add the avidin of horseradish peroxidase-labeled, selectively combine with biotin, then flushing utilizes the substrate colour developing again.After the cessation reaction, 450nm reads absorbance in the place under ELIASA, and 25 (OH) D concentration and color intensity are inversely proportional to.
Above-mentioned 25 hydroxylated cellulose D detection kit are compared with present normally used similar detection kit, can increase work efficiency, and significantly practice thrift cost.The present invention adopts the monoclonal antibody coated slab; Sensitivity is about 5nmol/L, and the range of linearity is greater than similar kit, and precision and accuracy are all in the kit claimed range; And serum sample need not dilute and get final product direct application of sample; Method of operating is simpler than similar kit, and those skilled in the art can operate, and the running time reduces half the than similar kit.And pollution-free, can be used as the substitute products of similar radioimmunoassay kit.
Embodiment
Below specify the present invention through specific embodiment.
Embodiment 1
The main composition of the described 25 hydroxy-vitamine D detection kit of present embodiment is following:
1, is coated with the microwell plate of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies
The package amount of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies is 0.2 μ g in each micropore of said microwell plate; Bar shaped microwell plate (12 * 8 hole) places framework.
2,25 hydroxy-vitamine D standard items 0.1.2.3.4.5
Can directly use, totally 6 bottles, every bottle of 300ul, the linear concentration of standard solution is respectively: A:0; B:6.5; C:26; D:60; E:180; F:360ng/ml, corresponding standard items 0.1.2.3.4.5 respectively.
3,25 hydroxy-vitamine D quality-control products 1.2
Can directly use, totally 2 bottles, every bottle of 300ul, concentration is respectively 30ng/ml and 100ng/ml.
4, biotin labeled 25 hydroxy-vitamine Ds
Can directly use, concentration is 0.1 μ g/ml, 1 bottle, and every bottle of 3ml.
5, enzyme conjugates
The avidin of the horseradish peroxidase-labeled that can directly use (be dissolved in and contain in detergent, antiseptic and the BSA buffer solution), working concentration is 1.0 μ g/ml, 1 bottle, 6.0mL.
6, substrate solution
The tetramethyl benzidine that can directly use (TMB) substrate (being dissolved in the acid solution), 1 bottle, 12.0mL.
7, stop buffer
The hydrochloric acid solution that can directly use (HCl, 100ml/L; Process water, 900ml/L).1 bottle, 12.0mL.
8, concentrate washing lotion (20 *)
The concentrated cleaning buffer solution that contains detergent and antiseptic.1 bottle, 50.0mL.
9, seal film
The adhesive membrane of sealed micro orifice plate when hatching.
The preparation of said kit is following:
(1) standard items and quality-control product
(1) preparation of standard items
Be to be that 25 hydroxyl VD mother liquors and artificial serum, the proclin300 of 1mg/ml is formulated by concentration.
Prepare 1000 person-portion standard items, concrete process for preparation (is joined from high concentration) as follows:
F: the artificial serum+0.7mlproclin300 of mother liquor 252 μ l+699.048ml that gets 25 hydroxyl VD 1mg/ml; Packing;
E: get the artificial serum of the standard solution F150ml+149.7ml for preparing+0.3ml proclin300; Packing;
D: get the artificial serum of the standard solution F50ml+249.7ml for preparing+0.3ml proclin300; Packing;
C: get the artificial serum+0.3mlproclin300 of the standard solution F21.65ml+278.05m for preparing; Packing;
B: get the artificial serum of the standard solution F5.4ml+294.3ml for preparing+0.3ml proclin300; Packing;
A: add the artificial serum of 300ml, packing.
(2) preparation of quality-control product
Quality-control product 1 (100ng/ml): the artificial serum of 25 hydroxyl VD standard items F (360ng/ml) 83.35ml+216.35ml+0.3ml proclin300, packing;
Quality-control product 2 (30ng/ml): the artificial serum of 25 hydroxyl VD standard items F (360ng/ml) 25ml+274.7ml+0.3ml proclin300, packing.
(2) encapsulate the microwell plate of high specific mouse 25 (OH) D monoclonal antibody
Outsourcing high specific mouse 25 (OH) D monoclonal antibody.
(coating buffer: antibody 2.0mg+ encapsulates damping fluid 1000ml with coating buffer by 100 μ L/ holes; Encapsulating damping fluid is: Na
2CO
30.423g/L, NaHCO
31.344g/L) add in the micropore of ELISA Plate, 4 ℃ spend the night (＞17h); Adopt 25 (OH) VD after diluting to concentrate washing lotion (* 20:Na
2HPO
4.12H
2O116g/L, NaH
2PO
4.2H
2O 11.84g/L, NaCl 180g/L, Tween-2010ml/L, proclin3001ml/L) to wash, 200 μ L/ holes are washed twice; In micropore, add confining liquid (Na by 200 μ l/ holes
2HPO
4.12H
2O 5.8g/L, NaH
2PO
4.2H
2O 0.592g/L, NaCl 9.0g/L, sucrose 50g/L, lactose 50g/L, BSA 10g/L, proclin3001ml/L filter), room temperature is placed (18～26 ℃), and 4 ℃ are spent the night and seal; After drying shed (18-26 ℃) was drained, vacuum seal (2-8 ℃) obtained encapsulating the microwell plate of high specific mouse 25 (OH) D monoclonal antibody, kept dry.
(3) enzyme conjugates
Fill a prescription as follows: Na
2HPO
4.12H
2O, 2.9g/L; KH
2PO
4, 0.4g/L; NaCL, 8g/L; KCL, 0.4g/L; BSA, 10g/L; Proclin300,1ml/L; The avidin of horseradish peroxidase-labeled (outsourcing raw material), 1g/L; Filter.
(4) other
Biotin labeled 25 hydroxy-vitamine Ds, substrate solution, stop buffer, concentrate washing lotion (20 *), seal film and provide by other commercial companies.
The kit of embodiment 2 utilization embodiment 1 detects 25 hydroxy-vitamine Ds in the sample
The requirement of tested sample
Adopt serum or blood plasma (EDTA, sodium citrate or heparin) sample.Should separate as early as possible after the sample collection, sample is no more than 4 hours 15-25 ℃ of placement; Be no more than 12 hours during 2-8 ℃ of placement;-20 ℃ or can preserve 2 months during with held, but-80 ℃ of long preservation down should be avoided the multigelation of sample, at most can only freeze thawing once.Haemolysis sample, microbial contamination sample can not be used for measuring.
The operation steps of the kit of embodiment 1 is following:
1) on lath, puts the micropore lath well according to the sample size and the layout of required mensuration.
2) add biotin labeled 25 HECs, 5 μ l.
3) add each 25 μ l of 25 hydroxy-vitamine D standard items, 25 hydroxy-vitamine D quality-control products and sample to treating in the gaging hole.
4) with sealing film the microwell plate plate hole is sealed, placed room temperature following 30 minutes.
5) wash plate five times with the good concentrated washing lotion of dilution.
A. wash plate automatically: setting is washed the plate machine and is distributed every hole at least 300 μ l washing fluids.Repeat to wash 5 times.
B. manual plate washing: put upside down rapidly and pour out thing in the hole.Every hole adds 250 μ l washing fluids.Repeat to wash plate 5 times.
Before getting into next step, on thieving paper, firmly pat inverted plate to remove unnecessary washing lotion.
6) every hole adds enzyme conjugates 50 μ l, places room temperature 30 minutes.
7) repeating step 5.
8) every hole adds tmb substrate solution 100 μ l, seals up and seals film, lucifuge incubation 15 minutes.
Annotate: tmb substrate solution is vulnerable to pollute.Pipette needed amount, the liquid after using please don't be refunded in the bottle again.
9) every hole adds stop buffer 100 μ l.
10) add the inherent 450nm wavelength of stop buffer 30 minutes (reference wavelength is 650nm) and locate to read absorbance.
Table 1-3 extracts the testing result of 25 hydroxy-vitamine Ds in adult and the child's serum sample immediately for the kit of utilization embodiment 1.Adult and child's normal value is 50-150nmol/L.
Table 1
Table 2
Table 3
The Performance Evaluation A sensitivity assessment of the 25 hydroxy-vitamine D detection kit of embodiment 3 embodiment 1
Get 10 parts of " 0 " standard items respectively, detect and the record 450nm OD of place value, computation of mean values and SD value with the OD value of average-2SD, calculate corresponding dosage from dosage-response curve, and this dose value is the LDL of this method.The testing result calculating mean value is 2.7164, and standard deviation is 0.1778, and its sensitivity is 5.03nmol/L.
The B range of linearity
It is 400nmol/L that standard solution is diluted to concentration, 200nmol/L, 100nmol/L, 50nmol/L; 26nmol/L, 12.5nmol/L, 6.25nmol/L; The standard items of 0nmol/L, each standard items detects the OD value twice at 450nm place, and testing result is as shown in table 4.
The OD value of table 4 standard items
According to table 4 drawing standard curve as a result, and calculate the range of linearity, it is rational that measurement range is decided to be 5-400nmol/L.
C criticizes interpolation
The kit of the same lot number of use three covers carries out duplicate detection each 12 times to high value quality-control product and the low value quality-control product of 25 (OH) VD in the 450nm wavelength, measures absorbance mean value, standard deviation, the CV value of quality-control product respectively.
Table 5 batch interpolation testing result
Can be found out that by table 5 Quality Control of high value and the total Variation Lines number average of low value Quality Control are less than 10%, batch interpolation meets the requirements.
The D difference between batch
Respectively 25 (OH) VD is detected Quality Control of high value and low value Quality Control respectively 12 times with the kit of continuous three lot numbers, calculate the high value Quality Control absorbance of continuous three lot number kits and the CV value of low value Quality Control absorbance respectively.If the CV value that detects gained explains that then betweenrun precision can reach requirement in 10% scope.The result sees table 6.
Table 6 difference between batch testing result
Can know that by table 6 result the interassay coefficient of variation of Quality Control of high value and low value Quality Control can reach the requirement of kit precision all less than 10%.
E stability
After microwell plate vacuum seal, placed 37 ℃ of biochemical incubators 7 days, to take out then, balance is to room temperature; After getting the vacuum seal of same lot number microwell plate, be kept at 4 ℃, take out then, balance is to room temperature; Use two microwell plates to do the immunoassay experiment, survey OD value result such as table 7.
Table 7 stability experiment result
Table 7 is the result show, 37 ℃ of kits of placing after taking out balance to room temperature in 7 days are compared with the kit of 4 ℃ of preservations, and it is fine that the sample determination value meets in the scope that error allows, and the result is more stable.
The F specificity
Respectively with 25-hydroxyvitamin D3; 25-hydroxy-vitamin D 2; 24, the 25-dihydroxy vitamin d3; Five kind of 25 hydroxy-vitamine D related substances of cholecalciferol and calciferol D2 is diluted to 100nmol/L.Detect 6 standard items and these related substanceses simultaneously, parallel detection 2 times, the OD value of record 450nm wavelength is also calculated cross reacting rate, and the result is as shown in table 8.
Table 8 specificity experimental result
Table 8 is the result show, 25-hydroxyvitamin D3,25-hydroxy-vitamin D 2 are detection targets of kit of the present invention, so cross reaction is big.And 24,25-hydroxyvitamin D3 is few because of content in serum, so even have cross reaction also can not influence the specificity of kit of the present invention.Cholecalciferol, calciferol D2 then do not have intersection.
The interference of other materials in the G blood sample
(1) interference of anti-coagulants
The concentration that in serum, adds sodium citrate during detection respectively is 0mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 350mmol/L; The concentration that adds EDTA is 0mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L; The concentration that adds heparin is respectively 0U/ml, 10U/ml, 20U/ml, 40U/ml, 50U/ml, 100U/ml.Judge according to testing result whether three kinds of anti-coagulants disturb detecting to some extent in the scope that is detected.Testing result is shown in table 9-11.
The interference of table 9 sodium citrate
The interference of table 10 EDTA
The interference of table 11 heparin
Generally use the sodium citrate about 10.9mmol/L to use as anti-coagulants clinically, table 9 is the result show, sodium citrate is in the scope of 0-350mmol/L.The detection method of kit of the present invention there is not obvious interference; Generally use 3.4-4.8mmol/LEDTA to use as anti-coagulants clinically, table 10 is the result show, EDTA concentration does not have obvious interference to the detection method of kit of the present invention in the blood sample in the scope of 3-5mmol/L; Adopt venous blood clinically, heparin is generally the whole blood of 25.330-30U/ml as the use amount of anti-coagulants, and table 11 is the result show, the heparin in the blood sample does not have obvious interference to the detection method of kit of the present invention in the 10-100U/ml scope.Therefore, the existence of three kinds of anti-coagulants sodium citrates, heparin and EDTA is not disturbed the detection method of kit of the present invention basically.
(2) influence of haemolysis and piarhemia
Claim that haemoglobin is dissolved in the normal human serum, with this hemoglobin solutions of quality controlled serum dilution, the concentration that obtains haemoglobin is respectively 0mg/L, 0.25mg/ml, and 0.5mg/ml, 1mg/ml, 1.5mg/ml and 2mg/ml are blank with the quality controlled serum.Detect the serum of 6 kinds of HCs, the result is as shown in table 12.
The interference of table 12 haemoglobin
The result shows, content of hemoglobin detects kit of the present invention during less than 1.5mg/ml does not have influence.Content of hemoglobin is very micro-in the serum clinically, but contains a certain amount of haemoglobin in the haemolysis sample serum, so the haemolysis sample can not adopt kit test method of the present invention to detect.
Claim that cholerythrin is dissolved in the normal human serum,, obtain bilirubinic concentration and be respectively 0mg/L with this bilirubin solution of quality controlled serum dilution, 25mg/l, 50mg/l, 100mg/l, 200mg/l is a blank with the quality controlled serum.Detect the serum of 6 kinds of bilirubin concentrations, the result is as shown in table 13.
The bilirubinic interference of table 13
The serum bilirubin normal value is about 3-10mg/L clinically; Therefore we add the 15mg/L cholerythrin its content is exceeded more than the normal value upper limit twice in normal human serum; The result shows exist (0-200mg/L) of blood sample mesobilirubin, and the detection method to kit of the present invention does not produce interference.If but the bilirubin concentration increase will exert an influence.
Claim that cholesterol is dissolved in the dioxane, with the solution dilution series concentration, the cholesterol dioxane of getting 40ul series respectively is to the 2ml serum sample with dioxane; Obtaining experimentalists and technicians concentration is 0g/L, 0.625g/L, 1.25g/L; 2.50g/L, the concentration of 5.0g/L and 10.0g/L.Detect the serum of 6 kinds of cholesterol concentrations, testing result is as shown in table 14.
The interference of table 14 cholesterol
The cholesterol in serum normal high limit is 2.29g/L clinically, and experimental result shows that the cholesterol in serum addition is 0-10g/L, and measured value does not have tangible tendency to change, and therefore, cholesterol in serum is little to the detection influence of kit of the present invention.
Claim that triglyceride is dissolved in the dioxane, with the solution dilution series concentration, the triglyceride dioxane of getting 40ul series respectively joins the 2ml serum sample with dioxane; Obtaining experimentalists and technicians concentration is 0g/L, 5g/L, 8g/L; 10g/L, 15g/L, the concentration of 20g/L and 35g/L.Detect the serum of 6 kinds of triglyceride concentrations, testing result is as shown in Tble 15.
The interference of table 15 triglyceride
The triglyceride normal high limit is 2.29mmol/L in the serum clinically, and this experimental result shows that noiseless when the content of triglyceride is from 0-15mmol/L in the serum, therefore, triglyceride is higher than normal not influence of detection to kit of the present invention in the serum.
Can find out that by table 12-table 15 the piarhemia sample can not influence the mensuration result basically.Bilirubinic existence generally can not influence Interference Detection.The sample content of haemoglobin is very micro-in the serum clinically, but contains a certain amount of haemoglobin in the haemolysis sample, so the haemolysis sample has interference to detection.
The above embodiment has only expressed several kinds of embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.
Claims (6)
1. a hydroxy-vitamine D detection kit is characterized in that, mainly includes:
(A) be coated with the microwell plate of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies, the package amount of high specific mouse 25 hydroxy-vitamine D monoclonal antibodies is 0.1～0.5 μ g in each micropore of said microwell plate;
(B) concentration is biotin labeled 25 hydroxy-vitamine Ds of 0.1 μ g/ml, and consumption is every micropore 25 μ l.
2. 25 hydroxy-vitamine D detection kit according to claim 1 is characterized in that, said 25 hydroxy-vitamine D detection kit comprise that also working concentration is the avidin of the HRPO mark of 0.5～2.0 μ g/ml.
3. 25 hydroxy-vitamine D detection kit according to claim 2 is characterized in that, the working concentration of the avidin of said HRPO mark is 1.0 μ g/ml.
4. according to each described 25 hydroxy-vitamine D detection kit of claim 1-3, it is characterized in that the said package amount that encapsulates the interior high specific mouse 25 hydroxy-vitamine D monoclonal antibodies of each micropore of plate is 0.2 μ g.
5. the preparation method of a hydroxy-vitamine D detection kit is characterized in that, may further comprise the steps:
(1) preparation encapsulates the microwell plate of high specific mouse 25 (OH) D monoclonal antibody
Package amount by 0.1～0.5 μ g/ hole encapsulates high specific mouse 25 (OH) D monoclonal antibody to micropore, and 4 ℃ are spent the night, washing, and sealing seals, and obtains encapsulating the microwell plate of high specific mouse 25 (OH) D monoclonal antibody;
(2) prepare biotin labeled 25 hydroxy-vitamine Ds by conventional method, the concentration of said biotin labeled 25 hydroxy-vitamine Ds is 0.1 μ g/ml.
6. the preparation method of 25 hydroxy-vitamine D detection kit according to claim 5; It is characterized in that; Also comprise by the concentration of 0.5～2.0 μ g/ml avidin being dissolved in the buffer solution that contains detergent, antiseptic and BSA, prepare enzyme conjugates the HRPO mark.
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| CN103163306A (en) * | 2013-02-05 | 2013-06-19 | 北京九强生物技术股份有限公司 | 25 hydroxyl vitamin D detection kit and preparation method thereof |
| CN103308621A (en) * | 2013-06-18 | 2013-09-18 | 广州金域医学检验中心有限公司 | Method for detecting 25(hydroxyl)vitamin D by using high-pass liquid chromatography-tandem mass spectrometry |
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