CN106645764A - Enzyme-linked immunosorbent assay kit for detecting diazepam and application thereof - Google Patents
Enzyme-linked immunosorbent assay kit for detecting diazepam and application thereof Download PDFInfo
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- CN106645764A CN106645764A CN201611215776.0A CN201611215776A CN106645764A CN 106645764 A CN106645764 A CN 106645764A CN 201611215776 A CN201611215776 A CN 201611215776A CN 106645764 A CN106645764 A CN 106645764A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention provides an enzyme-linked immunosorbent assay kit for detecting diazepam. The enzyme-linked immunosorbent assay kit comprises an ELISA (Enzyme Linked Immunosorbent Assay) plate coated with a diazepam coupled antigen, a diazepam monoclonal antibody, an enzyme-labeled anti-antibody, a diazepam standard product solution, a substrate color developing solution, a stopping solution, a washing solution and a dissolution solution. The invention further discloses a method for utilizing the enzyme-linked immunosorbent assay kit to detect the diazepam. The method comprises the following steps: firstly, pre-treating a sample; secondly, detecting by utilizing the kit; finally, analyzing a detection result. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the content of the diazepam in animal tissues, urine, feed and water, is simple and convenient to operate, low in cost and high in sensitivity, can be monitored in field and is suitable for screening a lot of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, and in particular to a kind of ELISA reagent for detecting diazepam
Box, it is particularly suitable for the detection of diazepam content in animal tissue, urine, feed, water quality.
Background technology
Diazepam, is a kind of central nervous system sedative also known as stable, and its chemical name is 1- methyl -5- phenyl -7-
Chloro- 1,3- dihydros-Isosorbide-5-Nitrae-Benzodiazepine -2- ketone, it is mainly used in humans and animals calmness, hypnosis, the treatment of epilepsy；Herding is given birth to
It is used as the medicine of the anti-transport stress of animal in product, animal weightening accelerator is also illegally used as in a large number.When human body intake is containing ground
West is dissolved after the food of residual, and burden of liver increases, and brains is had dizzy spells, memory impaired, and fash, Neuroleptic Leukocytopenia occur in one or two people
And kinesitherapy nerve and muscle function are suppressed, be this Ministry of Agriculture specify must not to be detected in all food animals diazepam and its
Salt and ester etc..
At present, the diazepam reported both at home and abroad and its assay method of metabolin have HPLC, HPLC-MS/MS, GC-MS,
GC-ECD, SPE-UV Derivative Spectrophotometry detection, TLC-UV etc..The methods such as HPLC, TLC are limited by sensitivity, it is impossible to met
The requirement of the detection of retention analysis at present；HPLC-MS/MS as reporting most methods at present, can simultaneously west dissolve and
Qualitative and quantitative analysis of its metabolin, but need expensive instrument and special technical staff, sample pretreatment process it is complicated and
Spend high, time-consuming length, it is difficult to meet the needs of a large amount of samples and field sample quick detection.Enzyme linked immunosorbent assay analysis method
(ELISA) there is the characteristics of simplicity is quick, specifically sensitive, sample capacity is big, analysis cost is low, can simplifies or even save sample
Purifying step, in great amount of samples and the quick selective mechanisms of field samples unique advantage is shown, can preferably meet China
Food enterprise, government function supervision department etc. carry out detection work, great development potentiality.
The content of the invention
It is an object of the invention to provide a kind of simple structure, easy to use, low price, it is portable for ground west
The enzyme linked immunological kit of detection is dissolved, and a kind of efficient, accurate, simplicity is provided, the qualitative, quantitative of high-volume screening sample is suitable to
Detection method.
Kit of the present invention, it includes：It is coated with ELISA Plate, diazepam monoclonal antibody, the enzyme of diazepam coupled antigen
Mark antiantibody, diazepam standard solution, substrate nitrite ion, terminate liquid, cleaning solution, redissolution liquid；The diazepam is coupled anti-
Original is obtained by diazepam haptens and carrier protein couplet, and the carrier protein is mouse haemocyanin, thyroprotein, ox blood
Pure albumen, rabbit serum proteins, human serum albumins, ovalbumin, hemocyanin or fibrinogen, the diazepam half is anti-
Original be by maleimide yl benzoic acid and oxalyl chloride reaction after, then with nordiazepam reaction obtain, molecular structural formula is：
The diazepam monoclonal antibody is prepared as immunogene using diazepam coupled antigen.
The antiantibody of the enzyme mark antiantibody is sheep anti mouse antiantibody.
The marker enzyme of the enzyme mark antiantibody is horseradish peroxidase；Enzyme mark antiantibody be using glutaraldehyde method or
Marker enzyme and antiantibody are carried out being coupled what is obtained by Over-voltage protection.
In order to be more convenient on-site supervision and great amount of samples examination, the kit also includes diazepam standard solution, bottom
Thing nitrite ion, terminate liquid, cleaning solution, redissolution liquid.
6 bottles of the diazepam standard solution, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.2 μ g/L, 0.4 μ g/L, 0.8 μ
g/L、1.6μg/L。
The substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is hydrogen peroxide or peroxidating
Urea, substrate solution B liquid is o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is the sulfuric acid solution of 1～2mol/L.
It is 7.4 that the cleaning solution is preferably pH value, containing 0.5%～1.0% Tween-20,0.01 ‰～0.03 ‰ nitrine
Change sodium, the phosphate buffer of 0.1～0.3mol/L.
The redissolution liquid is preferably the phosphate buffer that pH value is 7.0,0.1mol/L.
Used coating buffer solution is carbonate that pH value is 9.6,0.05mol/L wherein in ELISA Plate preparation process
Buffer solution, it is 7.1～7.5 that confining liquid is pH value, the phosphate buffer containing 1%～3% casein, 0.1～0.3mol/L.
The preparation process of ELISA Plate is in the present invention：Coating antigen is diluted to into 20 μ g/mL with coating buffer solution, is added per hole
100 μ L, 37 DEG C of lucifuges are incubated 2h or 4 DEG C overnight, and liquid in hole of inclining is washed 2 times with cleaning solution, and each 30s is patted dry, then
150～200 μ L confining liquids, 37 DEG C of lucifuges are added to be incubated 1～2h in every hole, liquid is patted dry in hole of inclining, and after being dried aluminium film is used
Vacuum sealing is preserved.
The present invention Cleaning Principle be：
The pre-coated diazepam coupled antigen on capillary strip, after adding sample solution or standard solution, adds ground west
Monoclonal antibody solution is dissolved, the diazepam in sample competes diazepam monoclonal with coated diazepam coupled antigen on ELISA Plate
Antibody, adds enzyme mark antiantibody to be amplified effect, is developed the color with nitrite ion, and sample absorbance is in negative with the content of diazepam
Correlation, the residual quantity that diazepam in sample is obtained is compared with calibration curve；Simultaneously according to the depth of color on ELISA Plate, with
The comparison of the standard solution color of series concentration can roughly in judgement sample diazepam residual quantity concentration range.
Present invention also offers a kind of method for detecting diazepam using above-mentioned enzyme linked immunological kit, it includes step：
(1) Sample pretreatment；
(2) detected with kit；
(3) testing result is analyzed.
The enzyme linked immunological kit of present invention detection diazepam is mainly qualitatively or quantitatively examined using indirect competitive ELISA method
The content of diazepam in test sample sheet；Pre-treatment requirement to sample is low, and Sample pretreatment process is simple, can be while quick detection is big
Batch sample；Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, high, smart with specific high, sensitivity
The features such as exactness is high, the degree of accuracy is high.The enzyme linked immunological kit of the present invention, simple structure, easy to use, low price, carrying
Convenient, detection method is efficient, accurate, easy, qualitative and quantitative analysis that is being suitable to high-volume screening sample.
Description of the drawings
Fig. 1：Diazepam hapten synthesis route map
Fig. 2：Kit standard curve map
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.
The preparation of the reagent constituents of embodiment 1
1st, the haptenic synthesis of diazepam (synthetic route is shown in accompanying drawing 1)
A, maleimide yl benzoic acid 1g is taken, add methylene chloride dissolving, plus oxalyl chloride 1.09g, and 2h is stirred at room temperature；It is on the rocks
Water shakes, layering, removes a layer organic phase anhydrous sodium sulfate drying, is evaporated, and obtains oil product, chloroform/n-hexane (1/1, v/
V) recrystallize, obtain maleimidobenzoyl chlorine product 0.93g, yield 86.92%；
B, 0.93g maleimidobenzoyl chlorine pyridinium dissolution, plus nordiazepam 1.0g, oil bath heating, backflow
Reaction 4h, stops reaction, and TLC detects that raw material is substantially without residue；Revolving, removes pyridine, obtains red oil, 1,2- dichloro
Ethane is recrystallized, and obtains haptens product maleimide diazepam 1.2g, yield 66.67%.
2nd, the synthesis and identification of diazepam coupled antigen
It is prepared by immunogene --- and diazepam haptens is coupled with bovine serum albumin(BSA) (BSA) and obtains immunogene.
BSA 50mg, plus the carbonate buffer solution dissolving of 5mL 0.05mol/L pH9.5, plus dithiothreitol (DTT) 10mg are taken,
It is stirred overnight at room temperature, obtains A liquid；Take haptens maleimide diazepam 12mg, plus DMF (DMF) is molten
Solution, in being added dropwise to the BSA albumin A liquid for activating, room temperature lucifuge stirring reaction 4h；With 0.02mol/L PBSs 3 days, daily
Liquid 3 times is changed, packing, -20 DEG C save backup.
It is prepared by coating antigen --- and diazepam haptens is coupled with human serum albumins (HSA) and obtains coating antigen.
Haptens maleimide diazepam 12mg, plus DMF dissolvings are taken, A liquid is obtained；Take HSA 50mg, plus 5mL
The carbonate buffer solution dissolving of 0.05mol/L pH9.5, plus dithiothreitol (DTT) 10mg, are stirred overnight at room temperature, and A liquid is added dropwise to
In protein activation liquid, room temperature lucifuge stirring 4h；With 0.02mol/L PBSs 3 days, liquid 3 times, packing, -20 DEG C of preservations were changed daily
It is standby.
In the ratio of synthesis diazepam coupled antigen reaction haptens, carrier protein and coupled product used, carry out ultraviolet
(200nm～400nm) sweep measuring, by compare three respectively the light absorption value of 260nm and 280nm calculate its combine than.It is even
The maximum absorption band of connection thing diazepam hapten-carrier albumen is compared with diazepam haptens, the maximum absorption band of carrier protein
Significantly change is there occurs, the synthesis for showing diazepam hapten-carrier albumen is successful.
3rd, the preparation of diazepam monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation：Diazepam haptens-BSA conjugate (immunogene) is fully newborn with the Freund's complete adjuvant of equivalent
Change, the Balb/c mouse of the week old of hypodermic injection 6, every 0.2mL；
2) booster immunization is twice：From the beginning of first immunisation, every two weeks booster immunization once, is replaced with not formula Freund's incomplete adjuvant
Freund's complete adjuvant, method and the same first immunisation of dosage；
3) eyeground vein blood sampling survey potency and the suppression after a week of last time booster immunization, has suppression and potency reaches 1:
Following final immunization is carried out when more than 10000：Lumbar injection is not added with the immunogen solution 0.1mL of any adjuvant, puts to death after three days
Mouse, takes its spleen and myeloma cell fusion；
4) cell supernatant, the positive hole of screening are determined using indirect competitive enzyme-linked immunosorbent analysis method.Using limiting dilution
Method carries out cloning to positive hole, obtains and set up the hybridoma cell strain of stably excreting diazepam monoclonal antibody, takes and is in
The hybridoma of exponential phase makes cell suspension with frozen stock solution, is sub-packed in cryopreservation tube, preserves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery：Diazepam monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, in being immediately placed in 37 DEG C of water-baths
Speed is melted, and centrifugation is removed after frozen stock solution, moves into culture culture in glassware；
2) ascites and antibody purification are prepared：Using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing stone
Only, pneumoretroperitoneum injects hybridoma 5 × 10 to wax oil 0.5mL/ within 7 days5Individual/only, gather ascites after 7 days.With octanoic acid-saturation sulfuric acid
Ammonium method is purified, and obtains diazepam monoclonal antibody solution (- 20 DEG C of preservations).
(3) measure of antibody titer
The potency for determining antibody with indirect competitive ELISA method is 1:(200000～500000).
Indirect competitive ELISA method：With diazepam haptens-HSA conjugate coated elisa plates, diazepam standard items are added
The sheep anti mouse antiantibody solution of solution, diazepam monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions
30min, pours out liquid in hole, is washed with cleaning solution 3～5 times, is patted dry with blotting paper；Add substrate nitrite ion, 25 DEG C of reactions
After 15min, terminate liquid terminating reaction is added；Setting ELIASA is determined per hole absorbance at wavelength 450nm.
(4) measure of monoclonal antibody specificity
Antibody specificity refers to the ability and the ratio with such antigen-analogues ability of its homospecificity antigen binding
Compared with conventional cross reacting rate is used as evaluation criterion.Cross reaction is less, and the specificity of antibody is then higher.
Diazepam, nitrazepam, Oxazepam are serially diluted in this experiment, carry out indirect competition with monoclonal antibody respectively
ELISA, makes calibration curve, and analysis obtains IC50, then it is calculated as follows cross reacting rate：
As a result the cross reacting rate for showing each analog is：Diazepam 100%, nitrazepam 7.6%, Oxazepam 8.8%.
Antibody of the present invention has specific binding to the analog no cross reaction such as nitrazepam, Oxazepam just for diazepam.
4th, the preparation of sheep anti mouse antiantibody
With sheep as immune animal, with mouse source antibody as immunogen immune pathogen-free domestic sheep, sheep anti mouse antiantibody is obtained.
5th, enzyme marks the preparation of antiantibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.Pass
The Over-voltage protection of system requires that enzyme and the molar concentration rate of antibody are 4 in reaction system:1, because horseradish peroxidase is strong
The site that many is combined with antibody is produced under oxidation, the horseradish peroxidase molecule of so activation act as each point of connection
The bridge of son, reduces the enzymatic activity of enzyme marker, and many condensates are mixed with the conjugate for making preparation.Ask to solve this
Topic, we are improved traditional method, i.e.,：
(1) closed process of amino is eliminated, because the amino that can produce the connection of itself amino is actual seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, the method after improvement is than traditional side
Method is easy, and the loss to enzymatic activity is reduced.
6th, the preparation of ELISA Plate
Coating antigen (diazepam haptens-HSA conjugates) is diluted to into 20 μ g/mL with coating buffer solution, 100 are added per hole
μ L, 37 DEG C of lucifuges are incubated 2h, and liquid in hole of inclining is washed 2 times with cleaning solution, and each 30s is patted dry, and then add in every hole
200 μ L confining liquids, 37 DEG C of lucifuges are incubated 2h, and liquid is patted dry in hole of inclining, and are preserved with aluminium film vacuum sealing after being dried.
The establishment of the enzyme linked immunological kit of the detection diazepam of embodiment 2
Set up the enzyme linked immunological kit of detection diazepam so as to comprising following components：
(1) it is coated with the ELISA Plate of diazepam coupled antigen；
(2) 6 bottles of diazepam standard solution, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.2 μ g/L, 0.4 μ g/L, 0.8 μ g/
L、1.6μg/L；
(3) diazepam monoclonal antibody working solution；
(4) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) cleaning solution be pH value be 7.4, containing 0.5%～1.0% Tween-20,0.01 ‰～0.03 ‰ sodium azide,
The phosphate buffer of 0.1～0.3mol/L；
(8) liquid is redissolved for phosphate buffer that pH value is 7.0,0.1mol/L.
The detection of diazepam in the sample of embodiment 3
1st, Sample pretreatment
Tissue samples：
The tissue samples after 2.0g ± 0.05g homogeneous are weighed into 50mL polystyrene centrifuge tubes, 8mL 0.1mol/ are added
L sodium hydroxide solutions, with oscillator 5min, (20～25 DEG C) centrifugation 10min of 3000g room temperatures are vibrated；Pipette 1mL supernatants extremely
In 50mL polystyrene centrifuge tubes, 10mL n-hexanes are added, with oscillator 5min, (20～25 DEG C) centrifugations of 3000g room temperatures are vibrated
10min；5mL upper organic phases are pipetted into 10mL clean dried glass tubes, is dried up under 50～60 DEG C of water-bath nitrogen streams；Add
1mL redissolves liquid, with vortex instrument whirling motion 2min；50 μ L are taken for analyzing.
Urine, water quality sample：
The limpid samples of 1mL are pipetted into 50mL polystyrene centrifuge tubes, 4mL 0.1mol/L sodium hydroxide solutions are added, is used
Oscillator vibrates 2min；1mL mixed liquors are pipetted into 50mL polystyrene centrifuge tubes, 10mL n-hexanes are added, is shaken with oscillator
Swing 5min, (20～25 DEG C) centrifugation 10min of 3000g room temperatures；5mL upper organic phases are pipetted into 10mL clean dried glass tubes,
Dry up under 50～60 DEG C of water-bath nitrogen streams；1mL is added to redissolve liquid, with vortex instrument whirling motion 2min；50 μ L are taken for analyzing.Note：
If sample is not limpid, (20～25 DEG C) centrifugation 5min of 3000g room temperatures are needed.
Feed sample：
1.0g ± 0.05g feeds sample is weighed into 50mL polystyrene centrifuge tubes, be separately added into 1mL deionized waters and
3mL0.1mol/L sodium hydroxide solutions, with vortex instrument whirling motion 1min, add 10mL n-hexanes, and with oscillator 10min is vibrated,
(20～25 DEG C) centrifugation 10min of 3000g room temperatures；1mL upper organic phases are pipetted into 10mL clean dried teat glasses, in 50～
Dry up under 60 DEG C of water-bath nitrogen streams；1mL is added to redissolve liquid, with vortex instrument whirling motion 2min；DIFFERENT FEED sample is again as follows
It is diluted：Batch：Sample liquid is pressed into 1 with liquid is redissolved:9 volume ratios are diluted detection (+9 parts of redissolution liquid of 1 part of sample liquid),
Concentrate feed/premix：Sample liquid is pressed into 1 with liquid is redissolved:19 volume ratios are diluted detection (+19 parts of redissolution liquid of 1 part of sample liquid)；
50 μ L are taken for analyzing.
2nd, detected with kit
Diazepam standard solution or premenstrual process are added in the ELISA Plate micropore for be coated with diazepam coupled antigen
The μ L/ holes of sample solution 50, are subsequently adding the μ L/ holes of diazepam monoclonal antibody working solution 50, and gently vibration is mixed, and uses cover plate membrane cover
30min is reacted in the rearmounted 25 DEG C of light protected environments of plate；Liquid in hole is poured out, adds 250 μ L cleaning solutions fully to wash per hole 4～5 times,
Per minor tick 10s, patted dry with blotting paper；The μ L/ holes of sheep anti mouse antiantibody 100 of horseradish peroxidase-labeled are added, gently
Vibration is mixed, and with 30min is reacted in the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate, is taken out and is repeated board-washing step；Bottom is added per hole
The μ L of thing liquid A liquid urea peroxide 50, substrate solution B liquid tetramethyl benzidine (TMB) 50 μ L, gently vibration is mixed, with cover plate membrane cover plate
15min is reacted in rearmounted 25 DEG C of light protected environments, the μ L of terminate liquid 2mol/L sulfuric acid 50 are added per hole, gently vibration is mixed, and uses enzyme mark
Instrument wavelength is set at 450nm, is determined per hole absorbance (OD values).
3rd, Analysis of test results
With the absorbance values (B) of the standard solution of each concentration for being obtained divided by first standard solution (0
Standard) absorbance (B0) 100% is multiplied by again, obtain percentage absorbance.With the right of diazepam standard concentration (μ g/L)
Numerical value is X-axis, and percentage absorbance is Y-axis, draws calibration curve, as shown in Figure 2.Sample solution is calculated with same method
Percentage absorbance, the diazepam content of corresponding each sample then can read from calibration curve.
The determination test of the diazepam enzyme linked immunological kit technical parameter of embodiment 4
1st, kit sensitivity and test limit
Kit sensitivity is conventionally determined, kit standard curve minimum point is 0.1 μ g/L, calibration curve
Scope is 0.1～1.6 μ g/L, IC50(50% inhibition concentration) domain of walker is 0.19～0.25 μ g/L；To blank pork, chicken,
Each 20 parts of urine, water quality, batch, concentrate feed, premix sample is detected, found from calibration curve corresponding to each percentage
The concentration of absorbance, test limit is represented with the mean value of 20 parts of concentration of specimens plus 3 times of standard deviations, as a result obtains the method to group
Knit, urine, the detection of water quality sample are limited to 1 μ g/kg, the detection to batch sample is limited to 10 μ g/kg, to concentrate feed, premix
The detection of material sample is limited to 20 μ g/kg.
2nd, sample preci-sion and accuracy test
It is inclined with the testing result relative standard of a certain concentration samples of replication using the rate of recovery as accuracy estimating index
Difference (RSD%) is used as precision evaluation index.Computing formula is：The rate of recovery (%)=actual measured value/theoretical value × 100%,
The wherein addition concentration of theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD be standard deviation, X
For the mean value of determination data.
Blank pork, chicken, urine, water quality sample are carried out by 1 μ g/kg, 2 μ g/kg, 4 μ g/kg, tri- concentration diazepams
Addition is reclaimed and determined, and 10 μ g/kg, 20 μ g/kg, 40 μ g/kg, tri- concentration diazepams are added back to blank batch sample
Receive and determine, 20 μ g/kg, 40 μ g/kg, 80 μ g/kg, tri- concentration diazepams are added back to blank concentrate feed, premix sample
Receive and determine, each sample do 4 it is parallel, be measured with three batches of different kits, calculate the sample mean rate of recovery and precision
As a result see the table below.
The precision of table 1 and accuracy test
Blank pork, chicken, urine, water quality sample are added with the diazepam of 1,2,4 tri- concentration of μ g/kg, 10,
20th, the diazepam of 40 tri- concentration of μ g/kg is added to blank batch sample, the ground of 20,40,80 tri- concentration of μ g/kg
West is dissolved and blank concentrate feed, premix sample is added, and average recovery rate is between 70%～110%；In batch, between criticizing relatively
Standard deviation is respectively less than 15%.
3rd, stabilization of kit test
Kit preservation condition is 2～8 DEG C, through the measure of 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, diazepam add actual measured value within normal range (NR).Consider during transport and use, to have
Improper preservation condition occurs, and kit is placed 7 days under 37 DEG C of preservation conditions, carries out accelerated aging tests, as a result shows
The kit indices comply fully with requirement.Occur in view of kit freezing situation, kit is put into into -20 DEG C of refrigerators cold
Freeze 7 days, measurement result also indicates that kit indices are completely normal.Can show that kit can be at 2～8 DEG C from result above
At least preserve more than 12 months.
Claims (7)
1. it is a kind of detection diazepam enzyme linked immunological kit, it is characterised in that include：It is coated with the enzyme of diazepam coupled antigen
Target, diazepam monoclonal antibody, enzyme mark antiantibody, diazepam standard solution, substrate nitrite ion, terminate liquid, cleaning solution,
Redissolve liquid；The diazepam coupled antigen is obtained by diazepam haptens and carrier protein couplet, and the carrier protein is mouse
Haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human serum albumins, ovalbumin, hemocyanin or
Fibrinogen, the diazepam haptens be by maleimide yl benzoic acid and oxalyl chloride reaction after, then with remove Jia Dixi
Dissolve reaction to obtain, molecular structural formula is：
2. kit as claimed in claim 1, it is characterised in that the diazepam monoclonal antibody be coupled with diazepam it is anti-
Original work are prepared for immunogene.
3. kit as claimed in claim 1, it is characterised in that the antiantibody of the enzyme mark antiantibody is that sheep anti mouse is anti-
Body.
4. kit as claimed in claim 1, it is characterised in that the marker enzyme of the enzyme mark antiantibody is horseradish peroxidating
Thing enzyme, the substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is hydrogen peroxide or urea peroxide,
Substrate solution B liquid is o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is the sulfuric acid solution of 1～2mol/L.
5. kit as claimed in claim 1, it is characterised in that it is 7.4 that the cleaning solution is pH value, containing 0.5%～
1.0% Tween-20,0.01 ‰～0.03 ‰ sodium azide, the phosphate buffer of 0.1～0.3mol/L；It is described redissolution liquid be
PH value is the phosphate buffer of 7.0,0.1mol/L.
6. kit as claimed in claim 1, it is characterised in that the concentration of the diazepam standard solution is respectively 0 μ g/
L、0.1μg/L、0.2μg/L、0.4μg/L、0.8μg/L、1.6μg/L。
7. in a kind of detection sample diazepam content method, including step：
(1) sample pre-treatments；
(2) detected with the kit described in any one of claim 1～6；
(3) testing result is analyzed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061168A (en) * | 2018-08-27 | 2018-12-21 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of diclazuril |
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| CN112924700A (en) * | 2021-01-26 | 2021-06-08 | 海南医学院 | Coating process of Keyhole Limpet Hemocyanin (KLH) and quantitative detection kit for mouse KLH specific IgG antibody |
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| CN109061168A (en) * | 2018-08-27 | 2018-12-21 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of diclazuril |
| CN109061168B (en) * | 2018-08-27 | 2022-10-21 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting diclazuril and application thereof |
| CN112375148A (en) * | 2020-11-04 | 2021-02-19 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Preparation method of anti-diazepam single-chain antibody, product and diazepam detection method |
| CN112375148B (en) * | 2020-11-04 | 2022-03-29 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Preparation method of anti-diazepam single-chain antibody, product and diazepam detection method |
| CN112924700A (en) * | 2021-01-26 | 2021-06-08 | 海南医学院 | Coating process of Keyhole Limpet Hemocyanin (KLH) and quantitative detection kit for mouse KLH specific IgG antibody |
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