CN102520181B - Colloidal gold assay kit for rapidly detecting dolantin and preparation of kit - Google Patents
Colloidal gold assay kit for rapidly detecting dolantin and preparation of kit Download PDFInfo
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- CN102520181B CN102520181B CN201110458002.1A CN201110458002A CN102520181B CN 102520181 B CN102520181 B CN 102520181B CN 201110458002 A CN201110458002 A CN 201110458002A CN 102520181 B CN102520181 B CN 102520181B
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Abstract
The invention provides a colloidal gold assay kit for rapidly detecting dolantin and preparation of the kit. The kit comprises a reagent strip sequentially provided with filter sample paper, an immunologic colloidal gold paper piece, an immunologic nitrocellulose membrane and absorbent paper from a sample adding end, wherein the immunologic colloidal gold paper piece is a glass fiber gasket coated with a colloidal gold labeled monoclonal dolantin antibody, and the immunologic nitrocellulose membrane is provided with a detecting line and a quality control line. Dolantin-carrier complex is spotted on the detecting line, and goat-anti-mouse IgG (immunoglobulin G) is spotted on the quality control line. The invention further provides a method for preparing the colloidal gold assay kit for rapidly detecting the dolantin. The kit for detecting the dolantin is high in sensitivity, simple and rapid in operation, accurate in detecting result, low in detecting cost and convenient in storage and transportation.
Description
Technical field
The present invention relates to a kind of gold-immunochromatographyreagent reagent for assay box and preparation technology, be specifically related to Sauteralgyl gold-immunochromatographyreagent reagent for assay box and preparation technology.
Background technology
The formal name used at school of Sauteralgyl, pethidine, is called again mark pyridine, demerol, is artificial synthetic morphine substitute, its hydrochloride is white crystals sprills, water-soluble and the ethanol of energy, is made generally in injection, is mainly used in the indications such as pain, cardiac asthma, internal organ angina.Sauteralgyl is similar to morphine to the mechanism of action of human body, glad, analgesic activity is little compared with morphine, but use continuously 1-2 week just can produce dependence, once after stopping, can producing similar in appearance to the withdrawal syndrome after morphine abstinence syndrome, main manifestations is One's spirits are drooping depressed, general malaise, the runny nose of shedding tears, vomiting, diarrhoea, insomnia, and severe patient also can produce collapse.The abuse of Sauteralgyl is one of drug issue that China is current faced, and has been put into national narcotics and has given strict supervision.
At present, in the detection method of Sauteralgyl, use is mainly the method that gas chromatography GC, high-efficient liquid phase chromatogram HPLC, gaseous mass spectrum GC/MS or GC/FID analyze, these detection methods need professional to operate, and complicated time-consuming, cost is high, the confirmation that is only applicable to Sauteralgyl detects, but can not reach domestic drug law enforcement, virus investigation, counterdrug operation required easily and fast, sensitive requirement.So far, the domestic Patents that does not still relate to Sauteralgyl quick detection reagent.
Summary of the invention
The object of the invention is to overcome defect of the prior art, from improving detection sensitivity, provide a kind of not only convenient but also Sauteralgyl colloidal gold fast detecting reagent kit and preparation method thereof fast.
An aspect of of the present present invention, a kind of Sauteralgyl gold-immunochromatographyreagent reagent for assay box is provided, comprise reagent strip, reagent strip starts to be followed successively by filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter and thieving paper from application of sample end, wherein, the described immune colloid gold scraps of paper are the glass fibre pad that is coated with the anti-Sauteralgyl monoclonal antibody of colloid gold label, immunity nitrocellulose filter is provided with detection line (T line) and nature controlling line (C line), on detection line, specking has Sauteralgyl-carrier complexes, and on nature controlling line, specking has sheep anti-mouse igg.
Preferably, in the anti-Sauteralgyl monoclonal antibody of described colloid gold label, collaurum is the spheric grain of particle diameter 39-42nm.
A second aspect of the present invention also provides the preparation method of above-mentioned Sauteralgyl gold-immunochromatographyreagent reagent for assay box, comprises the following steps:
1) with two step reduction method, prepare the collaurum that mean grain size is 39-42nm;
2) the anti-Sauteralgyl monoclonal antibody of the colloid gold label adaptive immune collaurum that adopts step 1 to make;
3) adopt metal spraying damping fluid dilution step 2) immune colloid gold adaptive immune colloidal gold solution, with the pretreated glass fibre pad of immune colloid gold solution spraying, make the immune colloid gold scraps of paper;
4) specking Sauteralgyl-carrier complexes and sheep anti-mouse igg respectively on the detection line of nitrocellulose filter and nature controlling line, make immune nitrocellulose filter;
5) immune nitrocellulose filter, the thieving paper of the immune colloid gold scraps of paper of filter sample paper, step 3 preparation, step 4 preparation are sticked on offset plate successively, cutting makes detection reagent strip; Finally pack detection reagent strip into plastic casing and make detection kit.
Step 1) to prepare mean grain size be that the collaurum of 39-42nm specifically comprises the following steps: for described two step reduction method
A) reduce for the first time chlorauric acid solution:
The HAuCl that preparation concentration is 0.0004-0.0005mol/L
4aqueous solution, 20-30 minute is boiled in heating, adds while stirring afterwards trisodium citrate aqueous solution, HAuCl
4with the mol ratio of trisodium citrate be 1: (8-9), after sonic oscillation 2-3 minute, be cooled to room temperature, both made the collaurum original solution of 14～16nm particle diameter.
B) reduce for the second time chlorauric acid solution:
The collaurum original solution that the first step is made is positioned over equilibrium temperature in 4.0-4.5 ℃ of constant temperature ice bath, presses subsequently collaurum original solution and HAuCl
4aqueous solution volume ratio 1: (1.9-2.0) add the 0.003-0.004mol/L HAuCl of 4.0-4.5 ℃
4aqueous solution, immediately drips the ascorbic acid of 4.0-4.5 ℃ and the mixed aqueous solution of PVP (polyvinyl pyrrolidone), the HAuCl that the ascorbic acid adding and this step add while stirring
4mol ratio be (25-26): 1, the HAuCl that the PVP adding and this step add
4weight ratio be 1: (2.0-2.1), keep temperature constant in reaction, be stirred to and react completely, solution is transparent claret, has both made the colloidal gold solution of 39-42nm particle diameter.
In two step reduction method, configure various aqueous solution and all adopt distilled water.
Step a) in, the concentration that adds trisodium citrate aqueous solution is 0.01-0.02mol/L.
Step a) in, the frequency of ultrasonic concussion is 20-30KHZ.
Step b), in, the rate of addition of the mixed aqueous solution of ascorbic acid and PVP is drip/s of 1-2.
Step b) in, described ascorbic acid mixes in distilled water aqueous solution with PVP's, and the concentration of ascorbic acid is 0.017-0.019mol/L, and the concentration of PVP is 0.137-0.139g/L.
Step b), in, stirring reaction is about 50-70 minute.
The collaurum that adopts said method to make is limpid transparent, and grain size homogeneous, without agglutinating particle, does not find nonspherical particle substantially.
Step 2) the anti-Sauteralgyl monoclonal antibody of described colloid gold label can adopt conventional method mark.
Concrete, step 3) preparation of the immune colloid gold scraps of paper comprises the following steps:
I. use metal spraying damping fluid dilution step 2) immune colloid gold, obtain OD
540value is 1.2～2.0 immune colloid gold solution;
II. with pretreatment fluid, soak all-glass paper, after being dried, then use immune colloid gold solution spraying all-glass paper, dry, make the immune colloid gold scraps of paper.
Preferably, described in step I, the component of metal spraying damping fluid is: 8～12v%1.0M Tris liquid, 0.2～0.4w/v% PEG 20000,0.15～0.25w/v% bovine serum albumin(BSA), 0.1～0.3w/v% skim milk, 0.25～0.35w/v% casein, with 0.02～0.08w/v% sodium azide, with salt acid for adjusting pH to 8.5 ± 0.05, surplus is water.
Optimum, in step I, metal spraying buffer formulation is: 10v%1.0M Tris liquid, 0.3w/v% PEG 20000,0.2w/v% bovine serum albumin(BSA), 0.2w/v% skim milk, 0.3w/v% casein, with 0.05w/v% sodium azide, with salt acid for adjusting pH to 8.5 ± 0.05, surplus is water.
Preferably, the component of pretreatment fluid comprises described in Step II: 0.5～0.8v%Tween-20, and 12～18w/v% sucrose, solvent is water.Optimum, in Step II, pretreatment fluid formula is: 0.7%Tween-20, and 16w/v% sucrose, surplus is water.
Preferably, in Step II, while soaking all-glass paper with pretreatment fluid, every 30ml pretreatment fluid soaks all-glass paper 261mm * 220mm; During with immune colloid gold solution spraying all-glass paper, on every 261mm * 220mm all-glass paper, spray 20ml immune colloid gold solution, dry, make the immune colloid gold scraps of paper.
In the present invention, wt% is weight percentage, and v% refers to percent by volume; W/v% refers to percent weight in volume, and 1w/v% is in 100ml solution containing 1g.
Sauteralgyl-carrier complexes (as Sauteralgyl-bovine serum albumin(BSA) compound) is commercially available or adopt conventional method by Sauteralgyl and carrier is compound makes.
The principle of work of Sauteralgyl gold-immunochromatographyreagent reagent for assay box of the present invention, is antibody-antigen-specific association reaction and the immune film chromatographic technique that utilizes high special, by Immune competition inhibition method, carrys out the Sauteralgyl occurring in qualitative detection human urine.
Detection reagent strip in kit is to realize the key that Sauteralgyl detects, and in ELISA test strip district, (T line) has been coated with Sauteralgyl-carrier complexes.The other end in test strips is fixed with anti-Sauteralgyl monoclonal antibody-collaurum scraps of paper.When urine adds from well, be penetrated on the application of sample pad of reagent strip, the tested sample first Sauteralgyl monoclonal antibody of the colloid gold label on glass fibre membrane is combined, and by capillary action, continue chromatography to reagent strip nitrocellulose filter, order by specking on nitrocellulose filter the detection zone of Sauteralgyl-carrier complexes and specking the Quality Control district of IgG.If contain Sauteralgyl in urine, they will compete antigen binding site limited on collaurum-anti-Sauteralgyl antibody with Sauteralgyl-carrier complexes, when Sauteralgyl concentration reaches the threshold concentration of product design when above, they will occupy whole antigen binding site in anti-Sauteralgyl monoclonal antibody, so just stoped Sauteralgyl-compound combination of anti-Sauteralgyl monoclonal antibody-collaurum and detection zone, detection zone can not capture colloid gold particle and the appearance of redfree colour band, and testing result is positive.If there is no the concentration of Sauteralgyl or Sauteralgyl in urine lower than threshold concentration, anti-Sauteralgyl monoclonal antibody-collaurum moves to detection zone in company with urine, and detection zone captures colloid gold particle and presents red ribbon, and testing result is negative.
Whether the Quality Control district in test strips (C line) is coated with sheep anti-mouse igg, normal with the work of indicator box reactive system.The existence of the appearance of nature controlling line and Sauteralgyl or its metabolin is irrelevant.The appearance of Quality Control district colour band shows: 1. 2. sample normal operation on paper slip of sample addition abundance.
The using method of kit of the present invention is:
1. kit is placed on level table.
2. with application of sample suction pipe, draw urine sample, then drip 3 (about 120ul) urine samples in the well of kit.The a different sample of every detection is noted using different suction pipes.
3. observations: 3-8 minute sentence read result after dripping sample.
The determination methods of testing result:
Negative: in result watch window, occur two colour bands, red lines respectively appear in detection line (T line) and nature controlling line (C line) position, represent without Sauteralgyl, to exist in sample.
Positive: only in nature controlling line (C line) position of result watch window, occur red lines, any lines do not appear in detection line (T line), representing has Sauteralgyl to exist in sample.
Invalid: nature controlling line (C line) does not occur.In any case, C line all should form, and represents that application of sample and operation are correct.C line does not occur showing that test result is uncertain, should reform.
Beneficial effect of the present invention:
(1) the present invention is when preparing Sauteralgyl gold-immunochromatographyreagent reagent for assay box, adopted two step reduction method to prepare collaurum molecule, make the collaurum molecule obtaining be uniform about 40nm spheric grain, than it published technology, although increased an operation steps, but because colloid gold particle is of moderate size, whole homogeneous, make the joint efficiency of itself and monoclonal antibody higher, effect is more stable, this guaranteed markers step controllability, reduced the differences between batches of kit.
(2) in immune colloid gold scraps of paper preparation process, by adopting the pretreatment fluid of rational formula to carry out pre-service and match suitable metal spraying damping fluid glass fibre membrane, can guarantee that immune colloid gold discharges completely on basis, the release chromatography of immune colloid gold while effectively slowing down detection, be conducive to the abundant reaction bonded of antigen-antibody, effectively raise the sensitivity of reaction, under same threshold value, also can reduce the consumption of immune colloid gold, cost-saving.
(3) the Sauteralgyl detection kit that prepared by the present invention, has highly sensitively, can detect the Sauteralgyl of 900ng/ml in urine; Simple to operate, testing result naked eyes are visible, without special experimental apparatus and professional and technical personnel; Detect fast, in 5-8 minute, can show testing result; And testing result expense is cheap, the advantages such as storage and convenient transportation.
Accompanying drawing explanation
Fig. 1: collaurum transmission electron microscope picture
Fig. 2: Sauteralgyl gold-immunochromatographyreagent reagent for assay box reagent strip schematic diagram
1: filter sample paper 2: immune colloid gold paper 3: immune nitrocellulose filter 4: thieving paper
5: detection line (T line) 6: nature controlling line (C line)
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for the present invention is described, but not limit the scope of the invention.
The preparation of embodiment 1 Sauteralgyl gold-immunochromatographyreagent reagent for assay box
Reagent source:
Anti-Sauteralgyl monoclonal antibody, sheep anti-mouse igg polyclonal antibody, Sauteralgyl-bovine serum albumin(BSA) compound (KET-BSA): purchased from U.S. Mei Di Bioisystech Co., Ltd (MAXMED LABORATORIES INC.)
Cellulose nitrate film, all-glass paper: purchased from Sartorius AG (SARTORIUS)
Reagent preparation:
The phosphate buffer of 0.1M pH7.6: take respectively 10.2gNa
2hPO
4, 3.4gNaH
2pO
4, add 900ml purified water and dissolve, by hydrochloric acid conditioning solution pH value, be 7.6, by purified water, be settled to 1000ml.
10% bovine serum albumin(BSA) (BSA) solution: take 100gBSA and dissolve by purified water and be settled to 1000ml.
The borate buffer of 0.1%BSA: take borax 9.535g and be dissolved in 900ml purified water, with hydrochloric acid adjust pH to 8.6 ± 0.05, be settled to 1000ml, obtain 0.1M borate buffer solution.Take 1gBSA and with 0.1M borate buffer solution, dissolve and be settled to 1000ml, be the borate buffer of 0.1%BSA.
Preparation process:
1, the preparation of collaurum: the collaurum of preparing particle diameter 40nm with sodium citrate-ascorbic acid two step reduction method
A) reduce for the first time chlorauric acid solution: by the HAuCl of 6ml 0.0164mol/L
4aqueous solution adds in 200ml distilled water, and heating is boiled 30 minutes, slowly stirs afterwards and also accurately adds 50ml 0.016mol/L trisodium citrate aqueous solution.25KHZ sonic oscillation was cooled to room temperature after 2 minutes, had both made the collaurum original solution of about 15nm particle diameter.
B) reduce for the second time chlorauric acid solution: get the collaurum protokaryon solution 26ml that the first step makes, and be positioned over equilibrium temperature in 4 ℃ of water-baths, adding subsequently 50ml precooling is the 0.0035mol/L HAuCl of 4.0 ℃
4aqueous solution and slowly drip immediately 250ml precooling be 4.0 ℃ containing the ascorbic acid of 0.018mol/L and the mixed aqueous solution of 0.138g/L PVP, rate of addition is drip/s of 1-2, stirring reaction 1 hour, solution is transparent claret, has both made the colloidal gold solution of 40nm particle diameter.
The collaurum making adopts ultraviolet spectrophotometer scanning to have single absorption peak, and peak shape is narrow, and λ max is about 525nm, adopts transmission electron microscope observing, and as shown in Figure 1, particle size range is about 40nm, and grain size homogeneous, without agglutinating particle, is not found nonspherical particle substantially.
2, immune colloid gold preparation
2.1 are diluted to the phosphate buffer of 0.1M pH7.6 the antibody-solutions that concentration is 1.0mg/ml by anti-Sauteralgyl monoclonal antibody.
2.2 get the colloidal gold solution of 500ml, add inward the 0.1MpH7.6 phosphate buffer of 1/10 times of collaurum volume to mix 3 minutes.Under rapid stirring, more slowly the anti-Sauteralgyl monoclonal antibody solution 4ml of dilution is added wherein.Room temperature reaction 5 minutes also slowly stirs frequently.
After 2.3 reactions finish, in above-mentioned reactant liquor, add fast 10% bovine serum albumin(BSA) (BSA) solution of 10ml.Room temperature reaction 5 minutes also slowly stirs frequently.
2.4 by centrifugal 20 minutes of 8000 revs/min of immune colloid golds preparing, and retained precipitation, and collected 12500 revs/min of supernatants centrifugal 30 minutes, abandoned supernatant.Collecting twice centrifugation redissolves to OD with the borate buffer that contains 0.1%BSA
540between 13-15.
The anti-Sauteralgyl monoclonal antibody that the 2.5 purifying immune colloid golds that make are colloid gold label.
3, immune colloid gold is made of paper standby
Adopt respectively method 1,2,3 preparations.
Method 1:
The preparation of 3.1 pretreatment fluids: accurately take 7ml Tween-20,160g sucrose, adds purified water and be settled to 1000ml.
The preparation of 3.2 metal spraying damping fluids: get 800ml purified water, add inward 100ml 1.0M Tris liquid, with salt acid for adjusting pH to 8.5.Accurately take 3.0g PEG 20000,2.0g bovine serum albumin(BSA), 2.0g skim milk, 3.0g casein solution and 0.5g sodium azide add in solution, fully dissolve, and mix, and add purified water to cumulative volume 1000ml.
The immune colloid gold of 3.3 use metal spraying damping fluid dilution step 2 preparations, to solution O D
540value is 1.5, adaptive immune colloidal gold solution.
3.4 use pretreatment fluids soak all-glass paper, and every 30ml pretreatment fluid soaks all-glass paper 261mm * 220mm, soak after 30min, dry at 37 ℃; Use again immune colloid gold solution spraying all-glass paper, on every 261mm * 220mm all-glass paper, spray 20ml immune colloid gold solution, dry, make the immune colloid gold scraps of paper.
Method 2:
The preparation of 3.1 pretreatment fluids: accurately take 5ml Tween-20,180g sucrose, adds purified water and be settled to 1000ml.
The preparation of 3.2 metal spraying damping fluids: get 800ml purified water, add inward 120ml 1.0M Tris liquid, with salt acid for adjusting pH to 8.5.Accurately take 2.0g PEG 20000,1.5g bovine serum albumin(BSA), 3.0g skim milk, 2.5g casein solution and 0.8g sodium azide add in solution, fully dissolve, and mix, and add purified water to cumulative volume 1000ml.
3.3 use metal spraying damping fluid dilution step 2) immune colloid gold, to solution O D
540value is 1.5, adaptive immune colloidal gold solution.
3.4 use pretreatment fluids soak all-glass paper, and every 30ml pretreatment fluid soaks all-glass paper 261mm * 220mm, soak after 25min, dry at 37 ℃; Use again immune colloid gold solution spraying all-glass paper, on every 261mm * 220mm all-glass paper, spray 20ml immune colloid gold solution, dry, make the immune colloid gold scraps of paper.
Method 3:
The preparation of 3.1 pretreatment fluids: accurately take 8ml Tween-20,120g sucrose, adds purified water and be settled to 1000ml.
The preparation of 3.2 metal spraying damping fluids: get 800ml purified water, add inward 80ml 1.0M Tris liquid, with salt acid for adjusting pH to 8.5.Accurately take 4.0g PEG 20000,2.5g bovine serum albumin(BSA), 1.0g skim milk, 3.5g casein solution and 0.2g sodium azide add in solution, fully dissolve, and mix, and add purified water to cumulative volume 1000ml.
3.3 use metal spraying damping fluid dilution step 2) immune colloid gold, to solution O D
540value is 1.5, adaptive immune colloidal gold solution.
3.4 use pretreatment fluids soak all-glass paper, and every 30ml pretreatment fluid soaks all-glass paper 261mm * 220mm, soak after 30min, dry at 37 ℃; Use again immune colloid gold solution spraying all-glass paper, on every 261mm * 220mm all-glass paper, spray 20ml immune colloid gold solution, dry, make the immune colloid gold scraps of paper.
4, the preparation of immune nitrocellulose filter
4.1 are diluted to 0.5mg/ml by sheep anti-mouse igg with phosphate buffer, make nature controlling line (C line) solution.
4.2 Sauteralgyl-BSA compound is diluted to protein concentration with phosphate buffer is that 0.1mg/ml makes detection line (T line) solution.
4.3 use point film machine specking C, T line solution, make immune nitrocellulose filter.The long nitrocellulose filter of every 1m is coated with C line and the T line solution of 1ml, and the spacing of detection line and nature controlling line is 5.0mm.
5, filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter, thieving paper are sticked on offset plate successively, it is the reagent strip (as shown in Figure 2) of 4mm that cutting becomes width.
6, detection reagent strip is packed into and in plastic casing, obtain detection kit.
The sensitivity experiment of embodiment 2 Sauteralgyl gold-immunochromatographyreagent reagent for assay boxes
Detection method:
1, get the Sauteralgyl gold-immunochromatographyreagent reagent for assay box that embodiment 1 makes, kit is placed on level table.
2, with application of sample suction pipe, draw sample, then drip 3 (about 120ul) samples in the well of kit.The a different sample of every detection is used different suction pipes.
3, observations: 3-8 minute sentence read result after dripping sample.
Detect sample:
Configuration Sauteralgyl concentration is that the Quality Control reference material of 450ng/ml, 900ng/ml, 1800ng/ml is as sample.
Testing result:
| Sauteralgyl concentration | Kit testing result |
| 450ng/ml | Negative |
| 900ng/ml | Positive |
| 1800ng/ml | Positive |
Result confirmation, the Sauteralgyl gold-immunochromatographyreagent reagent for assay box sensitivity making meets the requirements.
The clinical testing of embodiment 3 Sauteralgyl gold-immunochromatographyreagent reagent for assay boxes
Gather 50 parts of human urine samples, wherein 20 parts is Sauteralgyl user's urine specimen, and 30 parts of urine specimens that are normal person, are used 50 parts of Sauteralgyl gold-immunochromatographyreagent reagent for assay boxes respectively urine sample to be detected.At kit well, drip 2-3 respectively and drip urine sample, after 5 minutes, kit all starts colour developing, and color stability after 6 minutes reaches the object developing the color in 8 minutes.Result shows that 20 parts of Sauteralgyl users' urine sample is all positive, and 30 parts of normal persons' testing result is all negative, without the kit not developing the color.
The testing result of gaseous mass analyzer GC/MS of take is added up clinical test results as reference reagent, and statistics sees the following form:
Clinical test results summary sheet
It is reference that gaseous mass spectrum GC/MS detects reagent, this Sauteralgyl gold-immunochromatographyreagent reagent for assay box:
Sensitivity=20/20 * 100%=100%
Specificity=30/30 * 100%=100%
Predictive value=20/20 * the 100%=100% of positive test
Predictive value=30/30 * the 100%=100% of negative test
50 * the 100%=100% of coincidence rate (crude agreement)=(20+30)
The specificity experiment of embodiment 4 Sauteralgyl gold-immunochromatographyreagent reagent for assay boxes
Get the detection kit that example 1 makes, by following table application of sample detection respectively, testing result is as follows:
Unit: ng/ml
| Sample | Concentration 1 | Concentration 2 | Result |
| Morphine | 300 | 3000 | Negative |
| Head-shaking pill | 500 | 50000 | Negative |
| Amphetamine | 1000 | 10000 | Negative |
| Meth | 1000 | 10000 | Negative |
| Cocaine | 300 | 3000 | Negative |
| Barbital | 300 | 3000 | Negative |
| Benzodiazepine | 300 | 3000 | Negative |
| Tricyclics | 1000 | 10000 | Negative |
| Ketamine | 1000 | 10000 | Negative |
As can be seen here, this kit and other common drugs are as no cross reactions such as morphine, amphetamine, meth, methamphetamines, and specificity is good.
Repeatability and the stability experiment of embodiment 5 Sauteralgyl gold-immunochromatographyreagent reagent for assay boxes
One, kit batch in and batch between repeated experiment
1. experimental technique:
Prepared by the method that adopts embodiment 1 same batch is detected respectively with the Sauteralgyl gold-immunochromatographyreagent reagent for assay box of different batches the standard items that Sauteralgyl concentration is 450ng/ml, 900ng/ml, 1800ng/ml, and each concentration repeats 3 times, observes the repeatability of kit.
2. experimental result: empirical tests, kit batch in and batch between repeatability be 100%, false positive rate and false negative rate are 0.
Two, the stability experiment of kit
1. experiment purpose:
The sealing of Sauteralgyl gold-immunochromatographyreagent reagent for assay box is preserved, and deposit under room temperature (25 ℃ of left and right), observe the stability of kit.
1. experimental technique:
The kit that is stored in room temperature takes out weekly 4 boxes, detects respectively the standard items that Sauteralgyl concentration is 450ng/ml, 900ng/ml, 1800ng/ml;
2. experimental result:
Empirical tests, paper box at room temperature can be preserved 18 months, and within the preservable time limit, kit all can reach the detection sensitivity of 900ng/ml.
Contrast experiment 1
One, the preparation of kit:
1. immune colloid gold is made of paper standby: get pH and be the immune colloid gold of 8.5 0.1M Tris damping fluid dilution embodiment 1 preparation to solution O D
540value is 1.5, adaptive immune colloidal gold solution, and the all-glass paper that adopts this immune colloid gold solution direct spraying to soak without pretreatment fluid, sprays 20ml immune colloid gold solution on every 261mm * 220mm all-glass paper, dry, makes the immune colloid gold scraps of paper.
2. immune nitrocellulose filter, the thieving paper of the immune colloid gold scraps of paper of filter sample paper, this experiment preparation, embodiment 1 preparation are sticked on offset plate successively, it is the reagent strip of 4mm that cutting becomes width.
3. detection reagent strip is packed into and in plastic casing, obtain detection kit.
Two, kit sensitivity detects:
(1) get respectively the detection kit that embodiment 1 method 1 and this experiment make, kit is placed on level table.
(2) configuration Sauteralgyl concentration is that the Quality Control reference material of 800ng/ml, 900ng/ml, 1000ng/ml, 1100ng/ml, 1200ng/ml is as sample.With application of sample suction pipe, draw sample, then drip 3 (about 120ul) samples in the well of kit.Each concentration is established 3 repetitions, and a different sample of every detection is used different suction pipes.
(3) observations: 3-8 minute sentence read result after dripping sample.
Three. testing result:
The sensitivity experiment result of detection kit prepared by table 3 distinct methods
As shown in Table 3, adopt the immune colloid gold solution of same amount, the detection sensitivity of the kit of the embodiment of the present invention 1 preparation is 900ng/ml, and kit sensitivity prepared by this contrast test is 1000ng/ml left and right, lower than the kit of embodiment 1 preparation.
Claims (7)
1. the preparation method of a Sauteralgyl gold-immunochromatographyreagent reagent for assay box, described Sauteralgyl gold-immunochromatographyreagent reagent for assay box, comprise reagent strip, reagent strip starts to be followed successively by filter sample paper from application of sample end, the immune colloid gold scraps of paper, immunity nitrocellulose filter and thieving paper, wherein, the described immune colloid gold scraps of paper are the glass fibre pad that is coated with the anti-Sauteralgyl monoclonal antibody of colloid gold label, immunity nitrocellulose filter is provided with detection line and nature controlling line, on detection line, specking has Sauteralgyl-carrier complexes, on nature controlling line, specking has sheep anti-mouse igg, described preparation method comprises the following steps:
1) with two step reduction method, prepare the collaurum that mean grain size is 39-42nm:
A) reduce for the first time chlorauric acid solution:
The HAuCl that preparation concentration is 0.0004-0.0005mol/L
4aqueous solution, 20-30 minute is boiled in heating, adds while stirring afterwards trisodium citrate aqueous solution, HAuCl
4with the mol ratio of trisodium citrate be 1:(8-9), after sonic oscillation 2-3 minute, be cooled to room temperature, make the collaurum original solution of 14～16nm particle diameter;
B) reduce for the second time chlorauric acid solution:
The collaurum original solution that the first step is made is positioned over equilibrium temperature in 4.0-4.5 ℃ of constant temperature ice bath, presses subsequently collaurum original solution and HAuCl
4aqueous solution volume ratio 1:(1.9-2.0) add the 0.003-0.004mol/L HAuCl of 4.0-4.5 ℃
4aqueous solution, immediately drips the ascorbic acid of 4.0-4.5 ℃ and the mixed aqueous solution of PVP, the HAuCl that the ascorbic acid adding and this step add while stirring
4mol ratio be (25-26): 1, the HAuCl that the PVP adding and this step add
4weight ratio be 1:(2.0-2.1), in reaction, keep temperature constant, be stirred to and react completely, solution is transparent claret, makes the colloidal gold solution of 39-42nm particle diameter;
In two step reduction method, configure various aqueous solution and all adopt distilled water;
2) the anti-Sauteralgyl monoclonal antibody of colloid gold label adaptive immune collaurum employing step 1) making;
3) adopt metal spraying damping fluid dilution step 2) immune colloid gold adaptive immune colloidal gold solution, with the pretreated glass fibre pad of immune colloid gold solution spraying, make the immune colloid gold scraps of paper;
4) specking Sauteralgyl-carrier complexes and sheep anti-mouse igg respectively on the detection line of nitrocellulose filter and nature controlling line, make immune nitrocellulose filter;
5) will filter sample paper, step 3) the immune colloid gold scraps of paper, the step 4 prepared) immune nitrocellulose filter, the thieving paper prepared sticks on offset plate successively, and cutting makes detection reagent strip; Finally pack detection reagent strip into plastic casing and make detection kit.
2. the preparation method of Sauteralgyl gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, is characterized in that, step a) in, the concentration that adds trisodium citrate aqueous solution is 0.01-0.02mol/L, the frequency of ultrasonic concussion is 20-30KHZ.
3. the preparation method of Sauteralgyl gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterized in that step b) in, described ascorbic acid mixes in distilled water aqueous solution with PVP's, the concentration of ascorbic acid is 0.017-0.019mol/L, and the concentration of PVP is 0.137-0.139g/L.
4. the preparation method of Sauteralgyl gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, is characterized in that step 3) preparation of the immune colloid gold scraps of paper comprises the following steps:
I. use metal spraying damping fluid dilution step 2) immune colloid gold, obtain OD
540value is 1.2～2.0 immune colloid gold solution;
II. with pretreatment fluid, soak all-glass paper, after being dried, then use immune colloid gold solution spraying all-glass paper, dry, make the immune colloid gold scraps of paper.
5. the preparation method of Sauteralgyl gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterized in that, described in step I, the component of metal spraying damping fluid is: 8～12v%1.0M Tris liquid, 0.2～0.4w/v% PEG 20000,0.15～0.25w/v% bovine serum albumin(BSA), 0.1～0.3w/v% skim milk, 0.25～0.35w/v% casein, with 0.02～0.08w/v% sodium azide, with salt acid for adjusting pH to 8.5 ± 0.05, surplus is water.
6. the preparation method of Sauteralgyl gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, is characterized in that, the component of pretreatment fluid comprises described in Step II: 0.5～0.8v%Tween-20, and 12～18w/v% sucrose, solvent is water.
7. a Sauteralgyl gold-immunochromatographyreagent reagent for assay box, by the preparation method described in the arbitrary claim of claim 1-6, prepared, described kit comprises reagent strip, reagent strip starts to be followed successively by filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter and thieving paper from application of sample end, wherein, the described immune colloid gold scraps of paper are the glass fibre pad that is coated with the anti-Sauteralgyl monoclonal antibody of colloid gold label, immunity nitrocellulose filter is provided with detection line and nature controlling line, on detection line, specking has Sauteralgyl-carrier complexes, and on nature controlling line, specking has sheep anti-mouse igg.
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