CN102539764A - Pethidine hydrochloride detection kit and preparation method thereof - Google Patents
Pethidine hydrochloride detection kit and preparation method thereof Download PDFInfo
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- CN102539764A CN102539764A CN2010106066432A CN201010606643A CN102539764A CN 102539764 A CN102539764 A CN 102539764A CN 2010106066432 A CN2010106066432 A CN 2010106066432A CN 201010606643 A CN201010606643 A CN 201010606643A CN 102539764 A CN102539764 A CN 102539764A
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Abstract
The invention belongs to the technical field of determination of biological immunity methods, and particularly relates to a pethidine hydrochloride detection kit and a preparation method thereof. The kit comprises a sample pad (1), a colloidal gold pad (2), a nitrocellulose membrane (3), a sample sucking pad (4) and a PVC support plate (5). The colloidal gold pad is a colloidal gold-labeled meperidine hydrochloride monoclonal antibody polyester film, the nitrocellulose membrane is sequentially coated with meperidine hydrochloride-bovine serum albumin coupled antigen as a detection line (T line), and the goat anti-mouse IgG polyclonal antibody as a quality control line (C line). The method for preparing the meperidine hydrochloride detection kit by adopting the colloidal gold immunochromatography technology is simple, can be used for detecting the meperidine hydrochloride possibly existing in a sample, and has the characteristics of convenience in use, simplicity in operation, rapidness in reaction, economy, practicality and the like.
Description
Technical field
The present invention relates to the determination techniques field of biology immunization method, particularly relate to a kind of a kind of pethidine hydrochloride detection kit of utilizing colloid gold immune layer fabrication techniques and preparation method thereof.
Background technology
Sauteralgyl formal name used at school pethidine is called mark pyridine, demerol, demerol again, claims pethidine hydrochloride again.Its hydrochloride can be water-soluble for white, nothing are smelt, crystalloid powder, is made generally in the form of injection.As the anaesthetic of synthetic, Sauteralgyl is used in clinical at large, and it is similar with morphine with mechanism to the effect of human body, but analgesia, anesthetic action is less, only is equivalent to the 1/10-1/8 of morphine, keeps action time about 2-4 hour.Mainly act on central nervous system, cardiovascular, smooth muscle are also had certain influence.Toxic and side effect is also corresponding less, feel sick, symptoms such as vomiting, constipation are all slighter, to the inhibiting effect of respiratory system a little less than, problems such as expiratory dyspnea and excessive use generally can not appear.
But Sauteralgyl uses habituation continuously, uses 1-2 week just can produce drug dependence continuously.Research shows that this dependence is main with psychology, and physiology is auxilliary, but a little less than the dependence of both than morphine.The withrawal symptom that occurs during drug withdrawal mainly contains One's spirits are drooping depressed, general malaise, the runny nose of shedding tears, vomiting, diarrhoea, insomnia, and severe patient also can produce collapse.In case after the drug withdrawal then can produce the withdrawal syndrome behind the morphine abstinence syndrome.The patient of Sauteralgyl habituation is addicted in part because treat some disease gradually, belongs to the prescription medicine habituation, does not belong to drug abuse.Sauteralgyl abuse will habituation, becomes drugs, serious harm health and life security.On November 28th, 1987, State Council issues " administration of narcotic drug way ", Sauteralgyl is listed in wherein carried out strict control.
The detection method of relevant pethidine hydrochloride has a lot; For example: high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), high performance thin layer chromatography methods such as (HPTLC); Though the sensitivity that these methods detect is higher, testing result is accurate, and existence needs expensive instrument and equipment; Requirement to test material is high, needs restrictions such as purification processes.Therefore the detection method of research with advantage such as quick, portable has realistic meaning.The present invention has introduced a kind of detection kit with colloidal gold immunity chromatography fast detecting pethidine hydrochloride and preparation method thereof; This method has simply, fast; Need not any instrument and equipment, characteristics such as economical and practical are applicable to fast qualitative detects in the sample whether contain pethidine hydrochloride.
The colloidal gold immunochromatographimethod technology is a novel vitro diagnostic techniques that on monoclonal antibody technique, immunochromatography technique and collaurum developing technology basis, has grown up since 20th century the nineties.Can prepare colloid gold test paper based on the colloidal gold immunochromatographimethod technology; The principle of colloid gold test paper is to use colloidal gold-labeled method; With the collaurum is tracer, utilizes the antigen and antibody specific association reaction, utilize specific chromogenic reaction on test paper colour developing and judge the result of detection; Have simple to operate, quick, highly sensitive, the high specificity of detection, need not complicated characteristics such as instrument.
Summary of the invention
The object of the present invention is to provide pethidine hydrochloride kit of a kind of simple, quick, highly sensitive, high specificity and preparation method thereof, be used for detecting sample to be checked and whether contain pethidine hydrochloride.
A kind of detection kit that is used to detect pethidine hydrochloride comprises sample pad, collaurum pad, nitrocellulose filter, suction appearance pad and PVC back up pad, and tight adhesion has sample pad, collaurum pad, nitrocellulose filter and suction appearance pad successively on the PVC back up pad.Said sample pad is a spun glass; The pethidine hydrochloride monoclonal antibody polyester film that said collaurum pad is a colloid gold label; Be coated with detection line (T line) and nature controlling line (C line) on the said nitrocellulose filter successively, wherein encapsulated pethidine hydrochloride-bovine serum albumin coupled antigen on the detection line (T line), encapsulated the sheep anti-mouse igg polyclonal antibody on the nature controlling line (C line); Said suction appearance pad is thieving paper.
The present invention adopts nano-colloid technology for gold and antigen and antibody specific reaction; The principle of using immunity competition inhibitory reaction is prepared from; The pethidine hydrochloride monoclonal antibody of the pethidine hydrochloride-bovine serum albumin coupled antigen competition association colloid gold mark that encapsulates through detection line (T line) on the pethidine hydrochloride that contains in the sample to be checked and the nitrocellulose filter judges whether contain pethidine hydrochloride in the sample to be checked through the colour developing of T line.
The invention provides a kind of preparation method of pethidine hydrochloride detection kit, may further comprise the steps:
(1) preparation pethidine hydrochloride coupled antigen
With pethidine hydrochloride and bovine serum albumin coupling, synthetic hydrochloric acid pethidine-bovine serum albumin coupled antigen is as the pethidine hydrochloride coupled antigen.
(2) preparation pethidine hydrochloride monoclonal antibody
Adopting pethidine hydrochloride-bovine serum albumin coupled antigen is the immunogen immune BALB/C mice, through hybridoma technology, obtains secreting the hybridoma cell strain of anti-pethidine hydrochloride monoclonal antibody; Prepare antibody in a large number to induce the ascites method in the body, use Protein G post to carry out purifying, obtain the pethidine hydrochloride monoclonal antibody.
(3) preparation collaurum
Get 0.01% aqueous solution of chloraurate, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of processing like this is 20-40nm.
(4) preparation collaurum pad
Get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-8.5, and room temperature was placed 30 minutes; In above-mentioned solution, add the pethidine hydrochloride monoclonal antibody, the concentration that makes the pethidine hydrochloride monoclonal antibody is 10-50 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; It is 10-50 μ l/ml that bovine serum albumin (BSA) solution of adding 10% makes its concentration, mixes, and room temperature was placed 30 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition is with the collaurum redissolution liquid dissolving of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition obtains pethidine hydrochloride monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of the initial collaurum volume of 30%-100%; Pethidine hydrochloride monoclonal antibody-colloid gold label thing is pressed 1mL shop 40-70cm
2The ratio of polyester film evenly is layered on the polyester film, puts drying room again, and 38 ℃ of temperature, humidity was processed the collaurum pad less than under 30% the condition dry 24 ± 2 hours.
Above-mentioned collaurum redissolution liquid is the Tris that contains 0.01-0.1%, the PBS of the 0.02M pH7.0-8.5 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
(4) encapsulate pethidine hydrochloride-bovine serum albumin coupled antigen, sheep anti-mouse igg polyclonal antibody
Set and draw film appearance coating parameters 1 μ L/cm; Use respectively microsyringe get concentration as pethidine hydrochloride-bovine serum albumin coupled antigen of 0.5-4.0mg/ml, get concentration and be 0.5-5.0mg/ml sheep anti-mouse igg polyclonal antibody, receive A, the B pipe joint of drawing the film appearance in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film appearance, open and draw the film appearance, on nitrocellulose filter, apply pethidine hydrochloride-bovine serum albumin coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line).Preserve line back in the baking oven of 38 ℃ of temperature dry 24 ± 2 hours, subsequent use.
(5) processing of sample pad
Spun glass is soaked 20-40min in the PBS of 0.01M pH 7.0-8.0, wherein contain 0.5-1.5%BSA in the PBS, 0.5-1.0%Tweeen-20,38 ℃ of oven dry in drying baker are preserved, and are subsequent use
(6) assembling kit
On the PVC back up pad, adhere to sample pad, collaurum pad, nitrocellulose filter and suction appearance pad in order successively, obtain the said test strips that is used to detect pethidine hydrochloride, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is a spun glass, inhales the appearance pad and is thieving paper.
The detection method of kit according to the invention is: with the test sample balance to room temperature; Take out the pethidine hydrochloride pick-up unit, horizontal positioned; In sample pad, add 2-3 and drip sample, observe and write down the colour developing situation of C, T line in the time of 10-15 minute, judge testing result.
Kit of the present invention adopts colloidal gold immunochromatographimethod technical measurement pethidine hydrochloride, during detection, sample is added on the sample pad on the kit, can observe directly immunoreactive result, accomplishes sample detection.The present invention can be used for detecting the pethidine hydrochloride that possibly exist in the sample, have easy to use, simple to operate, be swift in response, characteristics such as economical and practical.
Description of drawings
Fig. 1 pethidine hydrochloride detection kit structural representation;
The reference numeral explanation:
1: sample pad;
2: collaurum pad (polyester film of colloid gold label pethidine hydrochloride monoclonal antibody)
3: nitrocellulose filter (T: the detection line that has encapsulated pethidine hydrochloride-bovine serum albumin coupled antigen; C: the nature controlling line that has encapsulated the sheep anti-mouse igg polyclonal antibody);
4: inhale the appearance pad;
The 5:PVC back up pad;
The testing result synoptic diagram of Fig. 2 kit of the present invention.
Be followed successively by line positive test symbol of C line from left to right; T, two line negative result of C; Invalid.
Embodiment:
Embodiment 1: the preparation of pethidine hydrochloride detection kit
1. prepare the pethidine hydrochloride coupled antigen
With pethidine hydrochloride and bovine serum albumin coupling, synthetic hydrochloric acid pethidine-bovine serum albumin coupled antigen is as the pethidine hydrochloride coupled antigen.
2. prepare the pethidine hydrochloride monoclonal antibody
Adopting pethidine hydrochloride-bovine serum albumin coupled antigen is the immunogen immune BALB/C mice, through hybridoma technology, obtains secreting the hybridoma cell strain of anti-pethidine hydrochloride monoclonal antibody; Prepare antibody in a large number to induce the ascites method in the body, use Protein G post to carry out purifying, obtain the pethidine hydrochloride monoclonal antibody.
3. preparation collaurum
Get 0.01% aqueous solution of chloraurate 100ml, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of processing like this is 20-40nm.
4. prepare the collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, adds 0.15ml 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 30 minutes; In above-mentioned solution, adding 0.033ml concentration is the pethidine hydrochloride monoclonal antibody of 3.8mg/ml, and after mixing, room temperature was placed 30 minutes; Add bovine serum albumin (BSA) solution of 0.2ml 10%, mix, room temperature was placed 30 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition is with the collaurum redissolution liquid dissolving of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition obtains pethidine hydrochloride monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of 50% initial collaurum volume; Pethidine hydrochloride monoclonal antibody-colloid gold label thing is pressed 1mL shop 56cm
2The ratio of polyester film evenly is layered on the polyester film, puts drying room again, and 38 ℃ of temperature, humidity was processed the collaurum pad less than under 30% the condition dry 24 hours.
Above-mentioned collaurum redissolution liquid is to contain 0.01% Tris, the PBS of the 0.02M pH 8.0 of 2.0% sucrose, 0.5% BSA.
4. encapsulate pethidine hydrochloride-bovine serum albumin coupled antigen, sheep anti-mouse igg polyclonal antibody
Set to draw film appearance coating parameters 1 a μ L/cm, use respectively microsyringe get concentration as pethidine hydrochloride-bovine serum albumin coupled antigen of 1.2mg/ml, get concentration and be 1.0mg/ml sheep anti-mouse igg polyclonal antibody, receive A, the B pipe joint of drawing the film appearance in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film appearance, open and draw the film appearance, on nitrocellulose filter, apply pethidine hydrochloride-bovine serum albumin coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line).Preserve line back in the baking oven of 38 ℃ of temperature dry 24 ± 2 hours, subsequent use.
5. the processing of sample pad
Spun glass is soaked 30min in the PBS treating fluid of 50ml 0.01M pH 8.0, wherein contain 1.0%BSA in the PBS, 0.5%Tweeen-2,38 ℃ of oven dry in drying baker are preserved, and are subsequent use
6. assembling kit
On the PVC back up pad, adhere to sample pad, collaurum pad, nitrocellulose filter and suction appearance pad in order successively, obtain the said test strips that is used to detect pethidine hydrochloride, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is a spun glass, inhales the appearance pad and is thieving paper.
Embodiment 2: the detection of pethidine hydrochloride detection kit
1. detection method:
Take out the pethidine hydrochloride detection kit, horizontal positioned; On sample pad, splash into 3 samples, observe and write down the colour developing situation of C, T line after 10 minutes, judge testing result.
2. the result judges
Positive: the T line does not develop the color, and only C line colour developing is judged to be positive findings;
Negative: T line, C line all develop the color, and are judged to be negative findings;
Invalid: the C line does not develop the color, and the rotten damage of maloperation or kit is described.
During test sample, sample fills up an end chromatography because of capillarity to inhaling appearance.If contain pethidine hydrochloride in the sample; They will and detection line (T line) on limited antibody combining site on the pethidine hydrochloride monoclonal antibody of the pethidine hydrochloride-bovine serum albumin coupled antigen competition association colloid gold mark that encapsulates; When the pethidine hydrochloride in the sample reaches finite concentration; Also saturated fully with the pethidine hydrochloride monoclonal antibody generation immune response of colloid gold label; This moment, colloidal gold composite did not have the pethidine hydrochloride-bovine serum albumin coupled antigen combination that encapsulates on vacant site and the detection line, and this moment, the T line did not develop the color this positive result.If not hydrochloric pethidine in the sample; Mark the colloid gold particle of pethidine hydrochloride monoclonal antibody will be to the T line position in company with the sample chromatography; Immune association reaction takes place with the pethidine hydrochloride that encapsulates on the T line-bovine serum albumin coupled antigen; Colloid gold particle is piled up at the T line position and is made the T line demonstrate a macroscopic red stripes, this negative result.No matter whether contain pethidine hydrochloride in the sample; The colloid gold label thing all can combine with the sheep anti-mouse igg polyclonal antibody on being coated on nature controlling line (C line) and develop the color; C line colour developing is to judge whether enough samples are arranged, and whether the chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
Embodiment 3: the applicating evaluating of pethidine hydrochloride detection kit
1. limit of identification
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration be 0,50,100, the pethidine hydrochloride standard items of 150ng/mL; Each concentration is carried out 10 parallel testings respectively; Observe testing result in the time of 10 minutes; The testing result of the pethidine hydrochloride standard items of 0ng/mL, 50ng/mL is that T line, C line all develop the color negative result; The testing result of the pethidine hydrochloride standard items of 100ng/mL, 150ng/mL is that the T line does not develop the color, the colour developing of C line, is judged to be positive test symbol; The limit of identification of final definite pethidine hydrochloride detection kit is 100ng/ml.
2. negative with reference to the article coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the morphine standard solution of 100ug/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T line, C line all present red stripes, and the result is negative.
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the ***e standard solution of 100ug/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T line, C line all present red stripes, and the result is negative.
3. positive with reference to the article coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is the pethidine hydrochloride standard items of 100ng/mL, 200ng/mL, 300ng/mL; Each concentration is carried out 10 parallel testings respectively; Observe testing result in the time of 10 minutes, the T line does not develop the color, the colour developing of C line, and the result is positive.
4. repeated
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is the pethidine hydrochloride standard items of 100ng/mL, carries out 10 parallel testings, observes testing result in the time of 10 minutes; The result is the T line and does not develop the color; The colour developing of C line, and colour developing degree homogeneous, positive result.
5. stable
Place after 20 days for 37 ℃, limit of identification, negative reference article coincidence rate, positive reference article coincidence rate, repeated each item index all meet above requirement, and this kit has good stable property.
Claims (6)
1. pethidine hydrochloride detection kit and preparation method thereof is characterized in that being made up of sample pad, collaurum pad, nitrocellulose filter, suction appearance pad and PVC back up pad; Said sample pad is a spun glass; The pethidine hydrochloride monoclonal antibody polyester film that said collaurum pad is a colloid gold label; Encapsulated pethidine hydrochloride-bovine serum albumin(BSA) coupled antigen on the said nitrocellulose filter successively as detection line (T line), the sheep anti-mouse igg polyclonal antibody is as nature controlling line (C line); Said suction appearance pad is thieving paper.
2. described pethidine hydrochloride detection kit of claim 1 and preparation method thereof is characterized in that described pethidine hydrochloride monoclonal antibody is obtained as the immunogen immune BALB/C mice by pethidine hydrochloride-bovine serum albumin coupled antigen.
3. described pethidine hydrochloride detection kit of claim 1 and preparation method thereof is characterized in that described collaurum is by gold chloride (HAuCl
4) under the effect of reductive agent citric acid trisodium, process size and be the colloid gold particle of 20-40nm.
4. described pethidine hydrochloride detection kit of claim 1 and preparation method thereof is characterized in that the preparation method of described collaurum pad is: get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-8.5, and room temperature was placed 30 minutes; In above-mentioned solution, add the pethidine hydrochloride monoclonal antibody, the concentration that makes the pethidine hydrochloride monoclonal antibody is 10-50 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; It is 10-50 μ l/ml that bovine serum albumin (BSA) solution of adding 10% makes its concentration, mixes, and room temperature was placed 30 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition is with the collaurum redissolution liquid dissolving of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining deposition obtains pethidine hydrochloride monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of the initial collaurum volume of 30%-100%; Pethidine hydrochloride monoclonal antibody-colloid gold label thing is pressed 1mL shop 40-70cm
2The ratio of polyester film evenly is layered on the polyester film, puts drying room again, and 38 ℃ of temperature, humidity was processed the collaurum pad less than under 30% the condition dry 24 ± 2 hours.
5. a claim 1 and the described pethidine hydrochloride detection kit of claim 5 and preparation method thereof; It is characterized in that described collaurum redissolution liquid is the Tris that contains 0.01-0.1%, the PBS of the 0.02M pH7.0-8.5 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
6. described pethidine hydrochloride detection kit of claim 1 and preparation method thereof; The method for coating that it is characterized in that two lines on the described nitrocellulose filter is: set and draw film appearance coating parameters 1 μ L/cm; Use respectively microsyringe get concentration as pethidine hydrochloride-bovine serum albumin coupled antigen of 0.5-4.0mg/ml, get concentration and be 0.5-5.0mg/ml sheep anti-mouse igg polyclonal antibody, receive A, the B pipe joint of drawing the film appearance in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film appearance, open and draw the film appearance, on nitrocellulose filter, apply pethidine hydrochloride-bovine serum albumin coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line).Preserve line back in the baking oven of 38 ℃ of temperature dry 24 ± 2 hours, subsequent use.
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Cited By (1)
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| CN103159669A (en) * | 2011-12-15 | 2013-06-19 | 曾立波 | Meperidine monoclonal antibodies, immunoassay plates and kits |
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Application publication date: 20120704 |