CN102565387A - Kit used for on-site rapid immunodetection and detection method thereof - Google Patents
Kit used for on-site rapid immunodetection and detection method thereof Download PDFInfo
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- CN102565387A CN102565387A CN2012100068471A CN201210006847A CN102565387A CN 102565387 A CN102565387 A CN 102565387A CN 2012100068471 A CN2012100068471 A CN 2012100068471A CN 201210006847 A CN201210006847 A CN 201210006847A CN 102565387 A CN102565387 A CN 102565387A
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Abstract
The invention relates to a kit used for on-site rapid immunodetection and a detection method thereof, which belongs to the technical field of immunodetection. The kit used for on-site rapid immunodetection comprises a test board, a reaction solution, a fluid drop bottle, positive control, negative control and a test strip. The detection method of the kit used for on-site rapid immunodetection comprises the steps of adding the reaction solution to different reaction grooves of the test board; respectively adding the positive control, the negative control and a sample to be tested to the different reaction grooves; adding the test strip for incubation and development or transferring the test strip to a development groove for development after the incubated test strip is rinsed; and comparing depths of colors of the positive control, the negative control and the sample to be tested on the test strip to determine whether the tested sample is positive or negative. The kit used for on-site rapid immunodetection and the detection method thereof have the advantages that the detection is high in efficiency, convenient and rapid, whether a detection result is valid can be determined correctly through one experience, concentration of target molecules in the tested sample can be detected semi-quantitatively and the like.
Description
Technical field
The invention belongs to technical field of immunoassay, particularly relate to a kind of on-the-spot with rapid immune detecting kit and detection method thereof.
Background technology
At present, when existing competition law immunochromatographydetecting detecting test strip is made, on support plate (like the PVC plate), be pasted with sample pad, gold mark pad, nitrocellulose filter, adsorptive pads etc. successively.Line encapsulates 1 antigen line as detection line (T line) on nitrocellulose filter, encapsulates 1 two anti-line in its downstream with giving capture antibody line line as a reference.Paste sample pad, gold mark pad, adsorptive pads then successively, promptly be combined as quick detection test paper.During detection, on sample pad, drip aqueous sample, observe color in 5～10min.If detection line presents red color, show that sample is negative; If detection line does not develop the color, show that sample is positive.No matter sample is the feminine gender or the positive, and reference line should develop the color, if reference line does not develop the color, shows that this test paper lost efficacy.
Existing competition law immunochromatographydetecting detecting test strip is that the nature controlling line (C line) on " single part " mensuration and the test strips is actually the existence that mensuration one resists, and resisting as for one has the inactivation of denying to survey to come out.If there be not (feminine gender) in object in the testing sample, and golden labeling antibody inactivation can not combine with the antigen of embedding on the T line but can be caught colour developing and draw positive findings by two on the C line is anti-in the same old way.And be actually the false positive that lost efficacy and to show because of test strips.On the contrary; If object exists in the testing sample; But capture antibody on the nature controlling line C line (two anti-) inactivation and golden labeling antibody is normal can not combine (this is normal positive sight) with the antigen of embedding on the T line but owing to can not develop the color and draw the conclusion of test strips inefficacy with the albumen/antibodies of catching of inactivation on the C line after so golden labeling antibody combines with object.And in fact should be positive.Therefore the result of the nature controlling line (C line) of existing competition law immunochromatographydetecting detecting test strip can not be used to estimate the quality of test strips quality fully.
Summary of the invention
The technical matters of the present invention for existing in the solution known technology, and provide a kind of on-the-spot with rapid immune detecting kit and detection method thereof.
The present invention discloses a kind of scene and uses the competition law immunity detection reagent.This kit comprises one three reactive tank test board or many reactive tanks test board, sample diluting liquid reactant liquor, drop bottle, one group of positive control and negative control, test strips and colour developing liquid.
Test board: the forming board that contains three or three above reactive tanks.Can be used for measuring simultaneously positive control, negative control and one or more testing sample.
But test board be have chemical property under solid shape carrying liquid, the normal temperature and pressure stable not with the forming board of sample to be measured and the material generation chemical reaction in the detection system.This test board can be disposable also can be reused after washing.The reactive tank of this test board can be horizontal arrangement, also can be vertical arrangement.
Test board is the plastic plate (such as polystyrene) or the papery plate that contain three or three above reactive tanks.
Reactant liquor: contain the good antibody that can discern testing molecule specifically of mark in advance.The mark of this specific antibody can be a collaurum, enzyme (like hydrogen peroxidase, alkaline phosphatase), and fluorescence molecule or other help to test directly or indirectly the molecule that is labeled antibody.
Test strips: contain good antigen molecule of embedding in advance or hapten conjugation molecule.This antigen or haptens are the molecules that can be discerned by the antibody that contains in the described reactant liquor.Also can be can with testing molecule competitively with said reactant liquor in the molecule that combines of antibody.
Positive control and negative control: the concentration of its contained target molecule to be measured of the positive control that has a concentration known at least in the kit just in time is the threshold concentration on clinical demand or product quality require.Such as: the milk that contains 1 mcg/ml melamine.Therefore, such positive control can be used for intuitively whether the comparative measurements sample exceeds standard.
Has a negative control that does not contain target molecule to be measured at least in the kit.Such as: the milk that does not contain melamine.Can calibrate the concentration range of the contained target molecule to be measured of true boundary of a piece of land location survey random sample article through the colour developing degree that compares negative control, positive control and location survey random sample article like this, and can be used for judging that detection system itself does not have not inefficacy.
It just in time is the threshold concentration on clinical demand or product quality to be measured require that the positive controls that an above variable concentrations can be arranged in the kit preferably wherein has a concentration that contrasts contained target molecule to be measured.Such as: the milk that contains 1 mcg/ml melamine.One group of positive controls like this can be used for the concentration of semiquantitative determination sample target molecule to be measured.
Colour developing liquid: be to come supporting according to the mark of the specific antibody in the dilution.
Drop bottle: the mouth of drop bottle is furnished with a cover lid inner cap and an enclosing cover.Inner cap is the lid that passage is arranged, be equipped with on the passage one consider film to prevent that bacterium from getting in the bottle, sediment is extruded and causes density unevenness in using bottle, preventing during liquid simultaneously bottle, also prevents liquid countercurrent, the aperture of said worry film is between 0.1-10u; Enclosing cover is can cover at the interior lid that covers in case liquid evaporation and change strength of fluid in the bottle.
Reactant liquor in the kit, positive control solution, negative control night, analyte sample fluid, dilution and colour developing liquid are to be contained in respectively in the drop bottle of same size, like this can be according to extruding dripping number and confirming its additions of liquid.The bore of said drop bottle is controlled at certain scope, is no more than+/-3 microlitres such as every 50 microlitre error.
One of the object of the invention provide a kind of have simple in structure; Easy to use; Detection efficiency is high; The positive control and the negative control that are implemented in once the sample of measuring unknown concentration in the experiment simultaneously and concentration known are so that correctly define testing result and validity, and rapid immune detecting kit is used at the convenient scene of defining the characteristics such as concentration of contained target molecule in the test sample with carrying out sxemiquantitative.
The on-the-spot technical scheme of using rapid immune detecting kit to be taked for solution prior art problem of the present invention is:
Rapid immune detecting kit is used at a kind of scene, is characterized in: kit comprises a test board, reactant liquor, drop bottle, one group of positive control and negative control, test strips.
The present invention is on-the-spot can also to adopt following technical measures with rapid immune detecting kit:
Rapid immune detecting kit is used at described scene, is characterized in: kit also comprises cleansing solution, colour developing liquid and plastic suction pipe.
Rapid immune detecting kit is used at described scene, is characterized in: test board is three reactive tank test boards or many reactive tanks test board; But test board has the solid shape carrying liquid, under the normal temperature and pressure chemical property stable not with the forming board of sample to be measured and the material generation chemical reaction in the detection system.
Rapid immune detecting kit is used at described scene, is characterized in: test board respond groove, colour developing groove and sink.
Rapid immune detecting kit is used at described scene, is characterized in: the arrangement mode of groove is horizontal arrangement or vertical arrangement on the test board; Groove on the test board is that fixed or removable is removed the type setting.
Rapid immune detecting kit is used at described scene, is characterized in: the filter membrane aperture of drop bottle is 0.1-10u.
Rapid immune detecting kit is used at described scene, is characterized in: the profile of the inner cap of drop bottle is cylindrical or taper, and the inboard shape of enclosing cover and the profile of inner cap are consistent.
Rapid immune detecting kit is used at described scene, is characterized in: the bore of drop bottle is consistent, every dropping liquid 40-60 microlitre, and error is less than 3 microlitres.
Two of the object of the invention provides a kind of simple operation; Detection efficiency is high; In once test with the competition law immune detection at the scene; Measure the sample of unknown concentration and the positive control and the negative control of concentration known (or a group) simultaneously, thereby can correctly define testing result and validity, or define the detection method of the scene of the characteristics such as concentration of contained target molecule in the test sample with carrying out sxemiquantitative with rapid immune detecting kit.
The technical scheme that the detection method of the on-the-spot use of the present invention rapid immune detecting kit is taked for solution prior art problem is:
On-the-spot detection method with rapid immune detecting kit, it is characterized in that: detection method comprises following process:
Add reactant liquor in each reactive tank on test board; Reactant liquor contains the good antibody that can discern testing molecule specifically of mark in advance;
In two reactive tanks, add positive control and negative control more respectively, add testing sample in other reactive tank, mixing;
Then, get test strips and mark, be respectively applied for the test positive control, negative control and testing sample place the reactive tank on the test board respectively;
At room temperature hatch after shaking mixing; Get cleansing solution or distilled water places sink, get colour developing liquid and place the colour developing groove;
When contained preliminary making antibody was golden labeling antibody in the reactant liquor, hatching had colour band appearance clearly on test strips; Take out test strips and transfer in the sink rinsing with color development stopping;
Or when contained preliminary making antibody in the reactant liquor is enzyme mark (like the hydrogen peroxide enzyme labeling) antibody, the test strips of hatching is transferred in the sink, transfer to the colour developing groove after the rinsing; Gently shake or leave standstill colour developing, on test strips, having clearly, colour band occurs; Take out test strips and transfer in the sink rinsing with color development stopping;
Relatively the colour band depth degree of positive control, negative control and testing sample is the positive or feminine gender with definite this test sample on the test strips.
The on-the-spot detection method with rapid immune detecting kit of the present invention can also adopt following technical measures:
Described on-the-spot detection method with rapid immune detecting kit, be characterized in: test strips contains good antigen molecule of embedding in advance or hapten conjugation molecule; Positive control is that the concentration of contained target molecule to be measured just in time is the threshold concentration on clinical demand or product quality require; Negative control is the negative control that does not contain target molecule to be measured.
Described on-the-spot detection method with rapid immune detecting kit, be characterized in: shaking the time of at room temperature hatching behind the mixing is 10-30 minute.
Advantage and good effect that the present invention has are:
On-the-spot with rapid immune detecting kit and detection method thereof owing to having adopted brand-new technology scheme of the present invention, compared with prior art, the present invention has simple in structure, and is easy to use, the high characteristics of detection efficiency.The present invention has realized in once test with the competition law immune detection at the scene; Measure the sample of unknown concentration and the positive control and the negative control of concentration known (or a group) simultaneously; The false positive that possibly hide when having avoided using existing competition law immuno-chromatographic test paper strip and the false result who lost efficacy (existing competition law immuno-chromatographic test paper strip is that " single part " is measured test strips so can't be realized measuring simultaneously one group of known positive and negative tester); Thereby can correctly define testing result and validity, or define the concentration of contained target molecule in the test sample with carrying out sxemiquantitative.
Description of drawings
Fig. 1 is the on-the-spot rapid immune detecting kit structural representation of using of the present invention;
Fig. 2 is the horizontal groove arrangement architecture of a three-flute test board synoptic diagram;
Fig. 3 is the left TV structure synoptic diagram of Fig. 2;
Fig. 4 is the vertical groove arrangement architecture of a multiple-grooved test board synoptic diagram;
Fig. 5 is the left TV structure synoptic diagram of Fig. 2;
Fig. 6 is a taper inner cap drop bottle structure synoptic diagram;
Fig. 7 is cylindrical inner cap drop bottle structure synoptic diagram.
Among the figure: the 1-test board; 1.1-reactive tank; 1.2-sink; 1.3-colour developing groove; The 2-reactant liquor; 3-drop bottle; 3.1-enclosing cover; 3.2-filter membrane; 3.3-inner cap; 3.4-bottleneck; 3.5-body; The 4-positive control; The 5-negative control; The 6-test strips; The 7-liquid that develops the color, the 8-plastic suction pipe.
Embodiment
For further understanding technology contents of the present invention, characteristics and effect, the following examples of giving an example now, and conjunction with figs. specifies as follows:
Rapid immune detecting kit is used at a kind of scene, comprises a test board 1, reactant liquor 2,3, one groups of positive controls of drop bottle 4 and negative control 5, test strips 6 and colour developing liquid 7; The drop bottleneck is provided with inner cap 3.1 and enclosing cover 3.2, and inner cap 3.1 has passage, and filter membrane 3.3 is housed on the passage, and the filter membrane aperture is 0.1-10u.Enclosing cover 3.2 covers on inner cap 3.1, and the profile of the inner cap of drop bottle is cylindrical or taper, and the inboard shape of enclosing cover and the profile of inner cap are consistent.The bore of drop bottle is consistent, every dropping liquid 40-60 microlitre, and error is less than 3 microlitres.
Test board is three reactive tank test boards or many reactive tanks test board; But test board has the solid shape carrying liquid, under the normal temperature and pressure chemical property stable not with the forming board of sample to be measured and the material generation chemical reaction in the detection system.Test board respond groove, colour developing groove and sink.The arrangement mode of groove is horizontal arrangement or vertical arrangement on the test board; Groove on the test board is that fixed or removable is removed the type setting.
On-the-spot detection method with rapid immune detecting kit comprises following process:
Add 0.5 milliliter of reactant liquor in each reactive tank on test board, reactant liquor contains the good antibody that can discern testing molecule specifically of mark in advance;
In reactive tank 1 and reactive tank 2, add 0.5 milliliter of positive control and negative control more respectively, add testing sample in other reactive tank, for use behind the mixing.
Then, appoint and get three (, then getting test strips number=2+ testing sample number) test strips (containing the good antigen of embedding in advance) and put on 1 with pencil if a plurality of testing samples are arranged; 2; 3 are respectively applied for the test positive control, and negative control and testing sample place 1 on the test board more respectively; In 2,3 three reactive tanks (sitting in the right seat).
At room temperature hatched behind the jog mixing 10-30 minute.In the meantime, get 10 milliliters of cleansing solutions (or distilled water) and place sink, 3 milliliters of colour developing liquid place the colour developing groove.
Then, take out and transfer in the sink with the test strips that pincet will be hatched, the paddling rinsing is transferred in the colour developing groove after 6 times (about 3 seconds) more back and forth.Gently shake or leave standstill colour developing, have on test strips clearly that colour band occurs, take out test strips and transfer in the sink rinsing with color development stopping.Compare test strips 1 (positive control), the colour band depth degree on 2 (negative controls) and 3 (testing samples) is to confirm that this test sample is the positive or feminine gender.
Testing result is analyzed:
Under the normal condition, positive control shows light colour band, and negative control shows dark colour band; If the colour band of this test sample is darker but more shallow than negative control than positive control, explain that this test sample is the weak positive but does not exceed standard; If the colour band of this test sample is same more shallow or lighter even do not have colour developing than positive control, explain that this test sample is positive to exceed standard.
On-the-spot with ammonia glycosides penicillin (ampicillin) test paper strip quick test kit.
1. starting material and reagent preparation:
Multiple-grooved test board: like Fig. 4 and shown in Figure 5.
Dilution: 1 gram BSA (bovine serum albumin is purchased the company in SIGMA) is dissolved in 100 milliliters of PBS damping fluids, just makes 1%BSA/PBS.It is subsequent use that aseptic filtration is placed on refrigerator.
Reactant liquor: with above-mentioned dilution hydrogen peroxidase-enzyme mark ammonia glycosides penicillin (Ampicillin-HRP purchases the company in RANDOX) is diluted by every milliliter 1 microgram, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Antibody-solutions: it is subsequent use with the PBS damping fluid goat-anti ammonia glycosides penicillin antibody (purchasing the company in RANDOX) to be placed on refrigerator by the dilution of every milliliter 10 microgram.
Test strips: nitrocellulose film (the NC film is purchased the company in FISHER) is cut into little of 0.3cmx2cm, prepares 10, and make a mark (as: Fu) to be decided to be positive upper end with mark's pen at each end of little.Then with above-mentioned antibody-solutions drop or setting-out in the middle of little NC film positive (about 0.5cm place below the numeral), every or every line 2 microlitres.After treating its air dry, soak 1 hour position with sealing NC film no antibody drop in surface or setting-out with 5 milliliters of above-mentioned dilutions.After 37 ℃ of dryings, with plastic film each test strips is wrapped respectively that to be placed on dry place subsequent use.
Positive control: with dilution or the same liquid (such as milk) of testing sample ammonia glycosides penicillin (ampicillin purchases the company in SIGMA) is made into 10ug/ml solution, prepares 5 milliliters, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Negative control: the dilution or the same liquid (such as milk) of testing sample that do not contain ammonia glycosides penicillin.Prepare 5 milliliters, be contained in after the aseptic filtration in the drop bottle of the present invention,, place refrigerator subsequent use.
Sample: simulate positive milk: 1 milliliter of fresh milk ammonification glycosides penicillin 20 microgram, join existing usefulness at present.
Cleansing solution: PBS damping fluid+0.1%Tween-20, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Colour developing liquid: DAB colour developing liquid (purchasing company) in SIGMA.Be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
(with above-mentioned reactant liquor, positive control solution, negative control night, dilution and colour developing liquid be contained in respectively in the drop bottle with same size of the present invention (such as: the bore of drop bottle is controlled at every 50 microlitre error and is no more than+/-2 microlitres).
Plastic suction pipe: 1 in suction pipe in 3.9 milliliters of plastics of 1-ml scale (purchasing company) in Lab Safety Supply.
2. detection method:
1) by following order; 6 (about 0.3 milliliter) negative controls of fine-still are transferred to the #1 reactive tank on the test board; The #2 reactive tank of 6 (about 0.3 milliliter) positive control solutions of fine-still to the test board, 6 (about 0.3 milliliter) testing samples of the fine-still #3 reactive tank to the test board; Then, the #1 of 6 (about 0.3 milliliter) reactant liquors of fine-still to the test board respectively, #2, #3 reactive tank; Light rolling mixing.
2) appoint and get three test strips, in the positive lower end of test strips with mark's pen # 1, #2, #3 on the mark respectively.Then it is immersed the #1 on the test board respectively, #2 is in the #3 reactive tank.After light rolling treated that the test strips completion is soaked, room temperature left standstill and rocks frequently 20 minutes.
3) in the meantime, get 10 milliliters of cleansing solutions (or distilled water) and place sink, 3 milliliters of colour developing liquid place the colour developing groove.After clock time arrived in 20 minutes, with pincet test strips is taken out and transferred in the sink, the paddling rinsing is transferred in the colour developing groove after 6 times (about 3 seconds) more back and forth, faces up.
4) draw 2 milliliters of colour developing liquid with suction pipe and be covered on the test strips, leave standstill colour developing, on test strips, having clearly, colour band occurs.Take out test strips and transfer in the sink rinsing with color development stopping.
5) compare test strips 1 (negative control), the colour band depth degree on 2 (positive controls) and 3 (samples) is to confirm that this test sample is the positive or feminine gender.
Testing result is following: positive control shows light colour band, and negative control shows dark colour band; The colour band of this test sample is slightly more shallow than positive control, explain that this test sample is positive and slightly exceed standard (＞10ug/ml).
On-the-spot with ammonia glycosides penicillin (ampicillin) colloidal gold test paper strip quick test kit.
1. starting material and reagent preparation:
Ammonia glycosides penicillin (ampicillin purchases the company in SIGMA)
Ammonia glycosides penicillin-BSA coupled antigen: be with haptens ammonia glycosides penicillin and carrier protein BSA coupling through the EDC mediation; Both EDC, ammonia glycosides penicillin, BSA were dissolved in the PBS damping fluid by molal quantity at 20: 20: 1; And, obtain after removing free EDC and ammonia glycosides penicillin through dialysis then room temperature reaction 2 hours.
Collaurum (purchasing company) in SPI Supplies.
Anti-ammonia glycosides penicillin antibody (purchasing company) in RANDOX
The anti-ammonia glycosides penicillin antibody of gold mark: to remove freshen, get aurosol 2ml with PBS dialysis antibody, use 0.1mol/L K
2CO
3Or 0.1mol/L HCl regulates aurosol to pH=9, adds the antibody-solutions of 2mg (about 0.5ml), stirs 2～3 minutes.Add 2ml 1%PEG20000 solution, centrifugal in 10000～100000g, the careful suction removed supernatant; Deposition is suspended in certain damping fluid, after the centrifugation, recovers with same damping fluid again, concentration is advisable about with A 1cm/540nm=1.5, puts 4 ℃ of preservations.Both made the anti-ammonia glycosides penicillin antibody of gold mark.
Multiple-grooved test board: like Fig. 4 and shown in Figure 5.
Dilution: 1 gram BSA (bovine serum albumin is purchased the company in SIGMA) is dissolved in 100 milliliters of PBS damping fluids, just makes 1%BSA/PBS.Be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Reactant liquor: with above-mentioned dilution gold is marked anti-ammonia glycosides penicillin antibody and dilute, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use by every milliliter 1 microgram.
The embedding antigenic solution: it is subsequent use with the PBS damping fluid ammonia glycosides penicillin-BSA coupled antigen to be placed on refrigerator by the dilution of every milliliter 10 microgram.
Test strips: nitrocellulose film (the NC film is purchased the company in FISHER) is definitely become little of 0.3cmx2cm, prepare 10, and make a mark (as: Fu) to be decided to be positive upper end with mark's pen at each end of little.Then with (about 0.5cm place below the numeral) in the middle of above-mentioned embedding antigenic solution drop or little NC film of setting-out what positive, every or every line 2 microlitres.After treating its air dry, soak 1 hour position with the no embedding antigen in sealing NC film surface with 5 milliliters of above-mentioned dilutions.After 37 ℃ of dryings, it is subsequent use with plastic film each test strips to be wrapped freeze-drying lyophilization place, back respectively.
Positive control: with dilution or the same liquid (such as milk) of testing sample ammonia glycosides penicillin (ampicillin purchases the company in SIGMA) is made into 10ug/ml solution, prepares 5 milliliters, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Negative control: the dilution or the same liquid (such as milk) of testing sample that do not contain ammonia glycosides penicillin.Prepare 5 milliliters, be contained in the drop bottle of the present invention, it is subsequent use that aseptic filtration is placed on refrigerator.
Sample: simulate positive milk: 1 milliliter of fresh milk ammonification glycosides penicillin 20 microgram, be contained in after the aseptic filtration in the drop bottle of the present invention, join existing usefulness at present.
(with above-mentioned reactant liquor, positive control solution, negative control night, analyte sample fluid be contained in respectively in the drop bottle with same size of the present invention (such as: the bore of drop bottle is controlled at every 50 microlitre error and is no more than+/-2 microlitres).
Plastic suction pipe: 1 in suction pipe in 3.9 milliliters of plastics of 1-ml scale (purchasing company) in Lab Safety Supply.
2. detection method:
1) 6 (about 0.3 milliliter) negative controls of fine-still are transferred to the #1 reactive tank on the test board, the #2 reactive tank of 6 (about 0.3 milliliter) positive control solutions of fine-still to the test board, 6 (about 0.3 milliliter) testing samples of the fine-still #3 reactive tank to the test board;
2) appoint and get three test strips, in the positive lower end of test strips with mark's pen # 1, #2, #3 on the mark respectively.Then it is immersed the #1 on the test board respectively, #2 is in the #3 reactive tank.Light rolling is treated that test strips is accomplished and is soaked.
3) #1 of 6 (about 0.3 milliliter) reactant liquors of fine-still to the test board respectively, #2, #3 reactive tank; Light rolling mixing, incubated at room was also rocked about 20 minutes frequently, and on test strips, having clearly, colour band occurs.
4) in the meantime, get 10 milliliters of cleansing solutions (or distilled water) and place sink, test strips is taken out and transferred in the sink rinsing with color development stopping with pincet.
5) compare test strips 1 (negative control), the colour band depth degree on 2 (positive controls) and 3 (samples) is to confirm that this test sample is the positive or feminine gender.
Testing result is following: positive control shows light colour band, and negative control shows dark colour band; The colour band of this test sample is slightly more shallow than positive control, explain that this test sample is positive and slightly exceed standard (＞10ug/ml).
Embodiment 5
On-the-spot with sxemiquantitative ammonia glycosides penicillin (ampicillin) test-strips quick testing reagent box.
1. starting material and reagent preparation:
Dilution: 1 gram BSA (bovine serum albumin is purchased the company in SIGMA) is dissolved in 100 milliliters of PBS damping fluids, just makes 1%BSA/PBS.Be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Reactant liquor: hydrogen peroxidase-enzyme is marked ammonia glycosides penicillin (Ampicillin-HRP with above-mentioned dilution; Purchase company in RANDOX) by being sub-packed in 2 milliliters every bottle in 3ml-drop bottle after the every milliliter 0.1 microgram dilution; Be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Antibody sandwich liquid: it is subsequent use with sodium carbonate/bicarbonate damping fluid (pH 9.2) goat-anti ammonia glycosides penicillin antibody (purchasing the company in RANDOX) to be placed on refrigerator by the dilution of every milliliter 1 microgram.
Multiple-grooved test board: two of the removable ELISA of the removing test-strips of 8 grooves (purchasing company) in Fisher Scientific.
Test-strips: prepare 12 the 8 removable ELISA of the removing test-strips of groove (purchasing company) in Fisher Scientific, an end of each bar do with mark's pen a mark (as: on) to be decided to be the upper end.In each groove, add the above-mentioned antibody sandwich liquid of 100 microlitres then; Spending the night encapsulates to place reefer (1-8 ℃); Remove antibody sandwich liquid also with cleansing solution washing trough 3 times; In each groove, add 200 microlitre dilutions then, place incubated at room to remove dilution after 2 hours and with cleansing solution washing trough 3 times, place 37-45 ℃ of dry back will pack well then with the plastics aluminum foil bag after the freeze-drying lyophilization place subsequent use.
Standard control liquid: with dilution pure ammonia glycosides penicillin (ampicillin) is made into 1ug/ml respectively, 0.1ug/ml, 0.01ug/ml and 0ug/ml solution; Be sub-packed in 3ml-drop bottle; 1 milliliter every bottle, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Sample: simulate positive milk: (sample a) and two groups of 10ug/ml (sample b) is joined existing usefulness at present to be made into 0.1ug/ml with fresh milk ammonification glycosides penicillin (ampicillin).
Cleansing solution: PBS damping fluid+0.1%Tween-20, be contained in after the aseptic filtration in the drop bottle of the present invention, place refrigerator subsequent use.
Colour developing liquid: TMB colour developing liquid (purchasing the company in SIGMA) is contained in after the aseptic filtration in the drop bottle of the present invention, places refrigerator subsequent use.
Stop buffer: 1N HCl solution (purchasing the company in SIGMA) is contained in the drop bottle of the present invention.
2. detection method:
1) gets (the seeing above-mentioned) test-strips that encapsulates in advance; Drip 2 (about 0.1 milliliter) ammonia glycosides penicillin standard control liquid respectively and (contain ammonia glycosides penicillin 1ug/ml respectively; 0.1ug/ml; 0.01ug/ml with 0ug/ml solution) A, B, C, D groove on the test-strips, drips E and F groove that 2 (about 0.1 milliliter) testing sample a and b arrive test-strips; (can be as required, each standard control liquid and sample repeat the 1-2 group).
2) drip 2 (about 0.1 milliliter) reactant liquors respectively to each reactive tank; Light rolling mixing, incubated at room was also rocked about 30 minutes frequently.
3) wash reactive tank 3 times with cleansing solution, each 0.3 milliliter/groove tips upside down on thieving paper to remove residual moisture with test-strips.
4) drip 4 (about 0.2 milliliter) colour developing liquid respectively to each reactive tank, incubated at room to blue-green manifests.
5) drip 4 (about 0.2 milliliter) stop buffers respectively to each reactive tank, show to stop look.
6) result judges: relatively the shade degree of test sample groove and each standard liquid bath contains the scope of ammonia glycosides penicillin concn to judge this test sample.Because be competitive reaction, color explains that more deeply to contain ammonia glycosides penicillin concn low more.Test sample E groove is close with the B groove, explains that this test sample a contains ammonia glycosides penicillin about 0.1.Test sample F groove is also more shallow than B groove, explains that this test sample b contains ammonia glycosides penicillin on 1ug/ml.
Determination methods as a result: if test sample color and negative control (the D groove contains ammonia glycosides penicillin 0ug/ml) are identical or slightly dark this test sample of explanation is negative.If going back bright this test sample of elementary introduction than A groove (containing ammonia glycosides penicillin 1ug/ml), the test sample color contains ammonia glycosides penicillin above 1ug/ml; If the test sample color is than A groove depth but contain ammonia glycosides penicillin between 0.1-1ug/ml than bright this test sample of B groove elementary introduction; If the test sample color is than B groove depth but contain ammonia glycosides penicillin between 0.01-0.1ug/ml than bright this test sample of C groove elementary introduction.
Claims (10)
1. on-the-spot use rapid immune detecting kit for one kind, it is characterized in that: kit comprises a test board, reactant liquor, drop bottle, one group of positive control and negative control, test strips; Described reactant liquor, positive control and negative control are to be contained in the drop bottle; Described drop bottle bottleneck is equipped with filter membrane.
2. use rapid immune detecting kit according to the described scene of claim 1, it is characterized in that: kit comprises cleansing solution, colour developing liquid and plastic suction pipe.
3. use rapid immune detecting kit according to the described scene of claim 1, it is characterized in that: the drop bottleneck is provided with inner cap and enclosing cover, in be stamped passage, filter membrane is housed on the passage, enclosing cover covers interior and covers.
4. use rapid immune detecting kit according to the described scene of claim 1, it is characterized in that: test board is three reactive tank test boards or many reactive tanks test board; But test board has the solid shape carrying liquid, under the normal temperature and pressure chemical property stable not with the forming board of sample to be measured and the material generation chemical reaction in the detection system.
5. use rapid immune detecting kit according to claim 1 or 4 described scenes, it is characterized in that: test board respond groove, colour developing groove and sink; The arrangement mode of groove is horizontal arrangement or vertical arrangement on the test board; Groove on the test board is that fixed or removable is removed the type setting.
6. use rapid immune detecting kit according to the described scene of claim 3, it is characterized in that: the filter membrane aperture of drop bottle is 0.1-10u.
7. use rapid immune detecting kit according to claim 3 or 6 described scenes, it is characterized in that: the profile of the inner cap of drop bottle is cylindrical or taper, and the inboard shape of enclosing cover and the profile of inner cap are consistent.
8. use rapid immune detecting kit according to claim 1,3 or 6 described scenes, it is characterized in that: the bore of drop bottle is consistent, every dropping liquid 40-60 microlitre, and error is less than 3 microlitres.
9. according to the described on-the-spot detection method with rapid immune detecting kit of claim 1, it is characterized in that: detection comprises following process,
Add reactant liquor in each reactive tank on test board; Reactant liquor contains the good antibody that can discern testing molecule specifically of mark in advance;
In two reactive tanks, add positive control and negative control more respectively, add testing sample in other reactive tank, mixing;
Then, get test strips and mark, be respectively applied for the test positive control, negative control and testing sample place the reactive tank on the test board respectively; At room temperature hatch after shaking mixing;
Get cleansing solution or distilled water places sink, get colour developing liquid and place the colour developing groove;
When contained preliminary making antibody was golden labeling antibody in the reactant liquor, hatching had colour band appearance clearly on test strips; Take out test strips and transfer in the sink rinsing with color development stopping;
Or when contained preliminary making antibody is enzyme labelled antibody in the reactant liquor, the test strips of hatching is transferred in the sink, transfer to the colour developing groove after the rinsing; Gently shake or leave standstill colour developing, on test strips, having clearly, colour band occurs; Take out test strips and transfer in the sink rinsing with color development stopping;
Relatively the colour band depth degree of positive control, negative control and testing sample is the positive or feminine gender with definite this test sample on the test strips.
10. according to the described on-the-spot detection method with rapid immune detecting kit of claim 9, it is characterized in that: test strips contains good antigen molecule of embedding in advance or hapten conjugation molecule; Positive control is the positive control solution of contained target molecule to be measured, and the concentration of target molecule to be measured just in time is the threshold concentration on clinical demand or product quality require; Negative control is the negative controls that does not contain target molecule to be measured.
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| CN102565387B (en) | 2014-11-19 |
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Effective date of registration: 20191224 Address after: 361000 Fujian Xiamen free trade pilot area Xiamen area (bonded port area) 1, 268 harbour view, 303. Patentee after: Chenggong (Xiamen) Biotechnology Co., Ltd. Address before: 300308 aviation industry support center of Tianjin Binhai New Area Airport Economic Zone (comprehensive bonded area) A246 Patentee before: Hudson (Tianjin) Biotechnology Co., Ltd. |
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