CN102135498B - Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof - Google Patents
Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof Download PDFInfo
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Abstract
The invention discloses an immune colloidal metal detection technology, which is a detection method performed by coating at least two capture agents in different capture strengths at different parts of a same detection film and reacting with a detected object in a same system, wherein the system comprises the solid-phase detection film, a support chip, a color developing agent, the capture agents and a standard color developing spectrum, and a variety of instant content detection products for the detected objects can be prepared, thereby being effectively used for clinical detection and content detection of the detected objects, such as foods, medicaments, experimental products and the like, and having good practical value and market prospects.
Description
[technical field] a kind of immune colloid gold belongs to detection technique, particularly a kind ofly can directly carry out colloidal metal detection technology of semi-quantitative analysis and its production and use.
[background technology]
Colloidal metal labelling technique is to using colloidal metal as tracer label thing or developer, is applied to a kind of Novel immune labelling technique of antigen-antibody reaction.Due to it, there is not the problems such as endogenous enzymes interference and radioisotope pollution, and utilize the colloidal metal of variable grain size can also make dual even multiple labelling, make location more accurate.Therefore become a kind of novel markings technology after fluorescein, enzyme, isotope and latex labelling technique.The fields such as manufacture that have now been widely used in Electronic Speculum, flow cytometer, Western blotting, external diagnosis reagent, food and medicine quality testing reagent.
Collaurum be by gold chloride reductive agent as the effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid under, polymerization becomes the gold grain of specific size, and because electrostatic interaction becomes a kind of stable colloidal state.Collaurum is electronegative under weak base environment, can form firmly and be combined with the positive charge group of protein molecule, because this combination is electrostatical binding, so do not affect the biological nature of protein.Collaurum except with protein bound, colloid gold particle can also with other multiple biomacromolecule material (as toxin, microbiotic, hormone, nucleic acid, polypeptide etc.) combinations.According to some physical behaviors of collaurum, as high electron density, grain size, shape and color reaction, add immunity and the biological characteristics of bond, thereby make collaurum be widely used in the fields such as immunology, histology, pathology and cell biology.
Immune colloid gold belongs to detection technique and can be divided into substantially and take liquid phase reactor and be main detection technique and take solid phase carrier reaction as main detection technique, wherein take liquid phase reactor as main detection technique, as fluidic cell detection technique with for the liquid phase SABC determination techniques of the distribution character of tissue, cell or the subcellular structure of light microscopic, Electronic Speculum horizontal location, qualitative or quantitative examination specific antigen material, the solid phase carrier reaction of take is main detection technique, as widely used colloidal metal immunochromatographic method etc.With other detection method comparisons, there are following advantages: 1) fast rapid: no matter be that immunochromatographic method or spot immune percolation all have feature fast, generally in minutes just can obtain a result, this is that other method for quick is beyond one's reach at present.2) highly sensitive: immune colloid gold belongs to detection method not because it has sacrificed its detection sensitivity fast, and limit of identification can reach below 0.1ng/ml, as serum cardiac troponin T quick detection reagent.3) safe and simple, because colloidal metal itself has color, qualitative detection does not need any instrument and equipment, and naked eyes get final product judged result.4) low production cost.
Yet, what be widely used at present take solid phase carrier reaction as main detection technique is not if being equipped with corresponding detecting instrument, only rely on naked eyes judged result, also still be confined to qualitative level, can only on a certain detection level, judge and have or nothing, if the urine human chorionic gonadotrophin limit of identification of the very early pregnancy colloidal gold method Test paper detecting for early pregnancy is 25U/L, and below the concentration 1U/L of normal urine human chorionic gonadotrophin, the limit of identification that is used for the blood cardiac muscle Troponin I gold-immunochromatographyreagent reagent for assay of early diagnosis of acute myocardial infarction is 1.5ng/ml, and normal blood cardiac muscle troponin I concentration is lower than 0.15ng/ml.Therefore, only have in the situation that has so large feminine gender and positive difference, the detection method of existing dependence naked eyes judgement could be judged test positive or feminine gender, but cannot determine that it has the dynamic change result of further clinical diagnosis and therapeutic value.Therefore, develop the judgement of a kind of easy to use, swift to operate relied on naked eyes carry out semi-quantitative analysis colloidal metal Fast Detection Technique will have great importance to improving the field service quality such as health care, animal husbandry, agricultural and quality control level.
[summary of the invention]
The object of this invention is to provide a kind of immune colloid gold and belong to detection technique, particularly a kind ofly do not need to rely on the immune colloid gold that instrument and equipment can be directly carries out semi-quantitative analysis by range estimation and belong to detection technique and its production and use.
For achieving the above object, the present invention has adopted following technical scheme.
A kind of immune colloid gold of the present invention belongs to detection technique, it is characterized in that described detection technique is the different parts that trapping agent that an above difference is caught to intensity is coated on same detection film, reacts the detection method of carrying out in same system with determinand.
Described detection technique, comprises the following steps:
1) trapping agent of an above difference being caught to intensity is coated on the different parts of same detection film;
2) determinand in testing liquid and colloidal metal developer are carried out to specific binding and react, form determinand developer compound;
3) described determinand developer compound is reacted in same system with described coated trapping agent, the coated site of now catching intensity trapping agent in difference presents different colored intensities, forms a specific colour developing pattern;
4) treat the concentration difference of determinand in test sample, the colour developing pattern presenting during detection is different;
5) with the standard solution of variable concentrations, carry out described detection reaction, obtain the standard coloration pattern of concentration dependent;
6) the colour developing pattern of sample detection gained and standard coloration pattern are compared, obtain the concentration interval range of determinand in testing sample.
Described standard coloration pattern is prepared with the standard solution of 2-20 variable concentrations, obtains the colour developing pattern that 2-20 kind is different, and a specific concentration in test sample is treated in the representative of every kind of colour developing pattern.The colour developing pattern of sample detection gained and standard coloration pattern are compared, just can obtain one or two standard coloration pattern immediate with it, according to the determinand sample concentration of this one or two standard coloration pattern representative, just can obtain the concentration interval range of determinand in testing sample.
Described detection technique detects film, support chip, developer, trapping agent and standard coloration pattern by solid phase and forms.
Described solid phase detection film has obvious protein bound characteristic and comprises nitrocellulose filter, pvdf membrane, nylon membrane, DEAE cellulose membrane.The conventional solid phase mould with obvious protein bound characteristic has nitrocellulose filter (Nitrocellulose Blotting Membranes, NC) and pvdf membrane (Polyvinylidene-Fluoride), nylon membrane, DEAE cellulose membrane etc.Nitrocellulose filter is the most widely used Protein transfer binding medium, and albumen is had to very strong binding ability, and is applicable to various coloration methods, comprises isotope, chemiluminescence (Luminol class), conventional colour developing, dyeing and fluorescence developing; Background is low, and signal to noise ratio (S/N ratio) is high.Polyvinylidene fluoride (PVDF) film is as the transfer film of matrix, compare with nitrocellulose filter, pvdf membrane is in protein retention ability, more superior performance all on physical strength and chemical compatibility, and the typical combination amount of commercially available nitrocellulose filter is 80-100 μ g/cm
2, and pvdf membrane binding capacity is 100-200 μ g/cm
2.Nylon membrane is also useful on protein combination, is compared to nitrocellulose filter, and its advantage is that adhesion is strong, and solid soft and be difficult for curlingly again, physical strength is large, convenient operation, and shortcoming is that background is high.
Described developer be colloidal metal mark can with the detection agent of determinand specific binding, wherein colloidal metal comprises collaurum, electroselenium, collargol, electrocuprol, CI; Comprise antigen, antibody, Avidin, streptavidin with the detection agent of determinand specific binding.
Described trapping agent comprises following material: 1) can with the antibody of described determinand specific binding; 2) can with the antigen of described determinand specific binding; 3) can be by the Avidin of biotinylated derivative and determinand specific binding; 4) can be by the streptavidin of biotinylated derivative and determinand specific binding.
Described can be the most frequently used biotin-avidin detection system of detection reagent by the Avidin of biotinylated derivative and determinand specific binding, be specially: when determinand is antigen, with colloidal metal mark specificity first antibody, the second antibody that can match with first antibody with biotin labeling, detects film by the coated solid phase of Avidin.During detection, antigen first with the first antibody specific binding of colloidal metal mark, by antigen, be combined with biotin labeled second antibody again, form colloidal metal-first antibody+antigen+second antibody compound, then described compound is combined with coated Avidin by biotin, produces chromogenic reaction.
Described can be the most frequently used biotin-streptavidin detection system of detection reagent by the streptavidin of biotinylated derivative and determinand specific binding, be specially: when determinand is antigen, with colloidal metal mark specificity first antibody, the second antibody that can match with first antibody with biotin labeling, detects film by the coated solid phase of streptavidin.During detection, antigen first with the first antibody specific binding of colloidal metal mark, by antigen, be combined with biotin labeled second antibody again, form colloidal metal-first antibody+antigen+second antibody compound, then described compound is combined with coated streptavidin by biotin, produces chromogenic reaction.
Described support chip is for fixing solid phase, to detect the stilt of film and other test material, the most conventional with PVC plate.
The trapping agent that described difference is caught intensity comprises following manner: 1) the same trapping agent of various dose, 2) dissimilar trapping agent and combination thereof, comprising more than one the composition coated coated detection of the potpourri film that detects film and be mixed and made into variable concentrations with more than one the composition in described antibody, antigen, Avidin, streptavidin respectively in the antibody with described, antigen, Avidin, streptavidin.The same trapping agent of described various dose comprises coated with the trapping agent of variable concentrations and coated with the trapping agents of the different coated volumes of same concentration, and net result is the difference that makes the dosage of the coated trapping agent of different parts on detection film or the absolute magnitude of coated trapping agent.Described dissimilar trapping agent and be combined as more than one the different combinations in described antibody, antigen, Avidin, streptavidin, for example that the different parts that detects film is coated with antibody, Avidin, the streptavidin of identical or different concentration or be coated with antigen, Avidin, streptavidin respectively; Or antibody and Avidin or streptavidin are mixed in proportion and be coated with or antigen mixes and is coated with in proportion with Avidin or streptavidin, consequently make to detect and when react from determinand in the coated site of trapping agents different on film, present different colored intensities.
The quantity that a described above difference is caught intensity trapping agent is selected 2-20, preferably 3-10, more selects 3-5.It can adopt line type in the coated mode detecting on film, also can adopt spot formula.
Described detection technique comprises immunochromatographic method and spotting method.Described detection technique also comprises two ball sandwich methods, microplate method etc.
Described detection technique is colloidal metal immunochromatographic method, by comprising that support chip, solid phase detect film, contain developer film pad, trapping agent, sample pad, standard coloration pattern, adsorptive pads, transfer pipet etc. and form, wherein detect film, adsorptive pads supporting on egative film the solid phase that sample pad is housed successively, contains developer film pad, is coated with trapping agent, comprise the following steps:
1) trapping agent of an above difference being caught to intensity is coated on the different parts of same detection film, is the solid phase detection film that is coated with trapping agent;
2) with colloidal metal solution mark can with the detection agent of determinand specific binding, prepare developer;
3) the immune developer after mark is sprayed on developer pad, dry, be and contain developer film pad;
4) dress successively pastes sample pad, contains developer film pad, the solid phase that is coated with trapping agent detects film, adsorptive pads supporting on egative film;
5) testing sample point is added in sample pad, enter and contain developer film pad, make determinand and colloidal metal developer carry out specific binding and react, form determinand developer compound;
3) described determinand developer compound is reacted in same system with described coated trapping agent, the coated site of now catching intensity trapping agent in difference presents different colored intensities, forms a specific colour developing pattern;
4) treat the concentration difference of determinand in test sample, the colour developing pattern presenting during detection is different;
5) with the standard solution of variable concentrations, carry out described detection reaction, obtain the standard coloration pattern of concentration dependent;
6) the colour developing pattern of sample detection gained and standard coloration pattern are compared, obtain the concentration interval range of determinand in testing sample, and then carry out the semi-quantitative analysis of thing content to be checked in testing sample.
Described detection technique, it is characterized in that combining and use multiple colloidal metal color signal amplifying technique to improve detection sensitivity, comprise that immunogold silver staining amplifying technique, biotin-avidin amplifying technique, biotin-streptavidin amplifying technique, second antibody are in conjunction with amplifying technique.The equipment such as described technology combined with fluorescent colour developing, colour developing quantitative test, can carry out the full quantitative test of determinand content.
The purposes of described detection technique in the product development of colloidal metal solid phase detection technique, comprises a plurality of fields such as health care, animal husbandry, agricultural.Current immune colloid gold belongs to detection technique and has been widely used in every field, the purposes of described detection technique in the target detection thing content detection of health care testing product and food, medicine and experimental article.The technology of the present invention can be made into multiple instant product, not only easy to use, be easy to storage, be easy to carry, and it is short to detect required time, as carries out semi-quantitative analysis, do not need specialized equipment, do not need special-purpose experiment condition and relevant professional knowledge yet.
[beneficial effect]
1, adopt first colloidal metal mark developing technology by determinand sample from be coated on same detection film on one of different parts above different trapping agent of catching intensity under same reaction system, react, by instrument and equipment, can directly not judge by visual inspection the content interval scope of determinand, carry out semi-quantitative analysis, improved the accuracy of this detection technique.
2, fast detecting is people to being widely used at present the detection reagent of health care medical diagnosis on disease and state of illness monitoring and for common requirement and the hope of the detection reagent of the production quality control in a plurality of fields such as animal husbandry and agricultural, all needing corresponding experimental facilities, experiment condition and corresponding personnel's technical requirement but existing immune colloid gold belongs to sxemiquantitative and the quantitative analysis method of detection technique.This makes troubles to detection, needs the detection time of growing simultaneously.And detect consuming time very crucial in some cases, as acute myocardial infarction patients from morbidity to completing necessary detection to clarify a diagnosis, implement to get involved or the time of thromboembolism treatment very crucial to disease prognosis, the time, shorter prognosis was better.And the comfort level detecting the directly universal use of influence technique product and the quality control of examined product as a rule.And fast detecting is the important feature of the technology of the present invention.
3, the sxemiquantitative that can carry out determinand content in sample by range estimation is directly measured, and overcomes the shortcoming that current colloidal metal detection technology can only carry out qualitative analysis by range estimation, has improved detection quality and the scope of application.The cardiac muscle troponin I gold-immunochromatographyreagent reagent for assay box of take is example, and cardiac muscle troponin I is the impaired important symbol thing of reflecting myocardium, and haemoconcentration is very low under normal circumstances, lower than 0.16ng/ml.During myocardial damage, haemoconcentration can raise to some extent, reaches as high as every milliliter of hundreds of nanogram (ng), relevant to the degree of myocardial damage.Most of myocardial damage diseases are if its blood cardiac muscle Troponin I concentration majorities such as myocarditis, heart failure, cardiomyopathy, angina pectoris are below 1.5ng/ml, be greater than the case less than 10% of 1.5ng/ml, but the case of 6 hours after 90% above acute myocardial infarction, its Blood Center flesh Troponin I concentration is greater than 1.5ng/ml, and changes along with change of illness state.The concentration of its Blood Center flesh Troponin I of case that most of cardiac muscular tissues are downright bad serious is greater than 5ng/ml.Use clinically at present without the instrument colloidal gold fast detecting reagent kit of judged result with the naked eye only, the testing result of positive or negative can only be provided, be that haemoconcentration develops the color while being greater than 1.5ng/ml, positive, when being less than 1.5ng/ml, haemoconcentration do not develop the color, negative, only to the case of acute myocardial infarction AMI values that provides assistance in diagnosis, to the change of illness state after other myocardial damage disease and myocardial infarction, dynamic and result for the treatment of cannot provide valuable detection data.The technology of the present invention without instrument only with the naked eye judgement can directly carry out the semi-quantitative analysis of blood cardiac muscle Troponin I concentration, and accomplish quick and conveniently, can effectively improve again monitoring quality, expand detect effective range.
4, easy to use.Product of the present invention is easy to use, easy to carry, for the use of product provide more easily by way of.
5, production technology is simply cheap, is beneficial to very much marketing.
Therefore, the technology of the present invention in a plurality of fields such as health care, animal husbandry, agriculturals to improving service quality and production quality control has great importance and good application prospect.
Accompanying drawing explanation
Fig. 1 is chromatography detection kit structural representation of the present invention
Fig. 2 is spotting method detection kit structural representation of the present invention
[specific implementation method]
By following concrete embodiment, can further understand the present invention, but following instance not limitation of the invention.Standard comparison product used are all determined its testing concentration through chemoluminescence method.
The making of embodiment 1-chromatography semi-quantitative detection kit of the present invention
As shown in Figure 1, the basic structure of chromatography of the present invention is by comprising that sample pad 1, developer 2, solid phase detection film 3, trapping agent 4, adsorptive pads 5, support chip 6, standard coloration pattern 7 form, wherein sample pad 1, the film pad that is coated with developer 2, the solid phase that is coated with trapping agent 4 detects film 3 and adsorptive pads 5 is attached on support chip 6 successively, the standard coloration pattern 7 of variable concentrations is also positioned in same packing as constituent.Detected sample is dropped in sample pad 1, then drive developer 2 swimming forward, the solid phase of flowing through detects film 3 and coated trapping agent 4, and is absorbed by adsorptive pads 5.Now solid phase detects on film 3 and can present a colour developing pattern, and this colour developing pattern and standard coloration pattern 7 are compared, and one or two standard coloration pattern that just can obtaining develops the color is adjacent and the sample concentration of representative, be testing sample concentration interval.
The making of embodiment 2-spotting method half-quantitative detection of the present invention kit
As shown in Figure 2, the basic structure of spotting method of the present invention is by comprising that developer 2, solid phase detection film 3, trapping agent 4, support chip 6, cleaning fluid 8, standard coloration pattern 7 form, the solid phase that is wherein coated with trapping agent 4 detects film 3 and is attached on support chip 6, the solid phase that is placed in the film pad that is coated with developer 2 detects on film 3, the standard coloration pattern 7 of variable concentrations and fill cleaning fluid 8 pipes and be also positioned in same packing as constituent.Detected sample is dropped on developer 2 film pads 1, then drive developer 2 to flow downward, the solid phase of flowing through detects film 3 and coated trapping agent 4, after reaction certain hour, remove developer 2 film pads, with cleaning fluid 8, repeatedly rinse solid phase detection film 3 close with standard coloration pattern 7 to background colour developing, discard whole feelings washing lotions 8, with the comparison of standard coloration pattern, one or two standard coloration pattern that just can obtaining develops the color is adjacent and the sample concentration of representative, be testing sample concentration interval.
The making of embodiment 3-single trapping agent cardiac muscle troponin I of the present invention colloidal gold chromatography semi-quantitative detection kit and experiment are observed
Experiment material: human cardiac troponin I, anti-human human cardiac troponin I monoclonal antibody, gold chloride, trisodium citrate, sal tartari, crystallization bovine serum albumin, nitrocellulose filter, PVC egative film, multi-polyester film, thieving paper, sample pad, micro sample adding appliance, cow's serum sample-loading buffer, normal human serum sample, acute myocardial infarction patients serum.
Method: with deionized water, gold chloride being made into concentration is 0.01% solution, be heated to boiling, every 100ml adds the citric acid three sodium solution of 100ul10%, stir rapidly, until color, become claret, stablize when constant, continue heating 10min, be cooled to room temperature, pack in reagent bottle, save backup.Get the colloidal gold solution that 10ml has been prepared, with 1% sal tartari, adjust pH to 8.2, add the anti-human human cardiac troponin I monoclonal antibody of 10ug/ml (final concentration), mix, standing 30 minutes, add 25ul/ml 10% crystallization bovine serum albumin aqueous solution, standing 30 minutes, 12000rpm, centrifugal 20 minutes, abandon supernatant, precipitation is dissolved in 100ul/ml containing in the PBS of 0.2%BSA, makes colloid gold label thing, be sprayed on multi-polyester film, 37 ℃ dry after, stand-by, this is developer film pad.
On nitrocellulose filter respectively successively the coated concentration of line be 0.05,0.1,0.2,0.4,0.8, the goat-anti human cardiac troponin I polyclonal antibody of 1.6mg/ml, concentration is that the sheep anti-mouse igg of 1.0mg/ml contrasts as detecting, 37 ℃ dry after, stand-by, this is the solid phase detection film that is coated with trapping agent.
The assembling of test-strips: successively sample pad, developer film pad, the solid phase that is coated with trapping agent are detected to film, thieving paper sticks on PVC egative film, is cut into the wide test-strips of 4mm with cutting cutter, kept dry is standby.
During detection, get 8 parts, normal human serum sample and 8 parts of each 100ul of acute myocardial infarction patients blood serum sample of the known Troponin I concentration that adopts the detection of immunochemiluminescence method, be added drop-wise to respectively in the sample pad of two test strips, through developer film pad, swimming forward, flow through and be coated with the solid phase detection film of trapping agent, determinand is caught by trapping agent specificity and is developed the color in the coated position of trapping agent.After point sample 15 minutes, solid phase detects on film can present a colour developing pattern, by this colour developing pattern and the comparison of standard coloration pattern, just can obtain sample develop the color one or two standard coloration pattern that pattern is adjacent and the sample concentration of representative, be testing sample concentration interval.
Preparation standard colour developing pattern: respectively configuration concentration be 0.15,0.5,1.0,1.5,5.0, the standard solution of the human cardiac troponin I of 7.5ng/ml, be added to respectively in the test-strips of having prepared, after 15 minutes, the colour developing pattern of each test-strips is taken pictures respectively, make standard coloration pattern.
The results are shown in Table 1, testing result of the present invention is consistent with the blood serum sample concentration that immunochemiluminescence method detects.
Table 1, serum cardiac troponin I concentration testing result comparison (ng/ml)
Making and the experiment of the multiple trapping agent combination of embodiment 4-the present invention myoglobins colloidal gold chromatography semi-quantitative detection kit are observed
Experiment material: human muscle hemoglobin, anti-human myoglobins monoclonal antibody, gold chloride, trisodium citrate, sal tartari, crystallization bovine serum albumin, nitrocellulose filter, PVC egative film, multi-polyester film, thieving paper, sample pad, micro sample adding appliance, normal human serum sample, acute myocardial infarction patients serum.
Method: with deionized water, gold chloride being made into concentration is 0.01% solution, be heated to boiling, every 100ml adds the citric acid three sodium solution of 100ul10%, stir rapidly, until color, become claret, stablize when constant, continue heating 10min, be cooled to room temperature, pack in reagent bottle, save backup.Get the colloidal gold solution that 10ml has been prepared, with 1% sal tartari, adjust pH to 8.8, add the anti-human myoglobins monoclonal antibody of 10ug/ml (final concentration), mix, standing 30 minutes, add 25ul/ml 10% crystallization bovine serum albumin aqueous solution, standing 30 minutes, 12000rpm, centrifugal 20 minutes, abandon supernatant, precipitation is dissolved in 100ul/ml containing in the PBS of 0.2%BSA, makes colloid gold label thing, be sprayed on multi-polyester film, 37 ℃ dry after, stand-by, this is developer film pad.
On nitrocellulose filter, the coated concentration of line is 0.05mg/ml goat-anti human muscle hemoglobin polyclonal antibody, 0.1mg/ml streptavidin, 0.2mg/ml goat-anti human muscle hemoglobin polyclonal antibody, 0.6mg/ml Avidin, 0.8mg/ml goat-anti human muscle hemoglobin polyclonal antibody successively respectively, concentration is that the sheep anti-mouse igg of 1.0mg/ml contrasts as detecting, 37 ℃ dry after, stand-by, the solid phase that is coated with trapping agent detects film.
Goat-anti human muscle hemoglobin polyclonal antibody biotin labeling: get goat-anti human muscle hemoglobin polyclonal antibody 5ml (1mg/ml), use 300mM NaHCO
3adjust pH to 8.0, add activation biotin 1mg, after mixing, room temperature is placed 2 hours, add 30% glycocoll 50mg, with 50mM phosphate buffer (pH7.4) dialysis three times, then with 0.5ul/cm, be coated with to sample film pad, 37 ℃ dry after, stand-by, i.e. biotin labeling antibody membrane pad.
The assembling of test-strips: successively sample pad, developer film pad, biotin labeling antibody membrane pad, the solid phase that is coated with trapping agent are detected to film, thieving paper sticks on PVC egative film, is cut into the wide test-strips of 4mm with cutting cutter, kept dry is standby.
During detection, get 8 parts, normal human serum sample and 8 parts of each 100ul of acute myocardial infarction patients blood serum sample of the known myoglobin concentration that adopts the detection of immunochemiluminescence method, be added drop-wise to respectively in the sample pad of two test strips, through developer film pad and biotin labeling antibody membrane pad, swimming forward, flow through and be coated with the solid phase detection film of trapping agent, determinand is caught by trapping agent specificity and is developed the color in the coated position of trapping agent.After point sample 15 minutes, solid phase detects on film can present a colour developing pattern, by this colour developing pattern and the comparison of standard coloration pattern, just can obtain sample develop the color one or two standard coloration pattern that pattern is adjacent and the sample concentration of representative, be testing sample concentration interval.
Preparation standard colour developing pattern: respectively configuration concentration be 25,50,100,250,300, the standard solution of the human muscle hemoglobin of 400ng/ml, be added to respectively in the test-strips of having prepared, after 15 minutes, the colour developing pattern of each test-strips is taken pictures respectively, make standard coloration pattern.
The results are shown in Table 2 testing results of the present invention consistent with the blood serum sample concentration that immunochemiluminescence method detects.
Table 2, serum myoglobin concentration testing result comparison (ng/ml)
Embodiment 5-the inventive method is observed the half-quantitative detection experiment of the blood serum sample cardiac muscle troponin I level of separate sources
Scheme described in experiment material and method employing embodiment 3.Testing sample comprises from each 15 parts of normal person, Q ripple acute myocardial infarction AMI, non-Q ripple acute myocardial infarction AMI, moderate slight, in heart failure in heart failure, severe in heart failure, acute myocarditis, unstable angina patients serums.Experiment represents testing result of the present invention as follows: < 0.15ng/ml is "+", 0.15-0.5ng/ml is " ++ ", 0.5-1.0ng/ml is " +++ ", 1.0-1.5ng/ml be " ++++", 1.5-5.0ng/ml be " +++ ++ ", 5.0-7.5 is " +++ +++ ", and > 7.5ng/ml is " +++ ++++".
The results are shown in Table 3, detection method acquired results of the present invention is consistent with immunochemiluminescence method detection acquired results, all present without the normal person of myocardial damage and with myocardial function, change into main and minimum without the slight patients serum's cardiac muscle troponin I of the heart failure concentration of obvious myocardial structural damages, moderate in heart failure with myocardium minor injury, acute myocarditis, unstable angina patients serum cardiac muscle troponin I concentration is taken second place, non-Q ripple acute myocardial infarction AMI and severe patients serum's cardiac muscle troponin I concentration in heart failure with slight cardiac muscular tissue's necrosis or structural damage obviously raise, with the Q ripple acute myocardial infarction patients serum cardiac troponin I concentration of the obviously cardiac muscular tissue's necrosis highly significant that raises, therefore, serum cardiac troponin I concentration is carried out to quantitative analysis, contribute to antidiastole and the analysis to pathology process of disease, adopt in time corresponding methods for the treatment of.
Testing result of the present invention illustrates that the most important part of the inventive method has been to provide a kind of fast, detection by instrument simultaneously and through range estimation, just can obtain the testing result consistent with instrumental quantitative analysis time with identical diagnostic value, has important clinical meaning.
Table 3, serum cardiac troponin I concentration testing result comparison (ng/ml)
Making and the experiment of the multiple trapping agent combination of embodiment 6-the present invention myoglobins colloidal gold dot method half-quantitative detection kit are observed
Experiment material: human muscle hemoglobin, anti-human myoglobins monoclonal antibody, gold chloride, trisodium citrate, sal tartari, crystallization bovine serum albumin, nitrocellulose filter, PVC egative film, multi-polyester film, sample pad, micro sample adding appliance, 50mM phosphate cleaning fluid, normal human serum sample, acute myocardial infarction patients serum.
Method: prepare developer film pad with the method described in embodiment 4.
First cellulose nitrate solid phase being detected to film sticks on PVC egative film, it is 0.05mg/ml goat-anti human muscle hemoglobin polyclonal antibody, 0.1mg/ml streptavidin, 0.2mg/ml goat-anti human muscle hemoglobin polyclonal antibody, 0.6mg/ml Avidin, 0.8mg/ml goat-anti human muscle hemoglobin polyclonal antibody that the spot formula of take is coated with concentration successively respectively on cellulose nitrate solid phase detection film, concentration is that the sheep anti-mouse igg of 1.0mg/ml contrasts as detecting, 37 ℃ dry after, stand-by, the solid phase that is coated with trapping agent detects film.
By the method described in embodiment 4, prepare biotin labeling goat-anti human muscle hemoglobin polyclonal antibody.
The assembling of test-strips: paste from top to bottom biotin labeling antibody membrane pad, developer film pad and sample pad successively on the cellulose nitrate solid phase detection film being pasted on PVC plate, be cut into the wide test board of 15mm with cutting cutter, kept dry is standby.
During detection, get 8 parts, normal human serum sample and 8 parts of each 100ul of acute myocardial infarction patients blood serum sample of the known myoglobin concentration that adopts the detection of immunochemiluminescence method, be added drop-wise to respectively in the sample pad of two beta versions, through developer film pad and biotin labeling antibody membrane pad, swimming downwards, flow through and be coated with the solid phase detection film of trapping agent, determinand is caught by trapping agent specificity and is developed the color in the coated position of trapping agent.After point sample 15 minutes, remove sample pad, developer film pad and biotin labeling antibody membrane pad, with 50mM phosphate cleaning fluid, clean solid phase and detect film to presenting the pattern that develops the color clearly.By this colour developing pattern and the comparison of standard coloration pattern, just can obtain sample develop the color one or two standard coloration pattern that pattern is adjacent and the sample concentration of representative, be testing sample concentration interval.
Preparation standard colour developing pattern: respectively configuration concentration be 25,50,100,250,300, the standard solution of the human muscle hemoglobin of 400ng/ml, be added to respectively on the beta version of having prepared, after 15 minutes, clean and the colour developing pattern of each beta version is taken pictures respectively, making standard coloration pattern.
The results are shown in Table 4, testing result of the present invention is consistent with the blood serum sample concentration that immunochemiluminescence method detects.
Table 4, serum myoglobin concentration spotting method testing result comparison (ng/ml)
Claims (8)
1. an immune colloid gold belongs to detection technique, it is characterized in that described detection technique is the different parts that trapping agent that an above difference is caught to intensity is coated on same detection film, in same system, react the detection method of carrying out with determinand, comprise following technical characterictic:
1) trapping agent of an above difference being caught to intensity is coated on the different parts of same detection film;
2) determinand in testing liquid and colloidal metal developer are carried out to specific binding and react, form determinand developer compound;
3) described determinand developer compound is reacted in same system with described coated trapping agent, the coated site of now catching intensity trapping agent in difference presents different colored intensities, forms a specific colour developing pattern;
4) treat the concentration difference of determinand in test sample, the colour developing pattern presenting during detection is different;
5) with the standard solution of variable concentrations, carry out described detection reaction, obtain the standard coloration pattern of concentration dependent;
6) the colour developing pattern of sample detection gained and standard coloration pattern are compared, obtain the concentration interval range of determinand in testing sample.
2. detection technique according to claim 1, is characterized in that described detection technique detects film, support chip, developer, trapping agent and standard coloration pattern by solid phase and forms, wherein:
1) the solid phase detection film described in has obvious protein bound characteristic and comprises nitrocellulose filter, pvdf membrane, nylon membrane, DEAE cellulose membrane;
2) described developer for colloidal metal mark can with the detection agent of determinand specific binding, wherein colloidal metal comprises collaurum, electroselenium, collargol, electrocuprol, CI; Comprise antigen, antibody, Avidin, streptavidin with the detection agent of determinand specific binding;
3) described trapping agent comprise can with the antibody of described determinand specific binding, can with the antigen of described determinand specific binding, can be by biotinylated derivative and determinand specific binding Avidin, can be by one or more the material in the streptavidin of biotinylated derivative and determinand specific binding;
4) the different colour developing pattern of 2-20 kind that described standard coloration pattern prepares for the standard solution with 2-20 variable concentrations, a specific concentration in test sample is treated in the representative of every kind of colour developing pattern;
5) described support chip be for the fixing stilt of solid phase detection film and other test material, the most conventional with PVC plate.
3. detection technique according to claim 1, is characterized in that described difference catches the trapping agent of intensity and comprise following manner:
1) the same trapping agent of various dose;
2) dissimilar trapping agent and combination thereof, comprising more than one the composition coated coated detection of the potpourri film that detects film and be mixed and made into variable concentrations with more than one the composition in described antibody, antigen, Avidin, streptavidin respectively in the antibody with described, antigen, Avidin, streptavidin.
4. detection technique according to claim 1, is characterized in that the quantity that a described above difference is caught intensity trapping agent selects 2-20.
5. detection technique according to claim 4, is characterized in that the quantity that a described above difference is caught intensity trapping agent selects 3-10.
6. detection technique according to claim 1, is characterized in that described detection technique comprises immunochromatographic method and spotting method.
7. detection technique according to claim 1, it is characterized in that combining and use multiple colloidal metal color signal amplifying technique to improve detection sensitivity, comprise that immunogold silver staining amplifying technique, biotin-avidin amplifying technique, biotin-streptavidin amplifying technique, second antibody are in conjunction with amplifying technique.
8. the purposes of detection technique claimed in claim 1 in the product development of colloidal metal immuno-chromatographic assay technology.
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