CN102890157A - Test strip and method for fast quantitative detection of human chorionic gonadotropin (HCG) - Google Patents
Test strip and method for fast quantitative detection of human chorionic gonadotropin (HCG) Download PDFInfo
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- CN102890157A CN102890157A CN2011102030277A CN201110203027A CN102890157A CN 102890157 A CN102890157 A CN 102890157A CN 2011102030277 A CN2011102030277 A CN 2011102030277A CN 201110203027 A CN201110203027 A CN 201110203027A CN 102890157 A CN102890157 A CN 102890157A
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Abstract
The invention relates to a test strip and a method for fast quantitative detection of human chorionic gonadotropin (HCG). The test strip comprises a sample loading pad, a marker pad, an NC membrane, a sample suction pad and a plastic plate. The sample loading pad, the marker pad, the NC membrane and the sample suction pad are orderly adhered to the plastic plate. The marker pad is coated with colloidal gold-marked mouse anti-human HCG-beta monoclonal antibodies. The NC membrane is coated with a detection line T composed of mouse anti-human HCG-alpha antibodies and a quality control line C composed of goat anti-mouse IgG antibodies. On the NC membrane, a detection line T side close to the marker pad is provided with a sample loading indication line for indicating a sample loading position. Through loading of a sample on the NC membrane, the test strip can solve the problem that the existing detection reagent produces a hook effect because of too high HCG content, and can realize quantitative detection of HCG in blood or urine by a matched immunochromatographic assay interpretoscope.
Description
Technical field
The present invention relates to the biologic applications technical field, particularly the test strips of a kind of Quantitative detection HCG and detection method.
Background technology
After embryonated egg is implanted the uterus, will secrete a kind of new hormone in pregnant woman's body, be called human chorionic gonadotrophin (Human Chorionic Gonadotrophin is called for short HCG).HCG was found in urine of pregnant women by Aschheim and zondek first in nineteen twenty-seven.HCG is a kind of hormone of cytotrophoblast and plamoditrophoblast secretion, and it at first is present in the placenta, is excreted in the urine by blood circulation.In placenta, blood and urine, all there are HCG and metabolic product thereof.After becoming pregnant, about 10 days, just can in pregnant woman's urina sanguinis, detect HCG.In the normal pregnant women serum, HCG concentration after corpus luteum reaches peak value 7~10 days or embryonated egg are implanted and are begun after postuterine 4~7 days to increase, and increase with exponential form, and average per 1.5~2 days concentration is just double, also varies with each individual but advance the speed.Gestation after 7~10 weeks the HCG concentration just can reach 20,000~100,000IU/L, decrease in gestation 13~15 when week concentration, begin again afterwards slowly to increase until 30~33 weeks of gestation, and slowly reduce until the gestation end.Ectopic pregnancy HCG rises then slower, or 3~6 conceived weeks, and HCG can not continue double increase, should consider the possibility of ectopic pregnancy or threatened abortion.
HCG is the main signal in the early pregnancy, and specificity marker has become the common counter of diagnosing clinically early pregnancy.Quantitatively dynamic monitoring HCG amount is also as the monitor control index of ectopic pregnancy.
The method of early detection gestation mainly contains three kinds: Ultrasonic Detection, examination of basal body temperature and immunochromatography detect.Ultrasonic Detection be owing to can only detect gestation situations in uterus after 6 weeks, and is normally used for the clinically monitoring of fetus.But although examination of basal body temperature is simple more loaded down with trivial details.Immunochromatography detect since it easy and simple to handle, have directly perceived, fast, the advantages such as detection efficiency is high, method is easy, pollution-free, expense is cheap, highly sensitive, high specificity, be widely used in clinical diagnosis, field condition and familial self and detect.
The reagent that immunochromatography detects HCG uses double antibody sandwich method.After test fluid was soaked or splashed into the sample pad of test strips, by the capillary action of water-absorption fiber, test fluid is drenched gold-marking binding pad rapidly, and the collaurum bond on the gold-marking binding pad is dissolved, and moves forward along chromatographic material with test fluid.If there is determined antigen Ag in the test fluid, then it will with the collaurum bond in antibody generation specific immune response, form the immune complex of collaurum bond-determined antigen.Then flow through with test fluid and be fixed on the capture agent of T line on the Nc film.The capture agent of T line is caught collaurum bond-determined antigen immune complex, namely with this immune complex generation specific immune response, form the immune complex of collaurum bond-determined antigen-capture agent, thereby make collaurum bond-determined antigen immune complex be trapped in T line place.Test fluid continues reach, when the Nc film of flowing through is fixed with the C line of sheep anti-mouse igg antibody, and sheep anti-mouse antibody generation immune response on the collaurum bond that does not react with determined antigen Ag and the C line, thus so that the collaurum bond that dissociates is trapped in C line place.This moment, T line and C line place all had the collaurum bond to be detained, and namely T line and C line place all can observe color (redness or purple), then represent the positive result of testing result, namely in the test fluid determined antigen are arranged; If only C line place observes color, and T line place does not observe color, then represent the negative result of testing result, namely in the test fluid without determined antigen; HCG content is different in the liquid to be measured, and the T line color depth that produces is not identical yet, and along with the increase of HCG content, the T line color will be darker.Yet, then can cause high-caliber " hook effect (hook effect) " at some sample middle and high concentration HCG, be that HCG concentration is when being far longer than the upper limit of detection of colloidal gold strip, HCG records the result and descends with the increase of concentration on the contrary, the T line color shoals on the contrary, in clinical diagnosis, cause the low-level illusion of HCG, make diagnostic result false problem occur.
Summary of the invention
The object of the present invention is to provide test strips and the detection method of a kind of Quantitative detection HCG, the test strips of HCG in a kind of energy Quantitative detection urine or the blood especially is provided.
Realize that technical scheme of the present invention is, the test strips of a kind of Quantitative detection HCG and detection method, comprise loading pad, labeling pad, NC film, inhale sample pad and plastic plate, described test strips and immunochromatography interpretoscope support the use the quantitative detection of carrying out HCG, and it is 10~15000mIU/ml that testing sample is diluted rear quantitative sensing range.
Described loading pad (1), labeling pad (2), NC film (3) and suction sample pad (4) adhere to plastic plate (8) successively, be coated with the mouse-anti HCG-β monoclonal antibody of colloid gold label on the labeling pad (2), be coated with respectively the detection line T line (6) of mouse-anti HCG-Alpha antibodies formation and the nature controlling line C line (7) that sheep anti-mouse igg antibody consists of on the NC film (3), draw the application of sample index line (5) that is useful on sign application of sample position near labeling pad (2) one sides at the upper detection line T line (6) of NC film (3) simultaneously.
Described reagent strip is prepared by following step:
(1) preparation of colloid gold label mouse-anti HCG-β labeling of monoclonal antibody thing pad: prepare the colloidal gold solution that diameter is 40nm with gold chloride-trisodium citrate reduction method, after finishing, preparation gets three parts of colloidal gold solutions, with 0.2MK2CO3 pH value of solution is transferred to pH7.5 respectively, pH8.5, pH9.5, then solution is placed on the magnetic stirring apparatus and slowly stir, by every 100ml solution 0.5mg, 1.0mg, 1.5mg mouse-anti HCG-β monoclonal antibody slowly is added drop-wise in the colloidal gold solution, continue to stir 1 hour, adding final concentration is 0.2%BSA again, 0.1% PEG 20000 sealed 30 minutes, 12000 left the heart 30 minutes, abandon supernatant, redissolve to 76.5ml with the collaurum working fluid, the ratio that spreads 16cm2 in 1ml solution is layered on the nonwoven fabrics equably, place again 20~25 ℃ of temperature, humidity was less than 30% drying room drying 2~4 hours, make labeling pad, for subsequent use;
(2) mouse-anti HCG-Alpha antibodies is coated: with 0.01M pH7.2PBS mouse-anti HCG-Alpha antibodies is diluted to respectively 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, then with spray film instrument mouse-anti HCG-Alpha antibodies coating buffer is pressed the 1.3ul/cm line at the NC film and be coated with, consist of detection line T line; Coated sheep anti-mouse igg antibody is as the C line simultaneously, and detection line T line is drawn near labeling pad one side and is useful on the application of sample index line that indicates the application of sample position on the NC film simultaneously, after coated the finishing the NC film is placed the drying room drying 3~4 hours, and is for subsequent use;
(3) assembling of test strips: 20~25 ℃ of temperature, humidity less than 30% hothouse in, get plastic support board, paste at the middle part that coated NC film is placed on plastic support board, in NC film T line one side overlap joint labeling pad, paste the loading pad at labeling pad opposite side overlap joint, inhale the sample pad in NC film C line one side apart from overlapping edges; Then with cutter the plastic plate that posts is cut into the test strips of proper width.
The method of a kind of quantitative detection HCG said method comprising the steps of:
(1) drafting of instrument memory standard curve: take National reference as reference, it is 0mIU/ml that the HCG standard solution is diluted to concentration with normal person's urine (feminine gender) or cow's serum, 10mIU/ml, 50mIU/ml, 250mIU/ml, 1000mIU/ml, 2500mIU/ml, 5000mIU/ml, 10000mIU/ml, 15000mIU/ml series standard product, balance is to room temperature, the reagent strip for preparing is kept flat, add 3~5ul standard items to be checked at NC film marking line, then on the loading pad, add 60~80ml sample dilution, put into supporting immunochromatography interpretoscope after static 10 minutes and carry out reading, to record GOD value (X) as horizontal ordinate, as ordinate, draw instrument memory standard curve with HCG content (y);
(2) method according to step (1) detects testing sample, and obtains the HCG concentration of described testing sample by typical curve.
Principle and the method for described immunochromatography interpretoscope integrated application machinery, electronics, optics, microcomputer and psychophysics, reach intelligent eye recognition interpretation function, visual signal to immunochromatography reagent response line is located automatically, background is rejected, color identifying, quantification treatment, the GOD value by a quantification shows.Namely in certain scope, the depth of line color and the concentration of measured object can present certain relation, are stored in instrument and carry in the IC-card as long as do a typical curve in the test reaction process, just can carry out quantitative test to immunochromatography reagent bar.
Described test strips not only can overcome the existing hook effect that reagent produces because of the HCG too high levels that detects, and can be to the HCG Quantitative Monitoring in blood or the urine, trouble and the error of having avoided the sample dilution to bring.Easy, accurate, fast quantification, sensitivity and specificity is high, economical and practical and with other hormone without intersecting, can better meet clinical requirement.
Compared with prior art, the test strips of the Quantitative detection HCG that the present invention relates to has following advantage and significant progressive:
(1) multifunctionality: the present invention both can be qualitative, can quantitatively detect again the content of HCG in urine or the blood;
(2) fast quantification: supporting immunochromatography interpretation registering instrument quantizes the shade degree that the people is observed visually the detection zone reaction solution, according to instrument memory standard curve, in certain scope, the depth of reaction line color and the concentration of measured object can present certain relation, can accurately carry out quantitatively HCG concentration in the sample;
(3) overcome hook effect: the present invention not only can overcome the existing hook effect that reagent produces because of the HCG too high levels that detects, and can be to the HCG Quantitative Monitoring in blood or the urine, be specially, add sample loading mode during detection for adding the tested sample of 3~5ul at the NC film first, then on the loading pad, add an amount of dilution, both overcome the hook effect that existing detection reagent produces because of the HCG too high levels, consequent erroneous judgement, false negative result have been avoided, improved again the detection sensitivity of reagent, sensitivity can reach 10mIU/ml;
(4) can preserve and review data and picture: existing market for detection of the Rapid test of HCG because the color that the characteristics of itself namely show within a certain period of time is effective, so great majority all are just reagent card have been abandoned after finishing watching the result, the supporting interpretation registering instrument of this reagent can not only be preserved the image document of result of determination in the product Validity Test time, also can preserve original whole image information.
Description of drawings
Fig. 1 is the colloidal gold strip structural representation of fast detecting HCG;
1, loading pad; 2, labeling pad; 3, cellulose nitrate (NC) film; 4, inhale the sample pad; 5, application of sample index line; 6, detection line T line; 7, nature controlling line C line; 8, plastic plate.
Embodiment
Below be the specific embodiment of the method for the Quantitative detection septicemia product that the present invention relates to, technical scheme of the present invention is done further the description, but protection scope of the present invention be not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
The preparation of embodiment 1HCG test strip and detection are used
(1) preparation of colloid gold label mouse-anti HCG-β labeling of monoclonal antibody thing pad
Prepare the colloidal gold solution that diameter is 40nm with gold chloride-trisodium citrate reduction method, get three parts of colloidal gold solutions after preparation is finished, use respectively 0.2MK
2CO
3PH value of solution is transferred to pH7.5, pH8.5, pH9.5, then solution is placed on the magnetic stirring apparatus and slowly stir, slowly be added drop-wise in the colloidal gold solution by every 100ml solution 0.5mg, 1.0mg, 1.5mg mouse-anti HCG-β monoclonal antibody, continue to stir 1 hour, adding final concentration is 0.2%BSA again, 0.1% PEG 20000 sealed 30 minutes, and 12000 left the heart 30 minutes, abandoned supernatant, redissolve to 76.5ml with the collaurum working fluid, press 1ml solution and spread 16cm
2Ratio be layered on equably on the nonwoven fabrics, put again 20~25 ℃ of drying room temperature, humidity is made labeling pad less than 30% drying 2~4 hours, and is for subsequent use.
(2) mouse-anti HCG-Alpha antibodies is coated
With 0.01M pH7.2PBS mouse-anti HCG-Alpha antibodies is diluted to respectively 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, then with spray film instrument mouse-anti HCG-Alpha antibodies coating buffer is pressed the 1.3ul/cm line at the NC film and be coated with, consist of detection line T line; Coated sheep anti-mouse igg antibody is as the C line simultaneously, and detection line T line is drawn near labeling pad one side and is useful on the application of sample index line that indicates the application of sample position on the NC film simultaneously.After coated the finishing the NC film was placed the drying room drying 3~4 hours, for subsequent use.
(3) assembling of test strips
20~25 ℃ of dry indoor temperatures, humidity is got plastic support board less than 30%, and paste at the middle part that coated NC film is placed on plastic support board, in NC film T line one side overlap joint labeling pad, pastes the loading pad at labeling pad opposite side overlap joint; Inhale the sample pad in NC film C line one side apart from overlapping edges.Then with cutter the plastic plate that posts is cut into the test strips of proper width.The test strips that cuts also can be packed in the plastic clip, forms to detect reagent card.
(4) drafting of instrument memory standard curve
Take National reference as reference, it is 0mIU/ml that the HCG standard solution is diluted to concentration with normal person's urine (feminine gender) or cow's serum, 10mIU/ml, 50mIU/ml, 250mIU/ml, 1000mIU/ml, 2500mIU/ml, 5000mIU/ml, 10000mIU/ml, 15000mIU/ml series standard product, balance is to room temperature, the reagent strip for preparing is kept flat, add 3~5ul standard items to be checked at NC film marking line, then on the loading pad, add 60~80ml sample dilution, put into supporting immunochromatography interpretoscope after static 10 minutes and carry out reading, to record GOD value (X) as horizontal ordinate, as ordinate, draw instrument memory standard curve with HCG content (y).
(5) clinical sample detects
Detect 30 parts of clinical samples according to above-mentioned detection method, take the general HCG quantitative reagent of Austria as contrast, the result shows that the sensitivity of this product reaches 10mIU/ml, specificity 100%, and general HCG quantitative reagent difficult to understand as a result coincidence rate be 98.1%.
Claims (4)
1. the test strips of a Quantitative detection HCG, comprise loading pad (1), labeling pad (2), NC film (3), inhale sample pad (4) and plastic plate (8), it is characterized in that, described test strips and immunochromatography interpretoscope support the use the quantitative detection of carrying out HCG, and it is 10~15000mIU/ml that testing sample is diluted rear quantitative sensing range.
2. test strips according to claim 1, it is characterized in that, described loading pad (1), labeling pad (2), NC film (3) and suction sample pad (4) adhere to plastic plate (8) successively, be coated with the mouse-anti HCG-β monoclonal antibody of colloid gold label on the labeling pad (2), be coated with respectively the detection line T line (6) of mouse-anti HCG-Alpha antibodies formation and the nature controlling line C line (7) that sheep anti-mouse igg antibody consists of on the NC film (3), draw the application of sample index line (5) that is useful on sign application of sample position near labeling pad (2) one sides at the upper detection line T line (6) of NC film (3) simultaneously.
3. reagent strip according to claim 1 is characterized in that, described reagent strip is prepared by following step:
(1) preparation of colloid gold label mouse-anti HCG-β labeling of monoclonal antibody thing pad: prepare the colloidal gold solution that diameter is 40nm with gold chloride-trisodium citrate reduction method, get three parts of colloidal gold solutions after preparation is finished, use respectively 0.2MK
2CO
3PH value of solution is transferred to pH7.5, pH8.5, pH9.5, then solution is placed on the magnetic stirring apparatus and slowly stir, slowly be added drop-wise in the colloidal gold solution by every 100ml solution 0.5mg, 1.0mg, 1.5mg mouse-anti HCG-β monoclonal antibody, continue to stir 1 hour, adding final concentration is 0.2%BSA again, 0.1% PEG 20000 sealed 30 minutes, and 12000 left the heart 30 minutes, abandoned supernatant, redissolve to 76.5ml with the collaurum working fluid, press 1ml solution and spread 16cm
2Ratio be layered on equably on the nonwoven fabrics, place again 20~25 ℃ of temperature, humidity less than 30% drying room drying 2~4 hours, make labeling pad, for subsequent use;
(2) mouse-anti HCG-Alpha antibodies is coated: with 0.01M pH7.2PBS mouse-anti HCG-Alpha antibodies is diluted to respectively 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, then with spray film instrument mouse-anti HCG-Alpha antibodies coating buffer is pressed the 1.3ul/cm line at the NC film and be coated with, consist of detection line T line; Coated sheep anti-mouse igg antibody is as the C line simultaneously, and detection line T line is drawn near labeling pad one side and is useful on the application of sample index line that indicates the application of sample position on the NC film simultaneously, after coated the finishing the NC film is placed the drying room drying 3~4 hours, and is for subsequent use;
(3) assembling of test strips: 20~25 ℃ of temperature, humidity less than 30% hothouse in, get plastic support board, paste at the middle part that coated NC film is placed on plastic support board, in NC film T line one side overlap joint labeling pad, paste the loading pad at labeling pad opposite side overlap joint, inhale the sample pad in NC film C line one side apart from overlapping edges; Then with cutter the plastic plate that posts is cut into the test strips of proper width.
4. a method that quantitatively detects HCG is characterized in that, said method comprising the steps of:
(1) drafting of instrument memory standard curve: take National reference as reference, it is 0mIU/ml that the HCG standard solution is diluted to concentration with normal person's urine (feminine gender) or cow's serum, 10mIU/ml, 50mIU/ml, 250mIU/ml, 1000mIU/ml, 2500mIU/ml, 5000mIU/ml, 10000mIU/ml, 15000mIU/ml series standard product, balance is to room temperature, the reagent strip for preparing is kept flat, add 3~5ul standard items to be checked at NC film marking line, then on the loading pad, add 60~80ml sample dilution, put into supporting immunochromatography interpretoscope after static 10 minutes and carry out reading, to record GOD value (X) as horizontal ordinate, as ordinate, draw instrument memory standard curve with HCG content (y).
(2) method according to step (1) detects testing sample, and obtains the HCG concentration of described testing sample by typical curve.
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CN110456041A (en) * | 2019-08-06 | 2019-11-15 | 珠海市医友生物科技有限公司 | A kind of norovirus GI type GII type combined detection reagent and preparation method thereof |
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