CN101592666A - Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof - Google Patents
Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof.It is in the test card shell according to test strips, case surface has detection window and application of sample window.This test strips is to catch line (7) and nature controlling line (8) by catching line (6) on the nitrocellulose membrane (NC film) (3) of the adsorptive pads of joint successively (2) on the base plate (1), specified packet quilt, the NC film, applies the glass fibre collaurum pad (4), sample pad (5) of colloid gold label specific antibody and the strip formed; It is S adenosyl homocysteine (SAH) monoclonal antibody and folic acid (Folate) monoclonal antibody that described glass fibre collaurum pad (4) applies the colloid gold label specific antibody.Use combined detection card provided by the present invention and detect homocysteine and folate level in the human serum, have characteristics such as simple to operate, quick, sensitivity and specificity are good, have a good application prospect.
Description
Technical field
The present invention relates to a kind of test card of utilizing homocysteine-folate level in the colloidal gold immunochromatographimethod technical tie-up detection blood and preparation method thereof, belong to field of medical examination.
Background technology
(homocysteine is the intermediate product of methionine metabolism in the human body Hcy) to homocysteine, has participated in changeing in the body sulfuration approach simultaneously.Hcy turns to methionine through the folic acid methyl that circulates again by B12 in the presence of the 5-methyl tetrahydrofolate, produce subsequently 5,10-dimethyl tetrahydro folic acid is reduced into the 5-methyl tetrahydrofolate by methyl tetrahydrofolate reductase (MTHFR).Hcy also can be turned to methionine by the heavy methyl of betaine.Heavily methylate and in liver and kidney, finish, in the methionine circulation, methionine is converted into S-adenosylmethionine (SAM) in the food, SAM is with the substrate as transmethylase methyl groups donor, produce unique product S-adenosyl homocysteine (SAH) under the transmethylase effect, SAH is hydrolyzed to Hcy and adenosine through the SAH hydrolytic enzyme.
As causing that because of a variety of causes blood Hcy concentration raises, Hcy urine disease or high Hcy mass formed by blood stasis can take place, and arteries is damaged.Research thinks that Hcy is the independent risk factor of a new vascular system disease, and is relevant with angiocardiopathy, cerebral infarction, peripheral vascular disease, and also the shortage with folic acid or cobalamin, B6 is relevant.Sixties end, systemic atherosclerosis and thrombosis can take place from pathology discovery homocystinuria and cystathioninuria patient in Mc Cully in early days, he can cause similar vascular lesion by accumulating in the animal model confirmation homocysteine blood again early seventies, the eighties people propose the independent hazard factor that hyperhomocysteinemiainjury is atherosclerotic and coronary heart disease, especially the dangerous index of coronary atherosclerosis and miocardial infarction, its concentration rising degree is directly proportional with the danger of disease, is described as " the new killer of heart ".Studies show that much that at present high HCY is cranial vascular disease hazards indexs, is the independent risk factor of ischemic cerebrovascular disease, cerebral infarction, and high Hcy and cerebral apoplexy disease take place closely related.
Folic acid (Folate) is that one group of chemical constitution is similar, the compound general designation that biochemical characteristic is close, be by the pyridine of talking endlessly, p-aminobenzoic acid and one or more glutamic acid be combined intos, in order to the synthetic necessary DNA of human body, shortage can cause megaloblastic anemia with dimension B12 for it; The pregnant preceding or pregnant early stage folic acid that lacks of women is one of important cause of disease of neural tube defects generation; Simultaneously, folic acid and homocysteine metabolism are in close relations, folic acid deficiency can make homocysteine obstacle occur to the methionine conversion, and then cause the homocysteine mass formed by blood stasis, thereby cause the cardiovascular morbidity risk, the generation of angiocardiopathy preventing is also had important biological significance so detect folate level.
Hcy detects the main at present technical method that adopts to be had: high performance liquid chromatogram (HPLC) method, fluorescence polarization immune detection (FPLA) method, Enzyme-multiplied immune technique (ELISA) and chemoluminescence method etc.The detection method of folic acid mainly contains at present: microbial method, isotope radioimmunology also have gas chromatography-mass spectrum detection, red, orange, green, blue, yellow (ROGBY), ion trap method, clone's enzyme donor immunoassay (CEDIA), enzyme connection part adsorption test (ELLSA) and the experiment of chemiluminescence acceptor etc. in addition.High performance liquid chromatogram (HPLC) method operation more complicated is tested consuming time longly, and the test figure variance ratio is bigger.During fluorescence polarization immune detection (FPLA) method check fee, must be equipped with special-purpose full-automatic fluorescence analyzer, apparatus expensive should not be popularized.Enzyme-multiplied immune technique (ELISA) major part is operating as manual operations, and more consuming time, detection by quantitative need be equipped with instrument, and special laboratory must be arranged.Chemoluminescence method, detection time is more than 2 hours, and costs an arm and a leg.Above method all is unsuitable for using in basic unit check place.The immune colloidal gold chromatography technical operation is simple, fast, sensitivity and specificity be good, the fast detecting that is used for homocysteine and folic acid, be of value to universal that homocysteine and folic acid basic medical unit detect, though this technology is a kind of comparatively proven technique, but the application to different antigen-antibody systems is variant, comprises raw material sources, the reagent preparation, production technologies etc. are all different, need carry out new design and test.At present all do not find the immune colloidal gold chromatography quick diagnosis combined detection card that is useful on joint-detection homocysteine-folic acid both at home and abroad, comprise bibliographical information and patent of invention.
Summary of the invention
For overcoming the deficiency of existing homocysteine and folic acid detection technique, the invention provides a kind of combined detection card of fast detecting homocysteine-folic acid.The present invention is according to colloidal gold immunochromatographimethod technical characterstic and antigen-antibody system characteristics, design new starting material, reagent and technological process, use combined detection card provided by the invention and detect homocysteine-folate level, have simple, quick, characteristics such as sensitive and specificity is good, went out the result with interior in 15 minutes, and price is low, is applicable to that basic unit detects and clinical primary dcreening operation.
The strip that combined detection card of the present invention is made up of the nitrocellulose membrane (3) that engages adsorptive pads (2), specified packet quilt on the base plate (1) successively, the glass fibre collaurum pad (4) that applies the colloid gold label specific antibody, sample pad (5); It is S adenosyl homocysteine monoclonal antibody and folic acid monoclonal antibody that described glass fibre collaurum pad applies the colloid gold label specific antibody.Test sample process sample pad and pad act on through chromatography and occur catching line and nature controlling line, with the naked eye judged result on the NC film.Only occur a nature controlling line on the NC film, the result is positive, occurs simultaneously catching line and nature controlling line on the NC film, and the result is negative, lines do not occur and represents that test strips or test card lost efficacy.Go out the result in 15 minutes.
Combined detection card adsorptive pads of the present invention is a kind of filter paper, comprises thieving paper and filter paper for oil, is cut into to require size directly to use.Be attached to the end of base plate, play water sorption.
The NC film of specified packet quilt is a kind of material that is specifically designed to colloidal gold strip on the combined detection card of the present invention, form by one deck filter membrane and glued membrane, the rolling wound packages, 3 lines of spray on the NC film, be respectively 2 and catch line and 1 nature controlling line, what catch line (6) spraying is S adenosyl homocysteine-bovine serum albumin(BSA) antigen, and what catch line (7) spraying is folic acid-bovine serum albumin(BSA) antigen, and what nature controlling line (8) sprayed is that the anti-mouse two of rabbit resists; The NC film is attached to the centre of base plate, and two ends are connected with adsorptive pads with pad respectively.
Pad is an all-glass paper on the combined detection card of the present invention, after the borate buffer immersion treatment that contains polyvinyl alcohol (PVA), 37 ℃ of dried overnight, the SAH monoclonal antibody of colloid gold label and folic acid monoclonal antibody in the spraying, freeze drying again, be connected with sample pad on the pad, be connected with the NC film down.
Combined detection card sample pad of the present invention is an all-glass paper, and with the borate buffer immersion treatment that contains polyvinyl alcohol (PVA), buffer solution ph is 7.2, after the immersion treatment, 37 ℃ of dried overnight play filtered sample in reagent strip, be positioned on the pad, be attached on the base plate.
The present invention also provides above-mentioned joint-detection to be stuck in application in preparation homocysteine-folic acid diagnosis test paper, diagnosis test paper also comprises the sign bar, this sign bar is two self-adhesive papers that have sign, stick on the two ends of test strips respectively, having the arrow end is to be attached on sample pad and the pad, end immerses testing sample thus, leans on the water sorption of the adsorptive pads of the other end, makes sample through the NC film.
The present invention also provides above-mentioned joint-detection to be stuck in application in preparation homocysteine-folic acid test card, test card also comprises plastic clip, this plastic clip be one with the special card of plastics, by backboard, cover plate is formed, backboard and cover plate can be entrenched togather, backboard mainly contains one and puts the groove of test strips and the latch that combines with cover plate, cover plate mainly comprises a detection window, an application of sample window and the latch that combines with backboard, the detection window next door is printed on T1 respectively, T2 and C printed words, T1 represents to catch the position of line (6), and T2 represents to catch the position of line (7), and C represents the position of nature controlling line (8), detection window is the window of observations, and the application of sample window is the position that drips sample.This plastic clip plays the protection test strips, and more attractive in appearance, reduces the safety of polluting and protecting the operator.
The preparation method of test strips of the present invention comprises the steps:
1) preparation of SAH-BSA antigen, Folate-BSA antigen:
A) with BSA, SAH and coupling agent room temperature reaction in reactant liquor, gel chromatography separation coupling thing, dialysis concentrates, and makes immunizing antigen and envelope antigen;
B) with BSA, Folate and coupling agent room temperature reaction in reactant liquor, gel chromatography separation coupling thing, dialysis concentrates, and makes immunizing antigen and envelope antigen;
2) preparation of SAH monoclonal antibody, Folate monoclonal antibody:
A) with synthetic SAH-BSA conjugate immunity Balb/c small white mouse, behind repeatedly fundamental immunity and booster immunization, get mouse boosting cell and Sp2/0 myeloma cell, utilize the PEG cell-fusion techniques to prepare SAHmAb, ascites increases, separation and purification;
B) with synthetic Folate-BSA conjugate immunity Balb/c small white mouse, behind repeatedly fundamental immunity and booster immunization, get mouse boosting cell and Sp2/0 myeloma cell, utilize the PEG cell-fusion techniques to prepare FolatemAb, ascites increases, separation and purification;
3) preparation of collaurum:
A) measure a certain amount of washing lotion in the flask of cleaning, boiled 5 minutes, change pickle again one time, boiled 5 minutes;
B) measure a certain amount of ultrapure water in this flask, boiled 5 minutes, change ultrapure water again one time, boiled 5 minutes;
C) discard ultrapure water, take by weighing an amount of tri-distilled water in this flask, be heated to boiling, add an amount of 1% gold chloride, be heated to boiling;
D) stir 6～10 milliliter of 2% trisodium citrate of adding down, seethed with excitement 5-10 minute;
4) colloidal gold probe mark: separately SAH monoclonal antibody, Folate monoclonal are used the NaCl dialysed overnight respectively, the centrifugal albumen precipitation of removing; Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir SAH monoclonal antibody, the Folate monoclonal antibody that adds 1 milliliter of dilution separately, slowly add 1 milliliter of abundant mixing of 10%BSA more respectively, stir, the centrifugal supernatant of abandoning adds the working fluid dissolving;
5) preparation detects the colloid gold immune test paper bar of homocysteine: earlier glass fibre membrane is put into mass percent concentration and be 1% bovine serum albumin(BSA), mass percent concentration is to seal 30min in the phosphate buffer of 1% Triton X-100,37 ℃ of oven dry, again glass fibre membrane is soaked in mark SAH monoclonal antibody respectively, in the colloidal gold probe of Folate monoclonal antibody, vacuum drying, NC film on the reagent batten for preparing is sprayed capture antigen (SAH-BSA coupled antigen) respectively with Membrane jetter, capture antigen (Folate-BSA conjugate) and Quality Control antibody (the anti-Balb/c globulin antibody of rabbit) have sprayed back 37 ℃ of dried overnight; Nitrocellulose membrane, glass fibre collaurum pad, the sample pad of wrapping quilt sticks on the base plate successively with adsorptive pads,, is cut into slice, a kind of colloid gold immune test paper bar that detects homocysteine.
In the above-mentioned Preparation of Colloidal Gold process, 200 milliliters of preferred tri-distilled waters, 10 milliliters of gold chlorides, 7.5 milliliters of trisodium citrates.
In the above-mentioned colloidal gold probe labeling process, preferably add 10%BSA after, add 1 milliliter of abundant mixing of 10%PEG20000 again, the centrifugal supernatant of abandoning adds the working fluid dissolving.
Beneficial effect of the present invention: use the combined detection card provided by the invention interior homocysteine-folate level of human body simultaneously, with low cost, simple to operate, quick, sensitive, and specificity is good, do not need the special detection instrument and equipment, therefore can be widely used in medical inspections at different levels place, especially basic medical unit, comprise that health clinics in towns and townships etc. all can carry out.The present invention is of value to popularizing of homocysteine and folic acid detection, and then the prevention that takes place for the cardiovascular and cerebrovascular incident has very important meaning.
Description of drawings
Fig. 1 is a structural representation of the present invention
1: base plate; 2: adsorptive pads; 3: the nitrocellulose membrane of specified packet quilt; 4: pad; 5: sample pad; 6: catch line; 7: catch line; 8: nature controlling line.
Fig. 2 is a testing result synoptic diagram of the present invention
A:Hcy-Folate is negative simultaneously; The b:Hcy feminine gender, the Folate positive; The c:Hcy positive, the Folate feminine gender; D:Hcy-Folate is positive simultaneously; E: invalid; 6:Hcy catches line; 7:Folate catches line; 8: nature controlling line.
Fig. 3 is a test strips schematic appearance of the present invention
A: sample pad; B: pad; 6:Hcy catches line; 7:Folate catches line; 8: nature controlling line; C: test strips title
Fig. 4 is a test card schematic appearance of the present invention
A: application of sample window; B: detection window; The c:ID tag slot; T1:Hcy catches line; T2:Folate catches line; C: nature controlling line.
Embodiment
The invention will be further described below in conjunction with embodiment, and all this areas of having done according to the disclosure of invention are equal to replacement, all belong to protection scope of the present invention.
Embodiment 1:
With homocysteine-folic acid colloid gold immune combined detection test paper preparation method is example, sets forth the preparation method of colloid gold immune test paper bar, and it comprises the steps:
1, the preparation of SAH-BSA antigen, Folate-BSA antigen:
A) get an amount of BSA and SAH and be dissolved in respectively in the 15mlPBS conical flask, magnetic stirrer is 10 minutes separately, and SAH solution is added in the BSA solution, the limit edged stirs, and adds 25% glutaraldehyde again, reacts 5 hours, gel chromatography separation coupling thing, the 24h that dialyses in PBS concentrates.
B) get an amount of BSA and Folate and be dissolved in respectively in the 15mlPBS conical flask, magnetic stirrer is 10 minutes separately, and Folate solution is added in the BSA solution, the limit edged stirs, and adds 25% glutaraldehyde again, reacts 5 hours, gel chromatography separation coupling thing, the 24h that dialyses in PBS concentrates.
2, SAH monoclonal antibody preparation:
A) with SAH-BSA conjugate immunity Balb/c mouse, behind repeatedly fundamental immunity and booster immunization, get mouse boosting cell, with splenocyte and Sp2/0 myeloma cell, utilize the PEG cell-fusion techniques to prepare SAH mAb.Hybridoma is injected mouse peritoneal, and preparation ascites is used the purification by chromatography monoclonal antibody.
B) with Folate-BSA conjugate immunity Balb/c mouse, behind repeatedly fundamental immunity and booster immunization, get mouse boosting cell, with splenocyte and Sp2/0 myeloma cell, utilize the PEG cell-fusion techniques to prepare Folate mAb.Hybridoma is injected mouse peritoneal, and preparation ascites is used the purification by chromatography monoclonal antibody.
3, the preparation of collaurum:
Measure a certain amount of washing lotion in the 250ml of cleaning Erlenmeyer flask, boiled 5 minutes, change pickle again one time, boiled 5 minutes; Measure a certain amount of ultrapure water in this flask, boiled 5 minutes, change ultrapure water again one time, boiled 5 minutes; Discard ultrapure water, take by weighing the 200ml ultrapure water in this flask, be heated to boiling, add 1% gold chloride 10ml, stir, be heated to boiling while boiling; Stir adding 2% trisodium citrate 10ml down, boiling water stirs down fast, and is slowly stable up to the color of chlorauric acid solution, and after taking on a red color, the granule collaurum is made in continuation boiling 7 minutes.
4, colloidal gold probe mark:
A) with SAH monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the SAH monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
B) with Folate monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the Folate monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
5, preparation detects the colloid gold immune combined detection test paper of homocysteine-folic acid:
The phosphate buffer of earlier glass fibre membrane being put into mass percent concentration and be 1% bovine serum albumin(BSA), mass percent concentration and be 1% Triton X-100 seals 30min, 37 ℃ of oven dry, again glass fibre membrane is soaked in the colloidal gold probe of mark SAH monoclonal antibody and Folate monoclonal antibody vacuum drying.NC film on the reagent batten for preparing is sprayed capture antigen (SAH-BSA coupled antigen), capture antigen (Folate-BSA coupled antigen) and Quality Control antibody (the anti-Balb/c globulin antibody of rabbit) respectively with Membrane jetter.Back 37 ℃ of dried overnight have been sprayed.Nitrocellulose membrane, glass fibre collaurum pad, the sample pad of wrapping quilt sticks on the base plate successively with adsorptive pads,, is cut into slice, a kind of colloid gold immune combined detection test paper that detects homocysteine-folic acid.
Embodiment 2:
Homocysteine-folic acid colloid gold immune combined detection test paper preparation method, it comprises the steps:
SAH-BSA antigen, Folate-BSA antigen, SAH monoclonal antibody and Folate monoclonal antibody are embodiment 1 preparation.
1, the preparation of collaurum:
Measure a certain amount of washing lotion in the 250ml of cleaning Erlenmeyer flask, boiled 5 minutes, change pickle again one time, boiled 5 minutes; Measure a certain amount of ultrapure water in this flask, boiled 5 minutes, change ultrapure water again one time, boiled 5 minutes; Discard ultrapure water, take by weighing the 200ml ultrapure water in this flask, be heated to boiling, add 1% gold chloride 10ml, stir, be heated to boiling while boiling; Stir adding 2% trisodium citrate 7.5ml down, boiling water stirs down fast, and is slowly stable up to the color of chlorauric acid solution, and after taking on a red color, medium sized colloid gold particle is made in continuation boiling 9 minutes.
2, colloidal gold probe mark:
A) with SAH monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the SAH monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, add 1 milliliter of abundant mixing of 10%PEG20000 again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
B) with Folate monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the Folate monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, add 1 milliliter of abundant mixing of 10%PEG20000 again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
3, preparation detects the colloid gold immune combined detection test paper of homocysteine-folic acid:
The phosphate buffer of earlier glass fibre membrane being put into mass percent concentration and be 1% bovine serum albumin(BSA), mass percent concentration and be 0.5% Triton X-100 seals 30min, 37 ℃ of oven dry, again glass fibre membrane is soaked in the colloidal gold probe of mark SAH monoclonal antibody and Folate monoclonal antibody vacuum drying.NC film on the reagent batten for preparing is sprayed capture antigen (SAH-BSA coupled antigen), capture antigen (Folate-BSA coupled antigen) and Quality Control antibody (the anti-Balb/c globulin antibody of rabbit) respectively with Membrane jetter.Back 37 ℃ of dried overnight have been sprayed.Nitrocellulose membrane, glass fibre collaurum pad, the sample pad of wrapping quilt sticks on the base plate successively with adsorptive pads,, is cut into slice, a kind of colloid gold immune combined detection test paper that detects homocysteine-folic acid.
Embodiment 3:
Homocysteine-folic acid colloid gold immune combined detection test paper preparation method, it comprises the steps:
SAH-BSA antigen, Folate-BSA, SAH monoclonal antibody and Folate monoclonal antibody are embodiment 1 preparation.
1, the preparation of collaurum:
Measure a certain amount of washing lotion in the 250ml of cleaning Erlenmeyer flask, boiled 5 minutes, change pickle again one time, boiled 5 minutes; Measure a certain amount of ultrapure water in this flask, boiled 5 minutes, change ultrapure water again one time, boiled 5 minutes; Discard ultrapure water, take by weighing the 200ml ultrapure water in this flask, be heated to boiling, add 1% gold chloride 10ml, stir, be heated to boiling while boiling; Stir adding 2% trisodium citrate 6.6ml down, boiling water stirs down fast, and is slowly stable up to the color of chlorauric acid solution, and after taking on a red color, bigger colloid gold particle is made in continuation boiling 9 minutes.
2, colloidal gold probe mark:
A) with SAH monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the SAH monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, add 1 milliliter of abundant mixing of 10%Casein again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
B) with Folate monoclonal anti body and function 0.005mol/L NaCl dialysed overnight, the centrifugal albumen precipitation of removing.Get 100 milliliters of collaurums, regulate colloidal gold solution pH value 9.0, stir the Folate monoclonal antibody that adds 1 milliliter of dilution, add 1 milliliter of abundant mixing of 10%BSA again, add 1 milliliter of abundant mixing of 10%Casein again, slowly add, stirred 15 minutes, centrifugal, 4 ℃ of 13000g/min of condition, 40 minutes; Abandon supernatant, add 100 milliliters of dissolvings of working fluid; Condition of storage: 4 ℃ of preservations.
3, preparation detects the colloid gold immune combined detection test paper of homocysteine-folic acid:
The phosphate buffer of earlier glass fibre membrane being put into mass percent concentration and be 1% bovine serum albumin(BSA), mass percent concentration and be 0.5% Triton X-100 seals 30min, 37 ℃ of oven dry, again glass fibre membrane is soaked in the colloidal gold probe of mark SAH monoclonal antibody and Folate monoclonal antibody vacuum drying.NC film on the reagent batten for preparing is sprayed capture antigen (SAH-BSA coupled antigen), capture antigen (Folate-BSA coupled antigen) and Quality Control antibody (the anti-Balb/c globulin antibody of rabbit) respectively with Membrane jetter.Back 37 ℃ of dried overnight have been sprayed.Nitrocellulose membrane, glass fibre collaurum pad, the sample pad of wrapping quilt sticks on the base plate successively with adsorptive pads,, is cut into slice, a kind of colloid gold immune combined detection test paper that detects homocysteine-folic acid.
Embodiment 4:
The colloid gold immune diagnosis combined detection test paper preparation method of homocysteine-folic acid, it comprises the steps:
Two self-adhesive papers (sign bar) that have a sign are sticked on the two ends of prepared test strips among the embodiment 2 respectively, having the arrow end is to be attached on sample pad and the pad, end immerses testing sample thus, leans on the water sorption of the adsorptive pads of the other end, makes sample through the NC film.A kind of colloid gold immune diagnosis combined detection test paper that detects homocysteine-folic acid, see Fig. 3.
Embodiment 5:
The colloid gold immune diagnosis combined detection card preparation method of homocysteine-folic acid, it comprises the steps:
Prepared test strips among the embodiment 2 is put into plastic clip backboard test strips groove, with last slice chimeric, a kind of colloid gold immune diagnosis combined detection card that detects homocysteine-folic acid, see Fig. 4.This plastic clip be one with the special card of plastics, form by backboard, cover plate, backboard and cover plate sheet can be entrenched togather, backboard mainly contains one and puts the groove of test strips and the latch that combines with cover plate, cover plate comprises that mainly a detection window (seeing Fig. 4 b), application of sample window (see that Fig. 4 a) and the latch that combines with backboard, the detection window next door is printed on T1, T2 and C printed words respectively, T1 represents to catch the position of line (6), T2 represents to catch the position of line (7), C represents the position of nature controlling line (8), detection window is the window of observations, and the application of sample window is the position that drips sample.
Embodiment 6:
Homocysteine-folic acid Immune colloidal gold by chromatography combined detection test paper using method:
As Fig. 1, shown in 2, test strips sample pad 5 is inserted in the testing sample, to soak into the back and take out, horizontal positioned after about 10～15 minutes, occurs catching line 6, catching line 7 and nature controlling line 8 on NC film 3 simultaneously, sees Fig. 2 a, and the result is that Hcy-Folate is negative simultaneously; Occur simultaneously catching line 6 and nature controlling line 8 on NC film 3, see Fig. 2 b, the result is the Hcy feminine gender Folate positive; Occur simultaneously catching line 7 and nature controlling line 8 on NC film 3, see Fig. 2 c, the result is Hcy positive Folate feminine gender; A nature controlling line 8 only occurs on NC film 3, see Fig. 2 d, the result is that Hcy-Folate is positive simultaneously; Lines do not occur on NC film 3, see Fig. 2 e, the expression test strips lost efficacy.The test strips detection sensitivity is 1 μ mol/l.
Embodiment 7:
Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card using method:
As shown in Figure 4, sample is added application of sample window a, application of sample amount 75 microlitres～80 microlitres, horizontal positioned after about 10～15 minutes, occurs catching line T1 simultaneously, catches line T2 and nature controlling line C in detection window b, and the result is that Hcy-Folate is negative simultaneously; Occur catching line T1 and nature controlling line C in detection window b simultaneously, the result is the Hcy feminine gender Folate positive; Occur catching line T2 and nature controlling line C in detection window b simultaneously, the result is Hcy positive Folate feminine gender; Only occur a nature controlling line C in detection window b, the result is that Hcy-Folate is positive simultaneously; Do not occur lines in detection window b, the expression test card lost efficacy.The test card detection sensitivity is 1 μ mol/l.
Claims (10)
1, a kind of homocysteine-folic acid Immune colloidal gold by chromatography combined detection card, in the test card shell of rectangular flat shell shape, test strips is arranged, the test card case surface has detection window and application of sample window, it is characterized in that: described test strips is by catching line (6) on the nitrocellulose membrane (3) of the adsorptive pads of joint successively (2) on the base plate (1), specified packet quilt, the nitrocellulose membrane, catch line (7) and nature controlling line (8), applies the glass fibre collaurum pad (4), sample pad (5) of colloid gold label specific antibody and the strip formed; It is S adenosyl homocysteine monoclonal antibody and folic acid monoclonal antibody that described glass fibre collaurum pad (4) applies the colloid gold label specific antibody.
2, according to the described combined detection card of claim 1, it is characterized in that: described adsorptive pads (2) is a kind of filter paper, comprises thieving paper and filter paper for oil; Adsorptive pads is attached to the end of base plate.
3, according to the described combined detection card of claim 1, it is characterized in that: the nitrocellulose membrane of described specified packet quilt (3) is made up of one deck filter membrane and glued membrane, and line (6) is caught in spraying on the NC film, and---the anti-mouse of S adenosyl homocysteine-bovine serum albumin(BSA) antigen,---folic acid-bovine serum albumin(BSA) and nature controlling line (the 8)---rabbit that catches line (7) two is anti-; Nitrocellulose membrane is attached to the centre of base plate, and two ends are connected with adsorptive pads with pad respectively.
4, according to the described combined detection card of claim 1, it is characterized in that: described pad (4) is an all-glass paper, after the borate buffer immersion treatment that contains polyvinyl alcohol (PVA), 37 ℃ of dried overnight, the SAH monoclonal antibody of colloid gold label and folic acid monoclonal antibody in the spraying, freeze drying again is connected with sample pad on the pad, is connected with nitrocellulose membrane down.
5, according to the described combined detection card of claim 1, it is characterized in that: described sample pad (5) is all-glass paper, soaks with the borate buffer that contains polyvinyl alcohol (PVA), and buffer solution ph is 7.2, after the immersion treatment, and 37 ℃ of dried overnight; Sample pad is positioned on the pad.
6, joint-detection as claimed in claim 1 is stuck in the purposes in preparation homocysteine-folic acid associating diagnosis test paper, it is characterized in that: diagnosis test paper also comprises the sign bar, this sign bar is two self-adhesive papers that have sign, stick on the two ends of test strips respectively, having the arrow end is to be attached on sample pad and the pad, end immerses testing sample thus, leans on the water sorption of the adsorptive pads of the other end, makes sample through nitrocellulose membrane.
7, purposes as claimed in claim 6, it is characterized in that: test card also comprises plastic clip, this plastic clip be one with the special card of plastics, by backboard, cover plate is formed, backboard and cover plate can be entrenched togather, backboard mainly contains one and puts the groove of test strips and the latch that combines with cover plate, cover plate mainly comprises a detection window, an application of sample window and the latch that combines with backboard, the detection window next door is printed on T1 respectively, T2 and C printed words, T1 represents to catch the position of line (6), and T2 represents to catch the position of line (7), and C represents the position of nature controlling line (8), detection window is the window of observations, and the application of sample window is the position that drips sample.The nitrocellulose membrane of specified packet quilt (NC film) (3) is over against detection window, and sample pad (5) is over against the application of sample window.
8, a kind of preparation method who detects the homocysteine-folic acid Immune colloidal gold by chromatography combined detection card comprises the steps:
1) preparation of S adenosyl homocysteine-bovine serum albumin(BSA) antigen, folic acid-bovine serum albumin(BSA) antigen;
2) preparation of S adenosyl homocysteine monoclonal antibody, folic acid monoclonal antibody;
3) preparation of collaurum;
4) colloidal gold probe mark;
5) preparation detects the colloid gold immune test paper bar of homocysteine.
9, described according to Claim 8 preparation method is characterized in that: 3) tri-distilled water described in the step is 200 milliliters, and described gold chloride is 10 milliliters, and described trisodium citrate is 7.5 milliliters.
10, described according to Claim 8 preparation method is characterized in that: after 4) adding 10%BSA described in the step, add 1 milliliter of abundant mixing of 10%PEG20000 again, the centrifugal supernatant of abandoning adds the working fluid dissolving.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101142095A CN101592666A (en) | 2008-05-30 | 2008-05-30 | Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101142095A CN101592666A (en) | 2008-05-30 | 2008-05-30 | Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof |
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| CN101592666A true CN101592666A (en) | 2009-12-02 |
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| CNA2008101142095A Pending CN101592666A (en) | 2008-05-30 | 2008-05-30 | Homocysteine-folic acid Immune colloidal gold by chromatography combined detection card and preparation method thereof |
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| CN102175878A (en) * | 2011-01-11 | 2011-09-07 | 南开大学 | ELISA (enzyme linked immunosorbent assay) kit of folic acid |
| WO2013071703A1 (en) * | 2011-11-17 | 2013-05-23 | 成都创宜生物科技有限公司 | Means for rapidly detecting adipsin for diagnosing preeclampsia, test kit and preparation method thereof |
| CN103134935A (en) * | 2013-02-28 | 2013-06-05 | 成都创宜生物科技有限公司 | Making method of immunochromatographic test paper for detecting premature rupture of fetal membranes |
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| CN103575921A (en) * | 2013-11-19 | 2014-02-12 | 宁波美康生物科技股份有限公司 | Homocysteine quality control product and preparation method thereof |
| CN110346575A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
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| CN102175878A (en) * | 2011-01-11 | 2011-09-07 | 南开大学 | ELISA (enzyme linked immunosorbent assay) kit of folic acid |
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| CN103134935B (en) * | 2013-02-28 | 2015-09-30 | 成都创宜生物科技有限公司 | A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper |
| CN103197068B (en) * | 2013-02-28 | 2015-10-14 | 成都创宜生物科技有限公司 | A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper |
| CN103575921A (en) * | 2013-11-19 | 2014-02-12 | 宁波美康生物科技股份有限公司 | Homocysteine quality control product and preparation method thereof |
| CN103575921B (en) * | 2013-11-19 | 2015-10-28 | 宁波美康生物科技股份有限公司 | Homocysteine quality control product and preparation method thereof |
| CN110346575A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
| CN113848330A (en) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
| CN113848330B (en) * | 2021-11-30 | 2022-03-15 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
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