CN1233828C - Antihuman hemoglobin detection reagent strips and monoclone antibody contained therewith - Google Patents
Antihuman hemoglobin detection reagent strips and monoclone antibody contained therewith Download PDFInfo
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- CN1233828C CN1233828C CN 03153440 CN03153440A CN1233828C CN 1233828 C CN1233828 C CN 1233828C CN 03153440 CN03153440 CN 03153440 CN 03153440 A CN03153440 A CN 03153440A CN 1233828 C CN1233828 C CN 1233828C
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- colloidal gold
- monoclonal antibody
- antihuman hemoglobin
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Abstract
The present invention discloses a hybridoma cell strain B9 with the preservation number of CGMCC 0970, a monoclonal antibody which is generated by the hybridoma cell strain B9 with the preservation number of CGMCC 0970 and resists human haemoglobin, a colloidal gold immunochromatographic detection reagent strip containing the monoclonal antibody which resists human haemoglobin, and a preparation method of the reagent strip. The sensitivity of the monoclonal antibody which is generated by the hybridoma cell strain B9 with the preservation number of CGMCC 0970 and resists human haemoglobin, and the reagent strip of the monoclonal antibody is equivalent to that of foreign similar products of top levels, but the specificity is better than that of foreign products. The hybridoma cell strain B9 completely accords with the standards and the requirements of medicolegal investigation, is favorable to the rapid and accurate inspection and identification of cases and material evidences, and rapid case solving, and benefits both the country and people.
Description
Technical field
The invention belongs to medical jurisprudence and immunological test technical field, can be directly used in the conclusive evidence of forensic to human blood, more specifically, the present invention relates to antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar, the antihuman hemoglobin monoclonal antibody that it contains and produce the hybridoma cell strain of this monoclonal antibody the present invention also relates to the preparation method of detection reagent bar.
Background technology
In public security legal medical expert work, the check as early as possible and the evaluation of case material evidence are for the timely detection of case, arrest the offender or discharge the suspect and have conclusive effect.Before the colloidal gold immunochromatographiassay assay reagent bar occurs, similar immunological test technology adopts immunoelectrophoresis, immunodiffusion(ID), Enzyme Linked Immunoadsorbent Assay methods such as (being ELISA) to carry out, these methods or experimental procedure are more relatively, complicated operation, to reviewer's technical requirements degree height, or check the used time longer, and all must carry out indoor.Therefore, it is necessary setting up that a novel method improves, overcome above-mentioned these problems and shortcoming.The development of colloidal gold immunochromatographiassay assay reagent bar and use make the case material evidence just can check and make evaluation at the scene even can determine the suspect thus, thereby can solve a case fast.But the colloidal gold immunochromatographiassay assay reagent bar of some detection human hemoglobin at present both domestic and external, because the antihuman hemoglobin antibodies specific does not reach public security or judicial inspecting standard, the situation of the check mistake that occurs now and then is as having identified the blood of animal into human blood.And well-known, public security or judicial check are not allow to occur false retrieval, because this is concerning someone's future or destiny.Therefore development only to human hemoglobin react, the antihuman hemoglobin monoclonal antibody and the reagent strip of coincidence method medical test standard and requirement fully, very urgent.
Summary of the invention
The purpose of this invention is to provide a kind of height sensitivity, special antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar, the present invention also provides the preparation method of this reagent strip.
Another object of the present invention provides a kind of antihuman hemoglobin monoclonal antibody to the human hemoglobin high special.
The present invention also provides a kind of hybridoma cell strain that produces above-mentioned antihuman hemoglobin monoclonal antibody.
For reaching above-mentioned purpose, technical scheme of the present invention is as follows:
The hybridoma cell strain B9 that the present invention uses is on July 1st, 2003, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is: CGMCC 0970.
The invention provides the monoclonal antibody of antihuman hemoglobin, can be with routine techniques from having the hybridoma cell strain B9 generation that preserving number is CGMCC 0970.
The invention provides antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar, this reagent strip comprises the antihuman hemoglobin monoclonal antibody that CGMCC 0970 hybridoma cell strain B9 of the present invention produces.
The invention provides antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar, this reagent strip comprises successively:
Sample application zone, the of the present invention anti-human blood red eggs that it comprises blank inert material district and comprises attached Radioactive colloidal gold
White monoclonal anti tagma;
Reaction zone, it comprises detection line and nature controlling line;
Adsorption zone;
Detection reagent bar of the present invention is characterized in that: blank inert material district one end of sample application zone overlaps with antihuman hemoglobin monoclonal antibody of the present invention district one end that comprises attached Radioactive colloidal gold.
Detection reagent bar of the present invention is characterized in that: an end of sample application zone and an end of reaction zone overlap, and an end of the other end of reaction zone and adsorption zone overlaps.
Detection reagent bar of the present invention is characterized in that: sample application zone, reaction zone and adsorption zone are fixed on the single face glue plastic mattress flaggy.
Detection reagent bar of the present invention, it is characterized in that: be stained with the single face adhesive tape layer on an end parts of reaction zone and the sample application zone, the other end of reaction zone partly with on the adsorption zone is stained with the single face adhesive tape layer, and liquid level can reach the MAX line of maximum height when wherein the single face adhesive tape layer that is stained with on an end parts of reaction zone and the sample application zone was marked with detection.
Detection reagent bar of the present invention, it is characterized in that: the single face adhesive tape layer that is stained with on an end parts of reaction zone and the sample application zone inflection of can postponing coats an end of sample application zone, and be adhered fixed the back side at single face glue plastic mattress flaggy, the single face adhesive tape layer that is stained with on the other end of reaction zone part and the adsorption zone inflection of can postponing coats the adsorption zone the other end, and is adhered fixed the back side at single face glue plastic mattress flaggy.
Detection reagent bar of the present invention is characterized in that: the antihuman hemoglobin monoclonal antibody of the present invention of the attached Radioactive colloidal gold of sample application zone bag is carried in the glass fibre, and the blank inert material district of sample application zone is a glass fibre.
Detection reagent bar of the present invention is characterized in that: detection line and nature controlling line are carried on the nitrocellulose filter.
Detection reagent bar of the present invention, it is characterized in that: the detection line of reaction zone is the many or monoclonal antibody of another antihuman hemoglobin, its with the antibody of the antihuman hemoglobin of the present invention of the attached Radioactive colloidal gold of bag at be identical antigen, but its antigen binding site or claim antigenic determinant inequality or identical.
Detection reagent bar of the present invention is characterized in that: what the detection line of reaction zone sprayed is rabbit or the goat-anti human hemoglobin polyclonal antibody that contains 0.2-1% sucrose.
Detection reagent bar of the present invention is characterized in that: the nature controlling line of reaction zone is the material that plays specific reaction with the antihuman hemoglobin monoclonal antibody of the present invention of the attached Radioactive colloidal gold of bag.
Detection reagent bar of the present invention is characterized in that: what the nature controlling line of reaction zone sprayed is the sheep anti-mouse igg antibody that contains 0.2-1% sucrose.
Detection reagent bar of the present invention is characterized in that: adsorption zone is a filter paper, and what the present invention was used is the thick filter paper of specific standard.
The method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention may further comprise the steps:
1) human hemoglobin MONOCLONAL ANTIBODIES SPECIFIC FOR: with preserving number is that the hybridoma cell strain B9 of CGMCC 0970 recovers according to a conventional method and cultivates, and injects in the mouse peritoneal, puts to death mouse, collects ascites;
2) separation and purification antihuman hemoglobin monoclonal antibody of the present invention: add ammonium sulfate method or affinity chromatography etc. with conventional n-caprylic acid and carry out;
3) preparation of Radioactive colloidal gold: with hydrochloro-auric acid (HAuCl
4) make colloidal gold solution with the reductive agent heating;
4) monoclonal antibody that makes Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances preparation: with step 2) stirs down and adds in the colloidal gold solution, and the antibody bag is attached on the colloid gold particle surface;
5) the raw-material pre-treatment of reagent strip: material glass fiber directly related with testing process and nitrocellulose filter are carried out pre-treatment;
6) glass fibre is handled and be written into to Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances: the Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances that makes with the diluted step 4), with the saturated immersion glass fibre of this binding substances after the dilution, dry then then;
7) spraying detection line and nature controlling line on the reaction zone nitrocellulose filter of handling well;
8) assembling reagent strip: on single face glue plastics flaggy, paste the glass fibre that Radioactive colloidal gold-the antihuman hemoglobin monoclonal antibody binding substances is saturated that blank glass pars fibrosa that the step 5) be fixed with sample application zone handled and step 6) make, the nitrocellulose filter of reaction zone and the filter paper of uptake zone; Glass fibre district one end that blank glass pars fibrosa one end of sample application zone and Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances that step 6) makes are saturated overlaps; One end of glass fibre one end of sample application zone and the nitrocellulose filter of reaction zone overlaps, and an end of the other end of the nitrocellulose filter of reaction zone and uptake zone filter paper overlaps; Wherein be stained with the single face adhesive tape layer on the glass fibre of an end parts of the nitrocellulose filter of reaction zone and sample application zone, the other end of the nitrocellulose filter of reaction zone partly with on the filter paper is stained with the single face adhesive tape layer;
9) 8) made mother matrix reagent strip is cut into the finished product reagent strip;
10) performance of test agent bar determines whether it is qualified.
A feature for preparing the method for antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is: after wherein step 4) stirs; identify its stable case with 10%NaCI; add BSA and PEG solution; 12; centrifugal 50 minutes of 000r/min; precipitation is to protect liquid: 1.0mmol/L sodium tetraborate-HCI, 0.1%BSA, 0.01%NaN
3, pH7.4 is resuspended, and it is standby to put 4 ℃ of preservations.
A feature for preparing the method for antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is: wherein step 5) is at 4 ℃, with the 50mM Tris-HCI that contains 0.5% tween 80, the pH8.0 damping fluid soaks glass fibre and spends the night, desalt with aseptic ultrapure water rinsing, dry up under the aseptic high efficiency filter; Soaked nitrocellulose filter 1 to 2 hour with the 20mM phosphoric acid salt normal saline buffer solution (pH7.4) that contains 0.05% polysorbas20, desalt with aseptic ultrapure water rinsing again, also existing with now steeping and dry up, and all damping fluids are all with 0.45 μ m filter filtration sterilization.
A feature for preparing the method for antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is: wherein the diluent in the step 6) is: 0.2～1.0% N of IgG, 0.1～0.5%PVP40 or 30,0.05～0.1%PEG20,000,0.5～2.0%BSA, 0.02%NaN
3Physiology PBS damping fluid (pH7.4).
The method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is characterized in that: what the detection line of reaction zone sprayed is rabbit or the goat-anti human hemoglobin polyclonal antibody that contains 0.2-1% sucrose.
The method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is characterized in that: what the nature controlling line of reaction zone sprayed is the sheep anti-mouse igg antibody that contains 0.2-1% sucrose.
Preserving number of the present invention is that the sensitivity of the antihuman hemoglobin monoclonal antibody that produces of the hybridoma cell strain B9 of CGMCC 0970 and the reagent strip made is suitable with the like product of external highest level; But specificity is better than external product, and coincidence method medical test standard and requirement fully helps rapidly, and the case material evidence is identified in check exactly, solves a case fast, benefits the nation and the people.
In order further to understand essence of the present invention, the present invention will be further described below in conjunction with the drawings and the specific embodiments.
Description of drawings
Fig. 1 is bar sensitivity of antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent and the specific test result that the embodiment of the invention 2 makes.
Fig. 2 is that the antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar that makes of the embodiment of the invention 2 is to the specific test result of monkey blood.
Fig. 3 is antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar sensitivity of U.S. Pan Probe company and specific test result.
Fig. 4 is Canadian IND company's antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar sensitivity and specific test result.
Fig. 5 is antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar sensitivity of U.S. Medyl company and specific test result.
Fig. 6 is U.S. Acon company (Hangzhou processing) bar sensitivity of antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent and a specific test result.
Fig. 7 is outmoded people's blood stain of preserving the different time limits (being respectively the people's blood stain in 1963,1974,1980,1989), the product (A) that makes with the embodiment of the invention 2, simultaneously with Canadian IND company (B), U.S. Acon (at the Hangzhou processing and fabricating) (C), the assay measured of the antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of Medyl (D), Pan Probe (E) company.
Fig. 8 is from people's blood stain, common ten kinds of animal (pig, sheep, ox, horse, donkey, dog, rabbit, chicken, duck, goose) blood stains and mixes blood stain, blank sample, people's seminal stain, people's salivary stain, people just milk spots, vaginal secretions and randomly draw 20 parts totally 32 parts from people's blood stain of case etc., the result of the product detection that makes with the embodiment of the invention 2.
Fig. 9 is the best synoptic diagram of antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention.
Embodiment
The preparation that embodiment 1 produces the CGMCC 0970 hybridoma cell strain B9 of human hemoglobin monoclonal antibody
1. immune mouse: to every BALB/C mice, earlier carry out four limbs, subcutaneous multiple spot and intraperitoneal injection with the 100 μ g human hemoglobins (U.S. Sigma company product) that add complete freund adjuvant, add the same dosage intraperitoneal injection of Freund after 4 weeks, after 4 all tail vein injections, kill after 3 days mouse get spleen according to a conventional method with the SP2/0 cytogamy.
2. screening and cloning: fused cell is observed with HAT selection, inverted phase contrast microscope, selects efficient hybrid cell.With 3 μ g/ml human hemoglobin solution bags by 96 hole enzyme plates (annotate: reserve a hole and made negative control) with 5% bovine serum albumin or 10% skim-milk bag, indirect elisa method, be about to bag and put 1hr by 37 ℃ of incubators of the enzyme plate of human hemoglobin, pour out liquid in the hole, wash each hole 1～3 time with 20mM phosphoric acid salt normal saline buffer solution (pH7.4) again, in each hole, add stoste respectively, the hybrid cell culture supernatant of 5 times of dilutions, 37 ℃ of incubators are put 1hr, wash each hole 3～6 times with the 20mM phosphoric acid salt normal saline buffer solution (pH7.4) that contains 0.05% polysorbas20, then each hole adds the sheep anti-mouse igg of the suitable working concentration of by specification regulation, react after 30 minutes and wash each hole 3～6 times with the 20mM phosphoric acid salt normal saline buffer solution (pH7.4) that contains 0.05% polysorbas20 equally, in each hole, add DAB or ABTS again, observe each hole colour-change situation, filter out human hemoglobin antigen positive hybrid cell.Antigen-positive cell with the limiting dilution assay cloning, is prepared the monoclonal antibody hybridoma cell strain of human hemoglobin after carrying out 1 milliliter of stable cultivation with nurse cell and HT nutrient solution.
3. induce ascites:, collect the ascites that produces after 10～15 days with the hybridoma cell strain inoculation BALB/C mice abdominal cavity of secretion antihuman hemoglobin monoclonal antibody.
4. secrete the foundation of the hybridoma cell strain of special human hemoglobin monoclonal antibody: n-caprylic acid is purified into the human hemoglobin monoclonal antibody in conjunction with the ammonium sulfate method from ascites, respectively with indirect ELISA, (antigen is 10% various animals and reaches the mixing hemoglobin solutions separately to suppress ELISA and spot ELISA method; Different body fluid of animal blood stain, people or corresponding spot extracting solution) detect animal blood stains such as animal blood haemoglobin solution such as pig, dog, rabbit, sheep, horse, ox, donkey, mule, mouse, monkey and pig, dog, chicken, turkey, rabbit, sheep, horse, ox, donkey, mule, mouse, monkey, cat, cavy, fish (soft-shelled turtle, carp, grass carp, crucian, eel, snakeheaded fish, yellow croaker), tortoises; The reactivity of people's body fluid such as people's seminal stain, saliva, vaginal secretions, serum, colostrum, urine and corresponding spot extracting solution.Identify the monoclonal hybridoma strain that only human hemoglobin is reacted thus, wherein a strain called after B
9, on July 1st, 2003, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is: CGMCC 0970.
5. tire and antibody type and subgroup identification: the cells and supernatant doubling dilution; Ascites 200 times of dilutions earlier, doubling dilution again, the indirect elisa method survey is tired, as a result CGMCC 0970 hybridoma cell strain B
9For: supernatant 512, ascites 1.28 * 10
5Doubly; Concentrate 10 times of cells and supernatant with double diffusion test and anti-mouse IgG (Fc section), IgG
1, IgG
2a, IgG
2b, IgG3
WithIgM antibody reacts, and antibody is mouse IgG as a result
1
The preparation of embodiment 2 antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bars
1. antihuman hemoglobin monoclonal antibody ascites preparation: from liquid nitrogen, take out CGMCC 0970 hybridoma cell strain B
9, the ordinary method recovery is also cultivated.The culturing cell of cultured proper amt is injected in the mouse peritoneal, after 7～10 days or observe and put to death mouse in the time of to slaughter, collect mouse ascites.
2. separation and purification antihuman hemoglobin monoclonal antibody: add the ammonium sulfate method or affinity chromatography carries out with conventional n-caprylic acid, the latter is specifically: the 1.0mol/L Tris-HCI that adds 1/10 volume in the mouse ascites, the pH8.0 damping fluid, then add saturated ammonium sulphate solution or solid ammonium sulfate to 50% saturation ratio again, after placing 2hr, centrifugal, precipitation is with physiology PBS (20mmol/L Sodium phosphate dibasic and SODIUM PHOSPHATE, MONOBASIC, 0.1mol/LNaCI pH7.4) damping fluid weighs molten and this damping fluid is fully dialysed.With dialysis buffer liquid balance HiTrap
TMProteinG HP post, dialysis back protein solution upper prop then earlier with the unconjugated foreign protein of the buffer solution elution of balance columns, with the albumen of 0.1mol/L glycine-HCI damping fluid (pH2.7) elution of bound, is surveyed A again
280Value, merge this value at the component more than 0.1, the antihuman hemoglobin monoclonal antibody that this CGMCC of the present invention 0970 hybridoma cell strain B9 that is purifying produces, SDS-PAGE (being sds page sex change electrophoresis) surveys purity, 5mM NaCI solution is dialysed, be concentrated into suitable concentration, stand-by.
3. Preparation of Colloidal Gold: take by weighing Sigma company and produce hydrochloro-auric acid (article No.: G-4022) and be made into 1% concentration, get in an amount of adding ultrapure water, heated and boiled adds 1% sodium citrate solution fast, boils to solution to become red-purple.Cooling is with 0.2mol/L K
2CO
3Transfer pH to 8.0～8.2,0.45 μ m to filter.Get 1 times of an amount of dilution, survey A
530Value is 1.912.
4. Radioactive colloidal gold-of the present invention antihuman hemoglobin monoclonal antibody binding substances preparation: by 30 μ g/ml with step 2. the antihuman hemoglobin monoclonal antibody of purifying preparation join in the colloidal gold solution, stirred 20～30 minutes, identify its stable case with 10%NaCI.Add BSA (bovine serum albumin) and PEG (polyoxyethylene glycol) solution is an amount of, 12, centrifugal 50 minutes of 000r/min, precipitation is with protection liquid (1.0mmol/L sodium tetraborate-HCI, 0.1%BSA, 0.01%NaN
3, pH7.4) resuspended, it is standby to put 4 ℃ of preservations.
5. the reagent strip starting material are handled: for guaranteeing accuracy, the specificity of assay, will carry out pre-treatment to the material directly related with testing process.Specifically: to contain the 50mM Tris-HCI of 0.5% tween 80, the pH8.0 damping fluid soaks glass fibre and spends the night (4 ℃), desalts with aseptic ultrapure water rinsing, dries up under the aseptic high efficiency filter; Soaked nitrocellulose filter 1 to 2 hour with the 20mM phosphoric acid salt normal saline buffer solution (pH7.4) that contains 0.05% polysorbas20, desalt with aseptic ultrapure water rinsing again, also existing with now steeping and drying up.(annotate: all damping fluids are all with 0.45 μ m filter filtration sterilization).
6. the processing of Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances and be written into glass fibre: (contain 0.2% N of IgG, 0.5%PVP40 (or 30), 0.1%PEG20,000,0.5%BSA, 0.02%NaN with diluent
3Physiology PBS damping fluid, pH7.4), be added to (wide about 15mm) on the glass fibre of handling 37 ℃ of oven dry equably with 6 times of Radioactive colloidal gold-monoclonal antibody binding substances dilutions.
7. on the reaction zone nitrocellulose filter of handling well, spray detection line and nature controlling line: will tire 20, more than 000, specificity conforms with purifying (carrying out with the affinity column of making in conjunction with the CNBr activated agarose gel of the human hemoglobin) rabbit of medicolegal examination standard and requirement or goat-anti human hemoglobin polyclonal antibody (content is more than the 3mg/ml) solution (containing 0.2 ~ 1% sucrose) 1 μ l point on nitrocellulose filter (wide about 20mm), point is apart from the about 6 ~ 8mm of the close width edge of nitrocellulose filter; Put the sheep anti-mouse igg (content 1mg/ml also contains 0.2 ~ 1% sucrose) of 1 μ l in the place of putting about 4 ~ 5mm apart from this; Behind the airing, film is put in the confining liquid (PBS preparation 1.0-10% skim-milk), and 37 ℃ of incubations 1 hour take out that (the 20mM phosphoric acid buffer pH7.0) washes twice, airing with washings again.(annotate: during with the machine point sample, two lines that point of sample forms for the wide spraying of about 1mm, the line that wherein sprays antihuman hemoglobin is called detection line, and the line of spray sheep anti-mouse igg is called nature controlling line.)
8. assemble reagent strip: on single face glue plastic mattress flaggy (wide about 80mm, thick about 0.8mm), paste the nitrocellulose filter of 7. handling well (noting not being pasted with the one side of sample) earlier, making its point or being sprayed with antihuman hemoglobin is the about 30mm of width edge that this backing plate of an end distance of detection line closes on, put or be sprayed with the also about 30mm of another width edge of end distance backing plate that sheep anti-mouse igg is a nature controlling line, form reaction zone; With material covering point of 6. handling well or the about 2mm of nitrocellulose filter that is sprayed with antihuman hemoglobin one end, rest part is pasted on the backing plate, make uniform with the backing plate width edge and upwards stickup with glass fibre (wide 25mm) end of 5. handling, rest part cover be pasted on backing plate by the material of 6. handling well, form sample application zone; At the other end of the nitrocellulose filter of having pasted, produce the thick filter paper of Xinhua (wide about 30mm) with Whatman 3M or Zhejiang and also cover about 2mm, rest part is pasted on and forms adsorption zone on the backing plate; With single face glue adhesive tape (the MAX line of the maximum height that liquid level can reach in the time of should indicating the indication detection on it, this line should close on the about 15mm of width edge apart from backing plate) cover about 33-35mm inflection and be pasted on the backing plate back side again at backing plate one end that is stained with glass fibre, cover about 33mm inflection and be pasted on the backing plate back side again at the backing plate the other end that is stained with filter paper with single face glue adhesive tape (being with different colours usually), note adhesive tape and nitrocellulose filter two join hold must paste solid.Make mother matrix colloid gold reagent bar therefrom, then cut along the backing plate width by 3～4mm width, gained is finished product antihuman hemoglobin gold-immunochromatographyreagent reagent for assay bar, as shown in Figure 9.
Experimental result:
The detection of finished product reagent strip
The normal man's hemoglobin solutions (article No. 527-30) that mixes human hemoglobin solution and SIGMA company with 6 person-portions of new preparation of in August, 2002 serves as to detect used antigen; The product (A) that makes with the embodiment of the invention 2, simultaneously with Canadian IND company (B), U.S. Acon (at the Hangzhou processing and fabricating) (C), the antihuman hemoglobin colloid gold reagent bar of Medyl (D), Pan Probe (E) company measures, and the results are shown in Figure 1, Fig. 3 to Fig. 6 and table 1.
Table 1 sensitivity reaches with external analogous products compares
| The antigen diluent multiple | A | B | C | D | E | |||||
| 10 | 30 | 10 | 30 | 10 | 30 | 10 | 30 | 10 | 30 | |
| 1000 2000 5000 10000 50000 100000 200000 300000 | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + + + | + + + + + + ± - | + + + + + + ± - |
| Antigen quantitative concentrations (ng/ml) | ||||||||||
| 1000 500 200 150 100 | + + + + ± | + + + + ± | + + ± - - | + + ± - - | + + + + + | + + + + + | + + + + + | + + + + + | + + + + - | + + + + - |
Annotate: "+" represents positive in the table; "-" represents negative; " ± " is like cloudy seemingly sun; 10 and 30 units are minute, expression observing time.
Conclusion: the sensitivity of antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention quite or be better than external like product.
Test 2 accuracys, specificity test
With ten kinds of common animals (ox, horse, sheep, donkey, dog, rabbit, pig, chicken, duck, goose) blood solution (anticoagulation of every kind of animal centrifuged supernatant after freeze thawing) serves as to detect used antigen, the product (A) that makes with the embodiment of the invention 2, simultaneously with Canadian IND company (B), U.S. Acon (at the Hangzhou processing and fabricating) (C), the antihuman hemoglobin colloid gold reagent bar of Medyl (D), Pan Probe (E) company measures, the product that makes with the embodiment of the invention 2 (A) has carried out specific assay to the monkey blood solution simultaneously, the results are shown in Figure 1 to Fig. 6 and table 2.
Table 2 accuracy, species specificity reach with external like product and compare
| Antigen and extension rate | A | B | C | D | E | ||||||
| 10 | 30 | 10 | 30 | 10 | 30 | 10 | 30 | 10 | 30 | ||
| Pig | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - |
| Sheep | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - |
| Ox | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | + + + ± | + + + ± | - - - - | - - - - |
| Horse | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | + + - - | + + - - |
| Donkey | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - |
| Dog | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - + | - - - + | - - - - | - - - - | - - - - | - - - - |
| Rabbit | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | + + + + | + + + + | + + + + | + + + + |
| Chicken | 100 500 1000 5000 | - - - - | - - - - | - - - + | - - - + | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - |
| Duck | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | + + + + | + + + + |
| Goose | 100 500 1000 5000 | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | - - - - | + + + + | + + + + |
| Monkey | 100 500 1000 5000 | - - - - | - - - - | ||||||||
| Physiological saline | - | - | - | - | - | - | - | - | - | - | |
Annotate: "+" represents positive in the table; "-" represents negative; " ± " is like cloudy seemingly sun; 10 and 30 units are minute, expression observing time.
Conclusion: the accuracy and the specificity of antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention are better than like product.
Test 3 outmoded people's blood stain checks
Preserve outmoded people's blood stain of the different time limits (being respectively the people's blood stain in 1963,1974,1980,1989), compiling respectively is one, two, three, No. four, the product (A) that makes with the embodiment of the invention 2, simultaneously with Canadian IND company (B), U.S. Acon (at the Hangzhou processing and fabricating) (C), the antihuman hemoglobin colloid gold reagent bar of Medyl (D), Pan Probe (E) company measures, experimental result is seen Fig. 7 and table 3.
Table 3 compares the detection of outmoded people's blood stain with external like product
| Various years people blood stain | A | B | C | D | E | |||||
| 5 | 30 | 5 | 30 | 10 | 30 | 10 | 30 | 10 | 30 | |
| One two three four | + + + + | + + + + | + + + + | + + + + | + + + - | + + + ± | - - - - | - - - - | - - - - | - - - - |
Annotate: "+" represents positive in the table; "-" represents negative; " ± " is like cloudy seemingly sun; 5,10 and 30 units are minute, expression observing time.
Conclusion: antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention is detecting aspect the outmoded blood stain performance quite or be better than like product.
Experiment 4 Blind Test at random
From people's blood stain, common ten kinds of animal (pig, sheep, ox, horse, donkey, dog, rabbit, chicken, duck, goose) blood stains and mix blood stain, blank sample, people's seminal stain, people's salivary stain, people just milk spots, vaginal secretions and randomly draw 20 parts totally 32 parts from people's blood stain of case etc., the product (A) that makes with the embodiment of the invention 2 detects, and the results are shown in Figure 8 and table 4.
Table 4 Blind Test assay
| Numbering | The sample title | The result | Numbering | The sample title | The |
| 1 2 3 4 5 6 7 8 9 10 | Common ten kinds of animals of the blank sample people of people's blood cake salivary stain vaginal fluid people seminal stain rabbit blood stain colostrum mix blood cake the dead Jia Ji soldier blood sample people seminal stain | + - - - - - - - + - | 11 12 13 14 15 16 17 18 19 20 | People's seminal stain dog blood stain chicken blood stain people blood cake pig blood stain Wang Hui blood sample people seminal stain sheep blood stain vaginal fluid ox blood trace | - - - + - + - - - - |
Conclusion: antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar of the present invention has been made accurate check to the Blind Test sample.
Experimental result shows that the like product of antihuman hemoglobin gold-immunochromatographyreagent reagent for assay bar sensitivity that the present invention is prepared and external highest level is suitable; But specificity then is better than external product, and all more or less there is non-specific responding in external product; And the check of outmoded people's blood stain is illustrated that also it reaches the international leading level; Blind Test to actual sample is entirely true.Proved its unique value on medical jurisprudence.
Claims (15)
1, has the hybridoma cell strain B9 that preserving number is CGMCC 0970.
2, antihuman hemoglobin monoclonal antibody, it is from having the hybridoma cell strain B9 generation that preserving number is CGMCC 0970.
3, the colloidal gold immunochromatographiassay assay reagent bar of antihuman hemoglobin is characterized in that: comprise the described antihuman hemoglobin monoclonal antibody of claim 2 in this reagent strip.
4, detection reagent bar as claimed in claim 3 is characterized in that it comprises:
Sample application zone, the described antihuman hemoglobin monoclonal antibody of claim 2 district that it comprises blank inert material district and comprises attached Radioactive colloidal gold;
Reaction zone, it comprises detection line and nature controlling line;
Adsorption zone.
5, detection reagent bar as claimed in claim 4 is characterized in that: the described antihuman hemoglobin monoclonal antibody of claim 2 of the attached Radioactive colloidal gold of bag is carried in the glass fibre in the sample application zone, and the blank inert material district of sample application zone is a glass fibre.
6, detection reagent bar as claimed in claim 4, it is characterized in that: detection line and nature controlling line are carried on the nitrocellulose filter.
7, as claim 4 or 6 described detection reagent bars, it is characterized in that: the detection line of reaction zone is the many or monoclonal antibody of another antihuman hemoglobin, its with the antibody of the described antihuman hemoglobin of claim 2 of the attached Radioactive colloidal gold of bag at be identical antigen, but antigen binding site or claim antigenic determinant inequality.
8, detection reagent bar as claimed in claim 7 is characterized in that: what the detection line of reaction zone sprayed is rabbit or the goat-anti human hemoglobin polyclonal antibody that contains 0.2-1% sucrose.
9, as claim 4 or 6 described detection reagent bars, it is characterized in that: the nature controlling line of reaction zone is the anti-antibody that plays specific reaction with the described antihuman hemoglobin monoclonal antibody of claim 2 of the attached Radioactive colloidal gold of bag.
10, detection reagent bar as claimed in claim 9 is characterized in that: what the nature controlling line of reaction zone sprayed is the sheep anti-mouse igg antibody that contains 0.2-1% sucrose.
11, detection reagent bar as claimed in claim 4 is characterized in that adsorption zone is a filter paper.
12, prepare the method for antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar, may further comprise the steps:
(1) preparation of antihuman hemoglobin monoclonal antibody: with preserving number is that the hybridoma cell strain B9 of CGMCC 0970 recovers according to a conventional method and cultivates, and injects in the mouse peritoneal, puts to death mouse, collects ascites;
(2) separation and purification antihuman hemoglobin monoclonal antibody of the present invention: add ammonium sulfate method or affinity chromatography etc. with conventional n-caprylic acid and carry out;
(3) preparation of Radioactive colloidal gold: with hydrochloro-auric acid (HAuCl
4) make colloidal gold solution with the reductive agent heating;
(4) Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances preparation: the antihuman hemoglobin monoclonal antibody stirring that makes in the step (2) is added in the colloidal gold solution down, the antibody bag is attached on the colloid gold particle surface.
(5) the raw-material pre-treatment of reagent strip: material glass fiber directly related with testing process and nitrocellulose filter are carried out pre-treatment.
(6) glass fibre is handled and be written into to Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances: the Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances that makes with diluted step (4), with the saturated immersion glass fibre of this binding substances after the dilution, dry then then;
(7) spraying detection line and nature controlling line on the reaction zone nitrocellulose filter of handling well; Detection line is the many or monoclonal antibody of another antihuman hemoglobin, its with the antibody of the antihuman hemoglobin of the attached Radioactive colloidal gold of bag at be identical antigen, but antigen binding site or title antigenic determinant are inequality; Nature controlling line is the anti-antibody that plays specific reaction with the antihuman hemoglobin monoclonal antibody of the attached Radioactive colloidal gold of bag.
(8) assembling reagent strip: on single face glue plastics flaggy, paste (5) be fixed with sample application zone and handled the glass fibre that Radioactive colloidal gold-the antihuman hemoglobin monoclonal antibody binding substances is saturated that blank glass pars fibrosa and step (6) make, the nitrocellulose filter of reaction zone and the filter paper of uptake zone; Reaction zone is in reagent strip central authorities, and adsorption zone and sample application zone are respectively at the reaction zone two ends; The contiguous sample application zone of the detection line of reaction zone, the contiguous adsorption zone of the nature controlling line of reaction zone; The contiguous reaction zone of the glass fibre that Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances is saturated; Glass fibre district one end that Radioactive colloidal gold-antihuman hemoglobin monoclonal antibody binding substances that blank glass pars fibrosa one end of sample application zone and step (6) make is saturated overlaps; One end of glass fibre one end of sample application zone band Radioactive colloidal gold and the nitrocellulose filter of reaction zone overlaps, and an end of the other end of the nitrocellulose filter of reaction zone and uptake zone filter paper overlaps; Wherein be stained with the single face adhesive tape layer on the glass fibre of an end parts of the nitrocellulose filter of reaction zone and sample application zone, the other end of the nitrocellulose filter of reaction zone partly with on the filter paper is stained with the single face adhesive tape layer;
(9) (8) are made mother matrix reagent strip is cut into the finished product reagent strip;
(10) performance of detection reagent bar determines whether it is qualified.
13, the method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 12, wherein step (5) is at 4 ℃, with the 50mM Tris-HCI that contains 0.5% tween 80, the pH8.0 damping fluid soaks glass fibre and spends the night, desalt with aseptic ultrapure water rinsing, dry up under the aseptic high efficiency filter; Containing the 20mM phosphoric acid salt normal saline buffer solution of 0.05% polysorbas20, pH7.4 soaked nitrocellulose filter 1 to 2 hour, desalted with aseptic ultrapure water rinsing again, and is also existing with now steeping and dry up, and all damping fluids are all with 0.45 μ m filter filtration sterilization.
14, the method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 12, wherein the diluent in the step (6) is: 0.2～1.0% N of IgG, 0.1～0.5%PVP40 or 30,0.05～0.1%PEG20,000,0.5～2.0%BSA, 0.02%NaN
3Physiology PBS damping fluid, pH7.4.
15, the method for preparing antihuman hemoglobin colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 12, it is characterized in that: what the detection line of reaction zone sprayed is rabbit or the goat-anti human hemoglobin polyclonal antibody that contains 0.2-1% sucrose, and what the nature controlling line of reaction zone sprayed is the sheep anti-mouse igg antibody that contains 0.2-1% sucrose.
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| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| CN102226171B (en) * | 2011-03-29 | 2012-08-15 | 成都华西海圻医药科技有限公司 | Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof |
| CN102841208B (en) * | 2011-06-24 | 2014-11-26 | 北京乐普医疗科技有限责任公司 | Colloidal gold test paper for quickly detecting troponin I and preparation method for colloidal gold test paper |
| DE102012013888A1 (en) * | 2012-07-09 | 2014-05-22 | Schebo Biotech Ag | Test kit (combi rapid test) for the synchronous detection of biomarkers in stool for the detection of pathological changes in the gastrointestinal tract, especially in the intestine |
| CN103364562A (en) * | 2013-08-01 | 2013-10-23 | 青岛宝依特生物制药有限公司 | Test strip for testing sulfanilamide drug residues and application method |
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| TW201708819A (en) * | 2015-08-28 | 2017-03-01 | 亞諾法生技股份有限公司 | Immunochromatographic analysis kit and a method of using the same |
| CN106872694A (en) * | 2017-02-16 | 2017-06-20 | 天津中新科炬生物制药股份有限公司 | The detection method and hemoglobin detection kit of a kind of hemoglobin |
| CN107656045A (en) * | 2017-11-01 | 2018-02-02 | 上海凯创生物技术有限公司 | Procalcitonin near-infrared fluorescent detection reagent card, kit and application thereof |
| CN112979799B (en) * | 2019-12-18 | 2022-11-08 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
| CN114478759A (en) * | 2020-11-12 | 2022-05-13 | 东莞市朋志生物科技有限公司 | Anti-human hemoglobin antibody, application thereof and diagnostic kit |
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