CN104422764A - Glycoprotein-type tumor marker immunochromatographic test strip as well as preparation method and application thereof - Google Patents
Glycoprotein-type tumor marker immunochromatographic test strip as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a glycoprotein-type tumor marker immunochromatographic test strip based on a phenylboronic acid marking technology as well as a preparation method and application thereof. The test strip adopts phenylboronic acid as a biological marker, and a result is reflected in a light signal transmission form under the irradiation of exciting light and can be judged by an instrument, so that the quantitative detection on a glycoprotein-type tumor marker can be realized. The test strip comprises a sample mat, a conjugate mat, an analysis film, a water absorption mat and a pad. The conjugate mat is fixedly provided with a phenylboronic acid marker, and a detection line and a quality control line are fixedly arranged on the analysis film. The invention also discloses a preparation method of the test strip and the application of the test strip in the quantitative detection on the glycoprotein-type tumor marker.
Description
Technical field
The present invention relates to a kind of glycoprotein tumor markers immuno-chromatographic test paper strip based on phenyl boric acid labelling technique and its preparation method and application, belong to technical field of immunoassay.
Background technology
Tumour is the disease of serious harm human health, and treatment of late stage effect is undesirable.Therefore, early detection, early diagnosis, early treatment are the keys of conquering tumour.Tumor markers checks convenient, fast, is one of important means checking tumour clinically.Tumor markers refers to the material that characteristic is present in malignant cell or is produced tumor response by material or the host of the abnormal generation of malignant cell.These materials are present in tumour cell and tissue, also can enter blood and other body fluid, and when tumorigenesis, these materials are obviously abnormal, indicate the existence of tumour.Tumor-marker in the body fluid of the overwhelming majority had both been present in tumour, was also present in normal population and non-tumour patient, and just the marker concentration of tumour patient is higher than non-tumour patient.Have the tumor markers of several only a fews such as PSA and specific organ only to be associated and to present organ specificity, most of tumor markers is positive on the kinds cancer of a certain organization type, but positive rate differs.Except minority tumour, most of tumour often has multiple positive tumor positive markers.A specific tumour, different tumor stage, different tumor cell types, during different prognosis, the tumor markers be positive may be not quite similar; Or identical mark positive rate is different, add the complicacy of tumor markers application.Some tumor-markers can be positive in kinds of tumors, are called broad-spectrum tumor mark (nonspecifictumor marker).
Glycoprotein tumor markers is the antigen formed due to cell membrane component Aberrant glycosylation.The name of this kind of antigen markers is random, some is the numbering of tumor cell line, some is the material numbering of antibody, conventional detection method is monoclonal antibody method, what have also makes dibit point immobilized enzyme immunization with the monoclonal antibody of two kinds of different loci simultaneously, and these improve a lot than the specificity of general chemical determination.And the heteroplasmon to some sugar antigens, then usual with different plant lectin usually carry out separation detect.
Nineteen eighty-three detects a kind of glycoprotein that can be combined by monoclonal antibody OC125 by Bast etc. from ovarian epithelial carcinoma antigen.Molecular weight is 200,000-100 ten thousand, and when being heated to 100 DEG C, the activity of CA125 is destroyed, and (RIA) the positive critical value in normal human serum CA125 is 35U/L.
CA125 is the mark of ovarian epithelial carcinoma and carcinoma of endometrium, and serosity endometrioid carcinoma, clear cell carcinoma, carcinoma of fallopian tube and the CA125 content not breaking up ovarian cancer patients can obviously raise.When Ovarian cancer, just can present CA125 increase at clinical definite earlier month, especially the Serum tumor marker CA125 of oophoroma transfer patient is more apparently higher than normal reference value.The combination of CA125 mensuration and pelvioscopy can improve the specificity of test.Dynamic observation Serum tumor marker CA125 concentration contributes to prognostic evaluation and the treatment control of oophoroma, and after treatment, CA125 content can obviously decline, if can not return to normal range, should consider the possibility having residual tumor.The Serum tumor marker CA125 concentration of the residual tumor patient of 95% is greater than 35kU/L.But, CA125 serum-concentration slightly rises and also sees 1% healthy women, 3%-6% benign ovarian illness or non-tumor patient, comprise initial 3 months of pregnancy period, menstrual period, mullerianosis, fibrosis of uterus, acute salpinitis, hepatopathy, pleuroperitoneum and pericardosis etc.
CA15-3 is the mouse monoclonal antibody (115-DB) made from glycoprotein MAM-6 HMFG's film in 1984; Within 1984, make monoclonal antibody (DF-3) from hepatic metastases breast cancer cell membrane, therefore be named as CA15-3.CA15-3 molecular weight is 400ku, and molecular structure is not yet clear.Normal health person change of serum C A15-3 content (RIA method) is less than 28kU/L.30%-50% is that the CA15-3 of patient with breast cancer obviously raises, and it is also the optimal parameter of monitoring patient with breast cancer postoperative recurrence, and when CA15-3 is greater than 100U/ml, can think there is metastatic pathology, change and the treatment results of its content are closely related.The change of serum C A15-3 of lung cancer, human primary gastrointestinal cancers, oophoroma and cervical cancer patient also can raise, and should give discriminating, and the content wanting exclusive segment gestation to cause especially raises.
CA19-9 uses colon cancer cell immune mouse in 1979, and hybridize gained 116NS19-9 monoclonal antibody with myeloma, the oligosaccharides Tumor associated glycoprotein of its to be a kind of molecular weight be 5000ku, its structure is the bond of Lea blood group antigens material and sialic acid Lexa.The CA19-9 serum content of normal population is (RIA method) 2-16U/ml.CA19-9 is the mark of cancer of pancreas and knot, the carcinoma of the rectum.The critical value of the change of serum C A19-9 positive is 37kU/L.Pancreas cancer patients 85%-95% is positive.When CA19-9 is less than 1000U/ml, have certain Surgical Significance, after tumor resection, CA19-9 concentration can decline, and as risen, then can represent recurrence again.The positive rate of colorectal cancer, carcinoma of gallbladder, cholangiocarcinoma, liver cancer and cancer of the stomach also can be very high, if detect CEA and AFP to can further improve positive detection rate simultaneously.As pancreatitis and jaundice during optimum illness, CA19-9 concentration also can increase, but often in " transient ", and its concentration is many lower than 120kU/L, must be differentiated.
CA50 is that the strain filtered out from a series of monoclonal antibodies of anti-human knot, the carcinoma of the rectum Colo-205 cell line nineteen eighty-three has kickback to knot, the carcinoma of the rectum, but the monoclonal antibody of not reacting with myeloma cell and blood lymphocyte, the antigen that can identify claims CA50.CA50 is present in cell membrane, and its antigenic determinant is sialic acid Lea blood group substance and sialic acid-N-four oxygen ceramide.At normal population, CA50 serum-concentration (RIA method) is less than 20U/ml.It is generally acknowledged, CA50 is the mark of pancreas and knot, the carcinoma of the rectum, because CA50 extensively exists pancreas, gall-bladder, liver, stomach, Colon and rectum, bladder, uterus, when malignant change of cell, because the inactivation of glycosyl invertase or some invertase that could enliven embryonic period, embryonic phase are activated, cell-surface carbohydrates structural property is caused to change and form CA50, therefore, it is again a kind of general tumor-marker related antigen, instead of refers in particular to the tumor markers of certain organ.So different positive rates can be detected in Several Kinds of Malignancy.Nineteen eighty-three, establish radio immunoassay, application CA50 monoclonal antibody in 1987, establish the early diagnosis of IRMA technology for tumour at home, the positive detection rate of cancer of pancreas, carcinoma of gallbladder reaches 90%, also has higher-value to liver cancer, cancer of the stomach, colorectal cancer and diagnosis on ovarian tumors, when pancreatitis, colitis and pneumonia are fallen ill, CA50 also can raise, but eliminates with inflammation and decline.
CA242 is a kind of sialylated glycosyl sphingolipid class antigen, almost always expresses together with CA50, but both are by different monoclonal antibody identification.All be used to the diagnosis of malignant tumor of digestive tract especially cancer of pancreas, colorectal cancer clinically, compared with CA19-9, CA50, the sensitivity of CA242 in cancer of pancreas, carcinoma of gallbladder and digestive system cancer, the specificity of a new generation are higher (CA50, CA19-9 are subject to liver function and cholestatic impact, often occur false positive in optimum obstructive jaundice and liver parenchyma infringement note disease).CA242 raises and sees Pancreas cancer patients positive rate and be about 72.4%, but thinks that the primary tumor markers of cancer of pancreas is still CA19-9 at present; To be 63.7%, CA242 detect in conjunction with CEA, CA19-9 PATIENTS WITH LARGE BOWEL CA242 positive rate is simultaneously the most responsive mark of colorectal cancer; Cancer of the stomach, liver cancer, cancer of the esophagus CA242 positive rate are respectively 40.9%, 37.6% and 30%; Non-neoplastic disease such as pancreatitis, ulcerative colitis, cirrhosis etc. can have CA242 slightly to raise.
CA72-4 is a kind of high molecular weight glycoproteins, content < 6U/ml in normal human serum, and abnormal rising all can produce in various tumor in digestive tract, oophoroma.Detection specificity for cancer of the stomach is higher, with > 6U/ml for critical value.Benign stomach disease only < raises, and cancer of the stomach rising person ratio can reach 42.6%, and as detected with CAl9-9 simultaneously, positive rate can reach 56%.The susceptibility of CA72-4 is not high, but it and CEA have complementation when diagnosing tumour, and both use the Sensitivity and Specificity that can improve diagnosis of gastric cancer simultaneously.
CA549 is also the mark of breast cancer, and it is a kind of acidoglycoprotein, most of healthy women < 11U/ml, and abnormal rising person's ratio is not high, is found in 50% breast cancer, oophoroma, 40% prostate cancer, 33% patients with lung cancer.Thus, as the early diagnosis of breast cancer, CA is then also comparatively short of, and answers other TM of use in conjunction.
Squamous cell related antigen (SCC) is purified by cervical cancer cell, is the good tumor markers of cervical carcinoma.SCC also exists in Normal squamous epithelium, is released into blood along with the propagation (pernicious) of squamous cell.Normal human serum horizontal < 2 μ g/L.Abnormal rising is found in SCC, and 21% adenocarcinoma of the uterine cervix also has rising.Lung squamous cancer has higher positive rate, and from 40%-100% not etc., small-cell carcinoma of the lung positive rate is lower (3.7%) then in each report.Esophageal squamous epithelial carcinoma, oral cavity squamous epithelioma all have higher positive rate, and change (20%-80%) with the difference that presents of tumour by stages.Visible SCC is the important symbol thing of squamous cell carcinoma.
The subunit that NMP-22 (nuclear matrix protein-22, NMP22) is caryomitosis device albumen (NuMAP 238KD).NMP22 is the important component part of paralinin, with the copying of DNA, transcribe, RNA synthesizes, the regulation and control of gene expression are relevant.When cell cancerates, in core, inhereditary material distributes extremely abnormal in mitosis telophase, and NuMAP synthesizes surge.Urothelial tumor cell discharges NMP22, as the biological markers of carcinoma of urinary bladder because apoptosis or other mechanism death enter urine.1996, U.S. food Drug Administration (FDA) have approved the clinical practice of NMP22 kit, the detection now common detection for carcinoma of urinary bladder of NMP22.Be a kind of new mark of carcinoma of urinary bladder, detect urine NMP22 and can differentiate good pernicious bladder disease.
Publication number is CN101324579, publication date is on Dec 17th, 2008, and what name was called " a kind of magnetic microparticle chemiluminescence enzyme immune analytic reagent kit and using method thereof detecting sugar antigen " application discloses a kind of chemical luminescent detecting technology.Although this technology adopts highly sensitive, operation steps is numerous and diverse, and needs expensive specialized equipment to complete detection, limits it in clinical application.
Publication number is CN101526535, publication date is on September 9th, 2009, and what name was called " multi-tumor marker liquid phase chip for joint detection and preparation method thereof " application discloses a kind of protein chip determination techniques.Although this technology adopts entry joint-detection, operating process needs expensive specialized equipment to complete detection equally, limits it in clinical application.
Several method in sum, all also exists many problems to a certain extent, urgently proposes a kind of new method and solves these problems.
Summary of the invention
The object of the invention is the prior art Problems existing proposed to solve above-mentioned background technology, and then a kind of glycoprotein tumor markers immuno-chromatographic test paper strip based on phenyl boric acid labelling technique and its preparation method and application is provided, and wherein phenyl boric acid label can adopt 4-NCS(diimide)-phenyl boric acid or 4-NHS(N-N-Hydroxysuccinimide)-phenyl boric acid makes as phenyl boric acid material.When using this test strips, only need the whole blood sample of trace, the content quantitatively detecting glycoprotein tumor markers can be realized in 3-5 minute, substantially increase the speed of examination, and highly sensitive, specificity good, test strips structure is simple, is easy to large-scale production.
The object of the invention is to be achieved through the following technical solutions:
Principle of the present invention please refer to accompanying drawing 1.
Glycoprotein tumor markers immuno-chromatographic test paper strip of the present invention comprises the sample pad, bond pad, analyzing film, the adsorptive pads that contact with each other successively, and bottom is provided with liner, and described analyzing film is provided with detection line and nature controlling line; Be fixed with the antibody for detectable antigens of phenyl boric acid label and fluorescent material marks in described bond pad, detection line contains the antibody reacted with testing sample, and nature controlling line contains antibody aitiogenic with phenyl boric acid label.
Further, described sample pad, bond pad, analyzing film, the adsorptive pads contacted with each other successively has the overlapping region of 1 ~ 2mm mutually.
Further, described analyzing film is arranged at the below of bond pad and adsorptive pads, and described sample pad is arranged at the top of described bond pad.
Further, described detection line and nature controlling line interval 5mm.
The invention also discloses a kind of application of glycoprotein tumor markers immuno-chromatographic test paper strip, it may be used for detecting biological sample, and detected object is the content detection of glycoprotein tumor markers in whole blood, serum, blood plasma.
Particularly, detection method, for add in sample pad by sample drop, makes liquid flow from adsorptive pads side by syphonic effect, after liquid comes into contact detection line and nature controlling line, is drawn the concentration of testing sample by the mode of instrument reading fluorescence intensity.
The invention also discloses a kind of preparation method of glycoprotein tumor markers immuno-chromatographic test paper strip, comprise the steps:
(1) preparation of phenyl boric acid label: by fluorescent dye compound respectively with damping fluid dilution, add phenyl boric acid material dissolves liquid respectively, stir evenly, room temperature reaction at least 1 hour, carry out separation and purification by gel column, collect label, preserve with after damping fluid dilution mixing;
(2) preparation of sample pad: with cellulose membrane as sample pad solid phase material, soak with 0.01 ~ 0.3M phosphate buffer of 0.01% ~ 0.5% polyglycol, 1% ~ 5% bovine serum albumin(BSA), 0.01% ~ 0.05% surfactant, described ph value of buffer solution is 7.2 ~ 7.6, after immersion treatment, take out after sample pad being put into vacuum drying chamber inner drying, vacuum seal is for subsequent use;
(3) preparation of bond pad: with glass fibre element film as bond pad solid phase material, with the 0.01 ~ 0.1MpH7.2 phosphate buffer dilution phosphor material label containing 1% ~ 5% bovine serum albumin(BSA), 0.1 ~ 2% polyglycol, 0.5 ~ 2% sucrose, 0.01% ~ 0.1% surfactant, make suspension, be sprayed on glass fibre element film, take out after bond pad is put into vacuum drying chamber inner drying, vacuum seal is for subsequent use;
(4) preparation of analyzing film: the antibody used with damping fluid dilution detection line and nature controlling line is to being applicable to concentration, adopt on the Membrane jetter detection line that is sprayed on analyzing film respectively and nature controlling line position, analyzing film after spray film is put into vacuum drying chamber, and after dry, taking-up vacuum seal is for subsequent use;
(5) preparation of adsorptive pads: select filter paper as adsorptive pads solid phase material, it is saved backup at dry environment;
Above step (1) is to step (5) not order restriction.
(6) preparation of finished product test strips: according to reaction sequence, first sticks to analyzing film on liner centre position, analyzing film upper end adhere to adsorptive pads, adsorptive pads above analyzing film, the two overlap 1 ~ 2mm; At analyzing film lower end adhesive bond thing pad, bond pad above analyzing film, the two overlap 1 ~ 2mm; Again bond pad lower end paste sample pad, sample pad above bond pad, the two overlap 1 ~ 2mm; Liner and the sample pad of pasting above, bond pad, analyzing film and adsorptive pads are together cut shaping.
Further, described antibody is anti-glycoprotein tumor markers monoclonal antibody, sheep anti-mouse igg antibody.
Further, described liner is made up of pet material.
Further, described phenyl boric acid material is phenyl boronic acid derivative, and described phenyl boronic acid derivative has the chemical group of mark fluorescent dyestuff, and the excitation light spectral limit of described fluorescent dye is 390 ~ 420nm, and wavelength of transmitted light scope is 600 ~ 700nm.
In the present invention, for quantitative test item, by setting up glycoprotein tumor markers standard items and fluorescence signal intensity typical curve, realize quantitative detection.
The present invention has following beneficial effect: the present invention adopts phenyl boronic acid derivative as biomarker, and result shows with the form of utilizing emitted light light signal under exciting light irradiates, and can carry out instrument interpretation, thus realizes the quantitative detection to target detected material.
Accompanying drawing explanation
Fig. 1 is for being principle schematic of the present invention;
Fig. 2 is the preferred structure schematic diagram of the test strips that the present invention obtains;
Fig. 3 is the quantitative test working curve diagram that test example 1 is made for embodiment 1;
Fig. 4 is that the contrast that test example 5 is made for embodiment 1 detects regression curve;
Reference numeral: 1, sample pad; 2, bond pad; 3, analyzing film; 4, glycoprotein tumor markers detection line; 5, nature controlling line; 6, adsorptive pads; 7, liner.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
As shown in Figure 2, the structure of product of the present invention comprises liner 7, be arranged at analyzing film 3 in the middle part of liner, be arranged at the adsorptive pads 6 of analyzing film upper one end, be arranged at the bond pad 2 of the analyzing film top other end and be arranged at the sample pad 1 of bond pad upper one end, analyzing film is provided with detection line 4 and nature controlling line 5.Adsorptive pads 1 ~ 2mm overlapping with analyzing film, bond pad 1 ~ 2mm overlapping with analyzing film, sample pad 1 ~ 2mm overlapping with bond pad.Detection line comprises glycoprotein tumor markers detection line 4 and nature controlling line 5.Glycoprotein tumor markers detection line, nature controlling line interval 5mm successively.
In the present invention, sample pad detects the position that glycoprotein tumor markers immuno-chromatographic test paper strip in use drips testing sample.Be fixed with phenyl boric acid material marking fluorescent material and fluorescent material marks antibody isoreactivity molecular bond in bond pad, after adding testing sample, start Ag-Ab immune response occurs at this.Analyzing film is the core of chromatographic test paper, is fixed with glycoprotein tumor markers detection line and nature controlling line respectively in its surface; Detection line contains, with glycoprotein tumor markers in testing sample, immunoreactive antibody occurs, and nature controlling line contains and the raw immunoreactive antibody of fluorescent material marks produce.Adsorptive pads, in whole testing process, provides liquid to flow through the power of whole test strips by syphonic effect.There is overlapping region between each several part, contribute to the continuity that liquid flows in test strips.
The principle of the detection glycosylated albumin immuno-chromatographic test paper strip that the present invention proposes is, when detecting, sample drop is added in sample pad, sample enters bond pad by infiltration and syphonic effect, makes fluorescent material marks thing wherein dissolve release, under the syphonic effect of adsorptive pads, liquid enters analyzing film, flow through glycoprotein tumor markers detection line and nature controlling line successively, and specific immune response occurs, produce and there is tell-tale fluorescence signal.
In in the manufacture process of product of the present invention; the amount of substance of each group of damping fluid, concentration, pH value and component can be identical; can be different; it is all within protection scope of the present invention, for different component out cited by specific embodiment be the present inventor do experiment in some of them preferred embodiment.
Application principle of the present invention is, by setting up determinand standard items and fluorescence signal intensity typical curve, to realize quantitative detection.When carrying out sample detection, be added in by sample drop in sample pad, sample enters bond pad by infiltration and syphonic effect, the bond that phosphor material is wherein marked dissolves again, and under the syphonic effect of adsorptive pads, discharge from bond pad and enter analyzing film, flow to adsorptive pads direction.In analyzing film in moving process, between fluorescent marker, target determinand, detection line, nature controlling line, specific immune response will be there is, and at detection line and nature controlling line generation, there is tell-tale light signal.
Embodiment 1
Product preparation method of the present invention is as follows:
1, the preparation of fluorescence luminescent material labeled monoclonal antibody: by anti-CA 125 monoclonal antibody and sheep anti-mouse igg antibody, be 0.1mol/L, pH with substance withdrawl syndrome be respectively that the sodium bicarbonate-carbonate solution dilution of 9.6 is to 1mg/ml, respectively get 5ml antibody-solutions, add 25mg fluorescence luminescent material metalloporphyrin lysate respectively, stir evenly, incubated at room 2 hours, every mixing in 15 minutes once.Finally cross column separating purification with the gel column that specifications and models are G50, collect the metalloporphyrin mark anti-CA 125 antibody and sheep anti-mouse igg antibody that have marked, with substance withdrawl syndrome be 0.01mol/L, pH value be 7.2 first phosphate buffer dilution, wherein the first phosphate buffer comprise containing mass percent be 0.15% polyglycol, the bovine serum albumin(BSA) of 2.5%, the first surface activating agent of 0.03%, pack with reagent bottle, preserve under 2 ~ 8 DEG C of conditions.
2, the preparation of sample pad: select cellulose membrane as the solid support material of sample pad, cut into the band of 5 × 300mm specification.By sample pad as in rectangular tank, with substance withdrawl syndrome be 0.05mol/L, pH value be 7.4 first phosphate buffer dilution soak 30min, wherein the first phosphate buffer comprise containing mass percent be 0.2% polyglycol, the bovine serum albumin(BSA) of 2.5%, the first surface activating agent of 0.03%.After immersion treatment, sample pad is taken out, is placed on clean network, the drying box inner drying putting into 60 DEG C after 80 minutes taking-up aluminium foil bag to vacuumize sealing for subsequent use.
3, the preparation of bond pad: select glass fibre element film as the solid phase carrier of bond pad, cut into the band of 5 × 300mm specification.Phenyl boric acid material marking fluorescent material, fluorescence labeling anti-albumin antibodies and sheep anti-mouse igg antibody that 2 ~ 8 DEG C are saved backup, with substance withdrawl syndrome be 0.01mol/L, pH value be 7.4 second phosphate buffer dilution make suspension, wherein the second phosphate buffer comprise containing mass percent be 0.35% polyglycol, the bovine serum albumin(BSA) of 2.8%, the sucrose of 2.5% and 0.05% second surface activating agent.With the line of Membrane jetter spray film, film liquid measure is 10ul/mm, is then placed on clean network, the drying box inner drying putting into 45 DEG C after 60 minutes taking-up aluminium foil bag vacuumize sealing and preserve.
4, the preparation of analyzing film: the preparation of (1) glycoprotein tumor markers CA125 detection line coating buffer: with 50ml substance withdrawl syndrome be 0.05mol/L, pH value is the phosphate buffer of 7.4, wherein, be calculated in mass percent, this phosphate buffer includes methyl alcohol 0.5%, trehalose 1.0%, bovine serum albumin(BSA) 1.5%, and dilution anti-CA 125 antibody is to final concentration 1.6mg/ml.(2) preparation of nature controlling line coating buffer: with 50ml substance withdrawl syndrome be 0.05mol/L, pH value is the phosphate buffer of 7.4, wherein, be calculated in mass percent, this phosphate buffer includes methyl alcohol 1%, bovine serum albumin(BSA) 0.8%, and dilution mouse IgG antibody is to final concentration 0.5mg/ml.(3) select nitrocellulose filter as solid phase carrier, be analyzing film, cut into the band of 25 × 300mm specification.Rule successively on the analyzing film that 25mm is wide with Membrane jetter, the line of the film of 10mm place spray from the bottom up, film liquid measure is 2ul/mm, as CA125 detection line detection line; Interval 5mm place again, the line of the film of 15mm place spray from the bottom up, film liquid measure is 1.5ul/mm, as nature controlling line.CA125 detection line detection line and nature controlling line be interval 5mm successively, and line fine uniform, places 37 DEG C of drying box process 50 minutes by analyzing film, vacuumizes pack sealing save backup after taking-up with aluminium foil bag.
5, the preparation of adsorptive pads: the filter paper selecting 1mm thick, as adsorptive pads solid phase material, is cut into the band of 25mmX300mm.Adsorptive pads saves backup at dry environment.
6, preparation detects the immuno-chromatographic test paper strip of glycoprotein tumor markers: first sticked to by analyzing film on liner centre position that pet material makes, adsorptive pads and bond pad is adhered in analyzing film upper end, sample pad is pasted again in bond pad upper end, by liner and be arranged at the sample pad on liner top, bond pad, analyzing film and adsorptive pads and together cut into strip, be and detect glycosylated albumin immuno-chromatographic test paper strip.First surface activating agent and second surface activating agent are the one in Tween20, TritonX-100 and tetronic1307, and preferably first surface activating agent and second surface activating agent are Tween20 in the present embodiment.
Embodiment 2
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA15-3 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.05mol/L, and pH value is 7.4, and it comprises: be calculated in mass percent, the polyglycol of 0.01%, the bovine serum albumin(BSA) of 1% and 0.01% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.5mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1%, the polyglycol of 0.1%, the sucrose of 0.5% and the second surface activating agent of 0.01%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA15-3 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are TritonX-100.
Embodiment 3
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA19-9 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.3mol/L, and pH value is 7.6, and it comprises: be calculated in mass percent, the polyglycol of 0.5%, the bovine serum albumin(BSA) of 5% and 0.03% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.1mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 5%, the polyglycol of 2%, the sucrose of 1% and the second surface activating agent of 0.1%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA19-9 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are tetronic1307.
Embodiment 4
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA50 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.25mol/L, and pH value is 7.4, and it comprises: be calculated in mass percent, the polyglycol of 0.8%, the bovine serum albumin(BSA) of 5% and 0.05% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.05mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 4%, the polyglycol of 1.5%, the sucrose of 1% and the second surface activating agent of 0.05%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA50 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are tetronic1307.
Embodiment 5
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA242 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.3mol/L, and pH value is 7.6, and it comprises: be calculated in mass percent, the polyglycol of 0.5%, the bovine serum albumin(BSA) of 5% and 0.03% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.1mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 5%, the polyglycol of 2%, the sucrose of 1% and the second surface activating agent of 0.1%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA242 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are Tween-20.
Embodiment 6
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA72-4 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.3mol/L, and pH value is 7.6, and it comprises: be calculated in mass percent, the polyglycol of 0.5%, the bovine serum albumin(BSA) of 2.5% and 0.05% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.01mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 3.5%, the polyglycol of 2.4%, the sucrose of 1.5% and the second surface activating agent of 0.1%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA72-4 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are Tween-20.
Embodiment 7
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-CA549 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.03mol/L, and pH value is 7.4, and it comprises: be calculated in mass percent, the polyglycol of 0.25%, the bovine serum albumin(BSA) of 5% and 0.04% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.03mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 2.5%, the polyglycol of 2%, the sucrose of 1% and the second surface activating agent of 0.05%.In step 4, change anti-CA 125 monoclonal antibody into anti-CA549 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are tetronic1307.
Embodiment 8
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-SCC monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.02mol/L, and pH value is 7.5, and it comprises: be calculated in mass percent, the polyglycol of 0.35%, the bovine serum albumin(BSA) of 1.5% and 0.02% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.03mol/L, and pH value is 7.4, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1.5%, the polyglycol of 2%, the trehalose of 1% and the second surface activating agent of 0.05%.In step 4, change anti-CA 125 monoclonal antibody into anti-SCC monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are tetronic1307.
Embodiment 9
On the basis of embodiment 1, difference is: in step 1 and step 2, change anti-CA 125 monoclonal antibody into anti-NMP22 monoclonal antibody; Damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.02mol/L, and pH value is 7.4, and it comprises: be calculated in mass percent, the polyglycol of 0.5%, the bovine serum albumin(BSA) of 3.5% and 0.01% first surface activating agent.In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.05mol/L, and pH value is 7.3, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1.5%, the polyglycol of 2%, the trehalose of 1% and the second surface activating agent of 0.05%.In step 4, change anti-CA 125 monoclonal antibody into anti-NMP22 monoclonal antibody; Damping fluid is identical.First surface activating agent and second surface activating agent are TrtonX-100.
Through test, its performance of glycoprotein tumor markers CA125 immuno-chromatographic test paper strip that embodiment 1 is prepared is best, it is highly sensitive, the method of the performance of test glycoprotein tumor markers CA125 immuno-chromatographic test paper strip is the method that those skilled in the art generally apply, and does not repeat at this.
Below the glycoprotein tumor markers CA125 immuno-chromatographic test paper strip that embodiment 1 is prepared is set forth further by the performance of test example to the glycoprotein tumor markers CA125 immuno-chromatographic test paper strip that the present invention proposes.
Test example 1
The glycoprotein tumor markers CA125 immuno-chromatographic test paper strip that embodiment 1 is prepared is detected, its concrete detection method is: pH value blood serum sample 20ul to be detected being added 500ul is 7.2, substance withdrawl syndrome is the phosphate buffer of 0.05mol/L, add in test card well again, after question response 3min, with the detection line in biological sensor interpretation detection window and nature controlling line, to obtain a result.
The drafting of standard working curve: the serum specimen getting artificial preparation be made into GA be 1000.0,500.0,200.0,100.0,50.0,20.0,10.0, the serum specimen of 0.0U/ml, measure its CA125 respectively again, to be made into value for X, actual measurement fluorescent value is Y drawing standard working curve, expression formula through statistical fit standard working curve lists regression equation: Y=2.032X-0.973, fitting coefficient square be R2=0.997.The results are shown in accompanying drawing 3, Fig. 3 is linearity test standard working curve, and it is 10 ~ 600U/ml that CA125 measures the range of linearity.
Test example 2
Replica test: replica test in batch: get same patients serum's sample, continuous duplicate detection 20 times, calculates CV value (value for coefficient of variation) simultaneously.Replica test between batch: same patients serum's sample is distributed into 20 parts, is stored in refrigerator, get 1 part of detection every day, totally 20 days, calculate CV value.In batch, CV value is 3.9%.Between batch, (in the daytime) CV value is 4.3%.According to the U.S. clinical trial room standardization council (NCCLS) documentation requirements batch in, batch between imprecision level should be less than 5%, the method meets the requirements.
Test example 3
Recovery test: according to interior addition method, adds respectively by the CA125 reference material of 100 μ l variable concentrations in 1000 μ l patients serum samples, and the recovery test carrying out high, medium and low variable concentrations detects.The recovery test result of high, medium and low variable concentrations is as shown in table 1, and average recovery rate is 97.0%, substantially meets clinical trial requirement.
Table 1
Test example 4
Interference test: measure CEA (560ng/ml), CA19-9 (400U/ml), CA15-3 (300U/ml) and CA50 (300U/ml) with test paper of the present invention, CA125 measured value is respectively 0.06U/ml, 0.28U/ml, 0.15U/ml, 0.24U/ml.Each numerical value is all lower than the normal contents 35U/ml of human body CA125 above, thus shows, measures in application in medical science, and above-mentioned interfering material measures CA125 without obvious interference to the inventive method.
Test example 5
Actual detection contrast test: the mensuration of the test strips of embodiment 1 being carried out to aspect of performance, get the fresh serum sample of 178 routine patients, every part is carried out double-blind study detection with Roche company CA125 electrochemiluminescence quantitative detecting reagent and the inventive method respectively.Result application correlation regression and paired t-test analytical approach are compared.Result shows, and two method correlativitys are good, no significant difference (n=178, Y=1.013X+0.766, r=0.973, t=0.0736, P>0.05).Regression curve is shown in accompanying drawing 4.
From above-mentioned detection, detection method has higher sensitivity, and realization batch in, batch between accurate quantification detection while there is good repeatability.
The detection glycoprotein tumor markers immuno-chromatographic test paper strip that the present invention proposes, it needs whole blood, serum, the plasma sample of trace, the content quantitatively detecting glycoprotein tumor markers can be realized in 3 ~ 5 minutes, substantially increase the speed of examination, have highly sensitive, specificity good and the simple advantage of structure, and its preparation method is simple, is easy to large-scale production.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a glycoprotein tumor markers immuno-chromatographic test paper strip, is characterized in that, comprise the sample pad, bond pad, analyzing film, the adsorptive pads that contact with each other successively, bottom is provided with liner, and described analyzing film is provided with detection line and nature controlling line; Be fixed with the antibody for detectable antigens of phenyl boric acid label and fluorescent material marks in described bond pad, detection line contains the antibody reacted with testing sample, and nature controlling line contains antibody aitiogenic with phenyl boric acid label.
2. glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 1, is characterized in that: described sample pad, bond pad, analyzing film, the adsorptive pads contacted with each other successively has the overlapping region of 1 ~ 2mm mutually.
3. glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 2, described analyzing film is arranged at the below of bond pad and adsorptive pads, and described sample pad is arranged at the top of described bond pad.
4. according to the glycoprotein tumor markers immuno-chromatographic test paper strip in claim 1-3 described in any one, it is characterized in that, described detection line and nature controlling line interval 5mm.
5. an application for glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 1, is characterized in that, it may be used for detecting biological sample, and detected object is the content detection of glycoprotein tumor markers in whole blood, serum, blood plasma.
6. the application of glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 5, it is characterized in that, detection method is for add in sample pad by sample drop, from adsorptive pads side by syphonic effect, liquid is flowed, after liquid comes into contact detection line and nature controlling line, drawn the concentration of testing sample by the mode of instrument reading fluorescence intensity.
7. a preparation method for glycoprotein tumor markers immuno-chromatographic test paper strip described in claim 1, is characterized in that comprising the steps:
(1) preparation of phenyl boric acid label: by fluorescent dye compound respectively with damping fluid dilution, add phenyl boric acid material dissolves liquid respectively, stir evenly, room temperature reaction at least 1 hour, carry out separation and purification by gel column, collect label, preserve with after damping fluid dilution mixing;
(2) preparation of sample pad: with cellulose membrane as sample pad solid phase material, soak with 0.01 ~ 0.3M phosphate buffer of 0.01% ~ 0.5% polyglycol, 1% ~ 5% bovine serum albumin(BSA), 0.01% ~ 0.05% surfactant, described ph value of buffer solution is 7.2 ~ 7.6, after immersion treatment, take out after sample pad being put into vacuum drying chamber inner drying, vacuum seal is for subsequent use;
(3) preparation of bond pad: with glass fibre element film as bond pad solid phase material, with the 0.01 ~ 0.1M pH7.2 phosphate buffer dilution phosphor material label containing 1% ~ 5% bovine serum albumin(BSA), 0.1 ~ 2% polyglycol, 0.5 ~ 2% sucrose, 0.01% ~ 0.1% surfactant, make suspension, be sprayed on glass fibre element film, take out after bond pad is put into vacuum drying chamber inner drying, vacuum seal is for subsequent use;
(4) preparation of analyzing film: the antibody used with damping fluid dilution detection line and nature controlling line is to being applicable to concentration, adopt on the Membrane jetter detection line that is sprayed on analyzing film respectively and nature controlling line position, analyzing film after spray film is put into vacuum drying chamber, and after dry, taking-up vacuum seal is for subsequent use;
(5) preparation of adsorptive pads: select filter paper as adsorptive pads solid phase material, it is saved backup at dry environment;
Above step (1) is to step (5) not order restriction;
(6) preparation of finished product test strips: according to reaction sequence, first sticks to analyzing film on liner centre position, analyzing film upper end adhere to adsorptive pads, adsorptive pads above analyzing film, the two overlap 1 ~ 2mm; At analyzing film lower end adhesive bond thing pad, bond pad above analyzing film, the two overlap 1 ~ 2mm; Again bond pad lower end paste sample pad, sample pad above bond pad, the two overlap 1 ~ 2mm; Liner and the sample pad of pasting above, bond pad, analyzing film and adsorptive pads are together cut shaping.
8. the preparation method of glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 7, is characterized in that, described antibody is anti-glycoprotein tumor markers monoclonal antibody, sheep anti-mouse igg antibody.
9. the preparation method of glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 7, it is characterized in that, described liner is made up of pet material.
10. the preparation method of glycoprotein tumor markers immuno-chromatographic test paper strip according to claim 7, it is characterized in that, described phenyl boric acid material is phenyl boronic acid derivative, described phenyl boronic acid derivative has the chemical group of mark fluorescent dyestuff, the excitation light spectral limit of described fluorescent dye is 390 ~ 420nm, and wavelength of transmitted light scope is 600 ~ 700nm.
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