CN104515859A - Hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit and preparation method and detection method thereof - Google Patents
Hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit and preparation method and detection method thereof Download PDFInfo
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- CN104515859A CN104515859A CN201410790838.5A CN201410790838A CN104515859A CN 104515859 A CN104515859 A CN 104515859A CN 201410790838 A CN201410790838 A CN 201410790838A CN 104515859 A CN104515859 A CN 104515859A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
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Abstract
The invention provides a hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit. The hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit comprises an upper shell, a lower shell, an immunostrip, test paper and the like; a gold-labeled composite containing a hemoglobin monoclonal antibody, a hemoglobin-haptoglobin composite monoclonal antibody and a transferrin monoclonal antibody is scribed on a nitrocellulose membrane; 3 test lines, a quality control line (15) and a gold-labeled composite membrane scribing line (8) are arranged on the nitrocellulose membrane in parallel. The invention further provides a preparation method and a detection method of the hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit. The hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit is convenient and simple in operation, stable in performance and accurate in result, has a relatively good reference value for early diagnosis and identification of colorectal cancer or colon cancer tumor or other tumors with lower gastrointestinal bleeding symptoms in clinic, significantly improves the positive detection rate of digestive hemorrhagic diseases, is suitable for clinical hospital examination and household self-examination, and provides measures for large-scale general examination of such diseases.
Description
Technical field
The invention belongs to clinical in vitro diagnosis in vitro reagent technique field, be specially a kind of haemoglobin, Hb-Hp, transferrins joint inspection kit, applied immunology principle, lateral chromatography technology and chemical ortho-aminotoluene development process, by detecting haemoglobin, Hb-Hp, the transferrins in human faecal mass, find hemorrhage of lower digestive tract symptom, the lower digestive tract tumours such as the early diagnosis carcinoma of the rectum or colon cancer.
Background technology
Haemoglobin (Hb, Hemoglobin) in ight soil, also known as just occult blood (FOB, Fecal Occult Blood), is counted as one of the tumor markers of Diagnose Rectal Cancer, colon cancer at present clinically.Hoptoglobin (Hp, Haptoglobin, also referred to as haptoglobin or pearl globulin) is extensively present in the mankind and much mammiferous blood and other body fluid, and intraindividual comparision contents is stablized.Hoptoglobin has the ability in conjunction with free hemoglobin, effect is similar to the transport protein of haemoglobin, haemoglobin easily stablizes irreversible compound with hoptoglobin in conjunction with formation, is called Hb-Hp (Hb-Hp Complex).Except as except haemolysis diagnosis index, going deep in recent years along with research, hoptoglobin is also relevant to nauseating tumour, ephrosis, tissue damage equal altitudes.Transferrins (TF), the susceptibility detected and specificity and haemoglobin detect quite, but stability is apparently higher than haemoglobin, haemoglobin hold-up time in intestines and stomach in detected sample long and false negative result of being decomposed or occurring after destroying can be overcome.Alimentary canal is hemorrhage on a small quantity can not cause stool color change, only has and leans on just occult blood test to determine.Every disease of digestive tract causes hemorrhage on a small quantity, all stool with occult blood can be had, therefore occult blood check for diagnosis multiple hemorrhage of digestive tract disease have important value, be generaI investigation screening disease of digestive tract effective means, just occult blood test become people deepen continuously research focus.
Just occult blood at present clinically main detection method have chemical method and immunological method: what chemical method was conventional have ortho-aminotoluene method, phenolphthalin method, benzidine method, pyramidon method, Yu creates wooden fat method etc.Its design concept is substantially identical, be all the effect having catalysis peroxide breakdown based on the hemosiderin part in haemoglobin, the hydrogen peroxide in energy catalytic reagent, decomposes release new ecological oxygen, be oxidized above-mentioned chromogen substance and colour generation, the depth of colour developing has reacted the number of haemoglobin.But in actual applications because ight soil composition difference is very large, each laboratory concrete operations details is different, there is very big-difference, and lack specificity and accuracy by chemical method testing result.If containing haemoglobin, myoglobins in exogenous diet, the effect of its protoheme all can make experiment be positive, also can catalysis peroxide breakdown containing activated plant superoxide in a large amount of salad vegetable, occurs false positive reaction.Blood overstays in enteron aisle, haemoglobin sex change, then there will be the negative findings be not inconsistent with the state of an illness.In addition, if patient takes a large amount of vitamin Cs or other have the medicine of reductibility, superoxide can be reduced in an experiment, thus can not chromogen substance be oxidized, experiment also can be made to occur false negative.Therefore, chemical method is subject to disturbing factor is many, false positive rate is high, glacial acetic acid is made into reagent and easily forms the condition restrictions such as crystal, fails to be used widely, need badly a species specificity good, highly sensitive, detect detection method of fast, simply occulting blood.
Immunological method detects conventional method of occulting blood at present, mainly comprises immune monoclonal antibody method, enzyme linked immunosorbent assay, immune spot-ing, latex Immunity transmission turbidity, radioimmunodiffusion, reverse indirect blood coagulation, colloid gold label sandwich immunoassay method of inspection etc.The index that immunization detects is generally human hemoglobin or human red blood cell matrix, antibody used is antihuman hemoglobin antibody or anti-human erythrocyte matrix antibody, detect human hemoglobin and can detect the hemorrhage of any position of alimentary canal, detect human red blood cell matrix and can only detect hemorrhage of lower digestive tract, but, easily there is false negative reaction in haemoglobin sex change due to the mucus composition influence being subject to the effect of bacterium in enteron aisle and colorectal mucosa and producing.
The limitation that haemoglobin detects: immunochromatography single stage method is carry out specific binding human hemoglobin antigen with antihuman hemoglobin antibody at present, this method is easy, quick, only specifically for human hemoglobin epitope, substantially eliminate the interference of the factor such as diet and medicine, recommended a kind of method comparatively confirmed as fecal occult blood experiment by gastrointestinal endoscopy association of the World Health Organization (WHO).Colloidal Gold occult blood test has certain sensitivity, all can occur false negative higher or lower than this scope.Monoclonal antibody colloidal gold method sometimes can be failed to pinpoint a disease in diagnosis in detection hemorrhage of digestive tract, and its reason mainly contains:
A. haemoglobin sex change through digestive ferment degraded, has not had original immunogenicity;
, there is Postzone phenomenon in B. excessive massive haemorrhage and cause antigen excess in reaction system;
C. the haemoglobin antigenic structure of patient changes also can affect haemoglobin or Rollet's stroma and monoclonal antibody affinity.
Up to the present, the sensitivity of single stool blood FOB diagnostic method and specificity all need to be improved further.Often need the method joint-detection of two kinds or more clinically.Existing relevant combined detection reagent occurs in the market.
The detection of transferrins has very important value for the diagnosis of hemorrhage of digestive tract.Transferrins detects, and susceptibility and specificity and haemoglobin detect quite, but stability is apparently higher than haemoglobin, can overcome haemoglobin hold-up time in intestines and stomach in detected sample long and be decomposed or the false negative result of appearance after destroying.
Hb-Hp detects: what reaction of occulting blood was surveyed is with or without occulting blood, instead of with or without tumor-marker, reaction of occulting blood is positive and must confirms with additive method, such as intestines mirror etc., this is because the bleeding positive once in a while that may be caused by other factors, same occult blood reaction feminine gender is also likely false negative, and this forms compound because infantile tumour is hemorrhage on a small quantity with hoptoglobin in stool, and the anti-hemoglobin antibodies of this compound can not to identification, testing result is negative.The black high will of order of Japan is to the ight soil of 48 routine colon cancers or rectal cancer patient, its Hb-Hp (Hb-Hp) and haemoglobin (Hb) is detected with radioimmunology, the result positive is respectively 44 examples and 35 examples, here 9 examples are had at least just not occult blood person for false negative, that is the positive coincidence rate of stool blood Diagnose Rectal Cancer or colon cancer is measured only about 70%, and the compound of Measuring hemoglobin-hoptoglobin to the positive coincidence rate of the carcinoma of the rectum or colon cancer more than 90%, mensuration is caused just to occult blood to the carcinoma of the rectum or colon cancer 20%-30% false negative main cause: because alimentary canal is hemorrhage on a small quantity, the haemoglobin (Hb) discharged is combined into compound at the Hp in longer enteron aisle and ight soil and causes false negative of occulting blood, it is seriously undetected that haemoglobin in obvious detection ight soil diagnoses the tumor markers of whether suffering from the carcinoma of the rectum or colon cancer to have.
Often need the method joint-detection of two kinds or more clinically, in the market existing haemoglobin-transferrins joint inspection kit, haemoglobin-Hb-Hp compound joint inspection kit etc.If Chinese invention patent application publication No. is that CN101965515A (date of publication is on February 2nd, 2011) provides a kind of method and apparatus for detecting occult blood, comprise the transferrins and haemoglobin that detect in GI sample, and the transferrins detected is compared with the respective predetermined value of haemoglobin with transferrins with the amount of haemoglobin, go thus to push up hemorrhage from GI, and the detection using cross flow sandwich immune determinator to realize transferrins and haemoglobin is provided, this technical scheme lacks Hb-Hp and detects, lower to the positive coincidence rate of the carcinoma of the rectum or colon cancer.
Summary of the invention
The present invention aims to provide a kind of haemoglobin, Hb-Hp, transferrins joint inspection kit, detect haemoglobin by one simultaneously, Hb-Hp, the test strips of transferrins, chemistry ortho-aminotoluene test paper, on get stuck and under get stuck composition kit, the structure of this kit is simple, detect rapidly and efficiently, there is fine sensitivity and specificity, good reference value is had clinically to the early diagnosis of tumor of the hemorrhage of lower digestive tract such as the carcinoma of the rectum or colon cancer symptom and discriminating, the positive rate of hemorrhage of digestive tract disease is significantly increased.
For above essential technique problem, the corresponding technical solution scheme of the present invention is:
A kind of haemoglobin, Hb-Hp, transferrins joint inspection kit, comprise and getting stuck, under get stuck, be placed in get stuck and under get stuck between immunity test strip and Test paper, on get stuck and be provided with well, watch window and chemical test paper watch window, under get stuck and have first groove corresponding with immunity test strip position and second groove corresponding with chemical method test paper position, described immunity test strip comprises thieving paper, nitrocellulose filter, sample pad and PVC base plate, sample pad edge pastes on nitrocellulose filter, thieving paper edge pastes on nitrocellulose filter, nitrocellulose filter is fixed on PVC base plate, containing haemoglobin monoclonal antibody, the gold mark compound of Hb-Hp monoclonal antibody and transferrins monoclonal antibody is drawn on nitrocellulose filter, nitrocellulose filter is arranged with 3 detection lines in parallel, the gold mark compound of nature controlling line and close nitrocellulose filter bottom draws film line, nature controlling line place is coated with sheep anti-mouse igg, article 3, detection line place is coated with haemoglobin monoclonal antibody simultaneously, hapto-hemoglobin monoclonal antibody and transferrins monoclonal antibody, gold mark compound is drawn film line place and is contained haemoglobin monoclonal antibody, the gold mark compound of Hb-Hp monoclonal antibody and transferrins monoclonal antibody.
Be provided with protruding partition in the middle of above-mentioned first groove and the second groove corresponding with chemical method test paper position, the bottom of Test paper loading section and sample pad is separated by partition, and well is positioned at directly over partition place.
Be chemical method test paper and sample pad below above-mentioned well, well spills chemical method test paper and sample pad for circle or ellipse are also simultaneously outer, described nitrocellulose filter correspondence watch window placement.
Get stuck on above-mentioned and be provided with ID enrollment or/and Quick Response Code.Quick Response Code is according to the chequered with black and white graphic recording data symbol information of certain rule in plane distribution with certain specific geometric figure, it is an important technology during information identifies automatically, also be one of the key and core technology of Internet of Things industry, it is widely applied in many fields such as industry, business, national defence, traffic, finance, health cares as a kind of timely, accurate, reliable, economic data input and transmission means, and planar bar code technology now uses relative maturity.
Above-mentioned PVC base plate is the PVC base plate with sticky gum layer, can select J-C6 base plate, 6cm*30cm, PVC plastic offset plate, can commercialization buy.
Described sample pad is a layer thickness is 355.6-508.0um, the glass fibre of commercial 8964.
Present invention also offers the preparation method of above-described haemoglobin, Hb-Hp, transferrins joint inspection kit.
Described Test paper is the preparation of following method: mass concentration is respectively 5% (W/W) cumyl hydroperoxide, 5% (W/W) ortho-aminotoluene, after 10% (W/W) superoxol in mass ratio (cumyl hydroperoxide: ortho-aminotoluene: hydrogen peroxide=1:1:2) mixing, thieving paper is tailored into slice, takes out after immersion treatment and be drying to obtain;
Described sample pad preparation method is: be respectively 10% (W/W) sodium borate buffer liquid (PH9.0), 5% (W/W) casein, 5% (W/W) PVP-10,1% (W/W) sodium taurocholate, 1.8% (W/W) RHODASURF ON-870 (commercial surfactant), the preparation of 0.02% (W/W) Sodium azide by mass concentration, uniform spreading is in sample pad and maintain 37 DEG C, and relative humidity is less than 20% dry.
Described gold mark compound is drawn the have colored particle of film line selection in colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, dye granule, latex particle or fluorescent grain and is lined on nitrocellulose filter.
To be 60nm collaurum obtain for the mark of antihuman hemoglobin monoclonal antibody, anti-human hoptoglobin monoclonal antibody and anti-human transferrin's monoclonal antibody above-mentioned gold mark compound.
The preparation method of gold mark compound is: every treated flask of 2000ml takes 1500g ultrapure water, magnetic force heating stirrer IKA Digital is set to 120 DEG C, temperature probe inserts in flask liquid, after instant heating reaches design temperature, seethes with excitement and adds 10% chlorauric acid solution 30g after 1 minute.Continue heating and fire 1 minute, adjusting knob is to maximum (top) speed, add rapidly 1% citric acid three sodium solution 20g, continuing heating stops heating stopping stirring after 5 minutes, naturally cool to room temperature and obtain 60nm colloidal gold solution, the process of described flask is with potassium dichromate: water: the concentrated sulphuric acid is according to after soaking 8 hours after the mass ratio preparation treating fluid of 1:2:18, then milli-Q water 3 times.
The preparation process of the described gold mark compound containing haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody is:
(1) get the colloidal gold solution 100mL being cooled to room temperature, add the solution of potassium carbonate of 1mL, 0.1mol/L, stir 30 minutes, add 0.1mg antihuman hemoglobin monoclonal antibody, continue stirring 30 minutes;
(2) in the solution of step (1), add the BSA solution (bovine serum albumin(BSA)) of 10% of 1mL, stir 30 minutes, condition is 4 DEG C, 12000rpm, after centrifugal 30 minutes, remove supernatant, by the resuspended precipitation of damping fluid containing 10%BSA (bovine serum albumin(BSA)), 0.05% tween, 20% sucrose, 5% trehalose, 0.02% Sodium azide, 5%Tris, form the colloid gold label compound of the high concentration multiple of 5ml;
(3) Hb-Hp monoclonal antibody and transferrins monoclonal antibody, marks to step (2) according to step (1) equally, obtains corresponding colloid gold label compound;
After (4) three kinds of monoclonal antibodies have marked, and be evenly coated on nitrocellulose filter with 2ul/cm by the mass ratio of 1:1.5:1, wrap by good nitrocellulose filter (9) at 37 DEG C, relative humidity be less than 20% oven drying 8 hours for subsequent use.
On described nitrocellulose filter, the step of antibody bag quilt is: with the PB (PH7.2) of the 0.01M containing 0.5%BSA (bovine serum albumin(BSA)), 3% trehalose, 0.02% Sodium azide, haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody are diluted to 1.0mg/ml, 0.5mg/ml, 1.5mg/ml respectively; Sheep anti-mouse igg is diluted to 0.8mg/ml, and spray film instrument parallel scribing in order respectively on nitrocellulose filter with BioDotXYZ3060, discharge rate is set to 1.8ul/cm, draw a film complete after immediately as 37 DEG C of drying rooms, dry 8 hours for subsequent use.
Use the detection method of above-mentioned haemoglobin, Hb-Hp, transferrins joint inspection kit, when it is characterized in that detecting, in circular or oval well, drip 3-4 drip the sample liquid handled well by sample collecting device, 5 to 8 minutes interpretation immunity test strips; Meanwhile, drip the superoxol of 10% at chemical test paper watch window, observe color change, the result finally in conjunction with two kinds of detection methods judges final detection result:
, in strip watch window, all there is purple red zone at detection line and nature controlling line position in the full positive: for immunological method.Represent in sample to there is haemoglobin, Hb-Hp, transferrins.For chemical method, there is blue reaction, show that result is positive;
Full feminine gender: for immunological method, in strip watch window, there is purplish red colour band in nature controlling line position, and all do not occur purplish red colour band in detection line position, represent in sample there is not haemoglobin, Hb-Hp, transferrins.For chemical method, there is not blue reaction, show that result is negative;
Compared to existing technology, joint inspection kit provided by the invention and preparation method thereof, beneficial effect is:
(1) the present invention is haemoglobin, Hb-Hp, transferrins joint inspection kit, adopt immunochromatography colloidal gold technique in conjunction with chemical colour reaction method, fast, easy haemoglobin, Hb-Hp, the transferrins simultaneously detected in stool, the drawback of prior art can be eliminated, it is hemorrhage on a small quantity that the canceration of accurate discovery alimentary canal causes, improve Early rectal tumor or diagnosis of colon cancer accuracy rate, also can for monitoring when this disease of early diagnosis provides quick, easy means and drug therapy.Testing result haemoglobin, Hb-Hp, transferrins are all positive or haemoglobin, Hb-Hp (Hb-Hp) are all positive or haemoglobin, transferrins is all positive, then strong suspicion is hemorrhage of digestive tract, probably suffer from the lower digestive tract such as the carcinoma of the rectum or colon cancer tumour, advise that further diagnosis is confirmed, such as intestines mirror; If Hb-Hp, transferrins are all negative and haemoglobin (Hb) positive may cause due to sudden inflammation; Haemoglobin (Hb), Hb-Hp (Hb-Hp), transferrins (TF) are all negative patients, illustrate that alimentary canal is not hemorrhage.
(2) kit of the present invention uses ID or Quick Response Code, on the one hand, ID place can hand filling sample information, avoids obscuring of testing result.On the other hand, Quick Response Code can high density be encoded, and information capacity is large, can stored in a large amount of parts informations.Coding range is wide, can stored in information such as word, picture, sound.Fault-tolerant ability is strong, and have error correcting capability, surface is subject to greasy dirt, loss etc. and can be distinguished in the same old way.Reliability of decode is high, and the bit error rate is no more than 1/10000000th.Also have cost low, easily make, the advantages such as durable, Quick Response Code optionally can carry out testing result typing according to condition, have a well monitoring to the healing of patient and rehabilitation course, result queries is convenient, and it is also more handy that doctor plans as a whole therapeutic scheme.Even also there is meaning statistically in pathology.
The joint inspection kit high specificity of the present patent application, highly sensitive, easily store, dedicated technician need not operate, do not need any instrument and equipment, and result readability.Good reference value is had to the early diagnosis of tumor of the hemorrhage of lower digestive tract such as the carcinoma of the rectum or colon cancer symptom and discriminating, better to the positive rate effect of hemorrhage of digestive tract disease.
Accompanying drawing explanation
Accompanying drawing 1 is the stereoscopic model schematic diagram of this kit.
Accompanying drawing 2 is fractionation structural representations of this kit.
In figure, get stuck 1., 2. get stuck down, 3. well, 4. watch window, 5. the first groove, 6. test strips, 17. chemical test paper watch windows, 18.ID record, 19. Quick Response Codes, 20. chemical test paper, 21. second grooves, 22. cut off.
Accompanying drawing 3 is structural representations of immunity test strip.
Accompanying drawing 4 is structural representations of immunity test strip.
In figure: 7. sample pad, 8. gold mark compound draws film line, 9. nitrocellulose filter, 10. thieving paper, 11.PVC base plate, 12. anti-human transferrin's monoclonal antibody detection lines, 13. antihuman hemoglobins-hoptoglobin compound monoclonal antibody detection line, 14. antihuman hemoglobin monoclonal antibody detection lines, 15. nature controlling lines.
Accompanying drawing 5 is formalness schematic diagram of this kit front plastic casing.
From top to bottom Quick Response Code respectively in figure, ID enrollment, " C " is the position of control line, and " Hb " is the position of haemoglobin detection line, " Hb-Hp " is the position of Hb-Hp detection line, and " TF " is the position of transferrins detection line.Ellipse is sample dropping place, and square window is chemical test paper color development window bottom.
Accompanying drawing 6 is full positive findings schematic diagram of this kit.
In figure, 6 haemoglobins, Hb-Hp, transferrins are all positive.
Accompanying drawing 7 is full negative findings schematic diagram of this kit
In figure, 7 haemoglobins (Hb), Hb-Hp (Hb-Hp), transferrins (TF) are all negative.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further, but it is pointed out that joint inspection kit of the present invention is not limited to this specific shape or structure.Obviously be understandable that for those skilled in the art, even if the following description content does not make any adjustments or revises, also can be directly applied for the test strips in these unspecified other types or structure.
As shown in Fig. 1-Fig. 7, a kind of haemoglobin, Hb-Hp, transferrins joint inspection kit, comprise immunity test strip 6, chemical test paper 20 (ortho-aminotoluene test paper), on get stuck 1, under to get stuck 2 compositions.
Surface design that joint inspection kit gets stuck has a circle or oval well 3, watch window 4, chemical test paper watch window 17, ID record 18, Quick Response Code 19, under get stuck that 2 inside are provided with first groove 5 corresponding with test strips 6, second groove 21 corresponding with chemical method test paper, the plastics that prevent reagent from obscuring cut off 22, immunity test strip 6 and chemical method test paper 20 be placed in get stuck 1 and under get stuck between 2.Immunity test strip 6 is made up of PVC base plate 11, thieving paper 10, nitrocellulose filter 9 and sample pad 7.
Nitrocellulose filter 9 is pasted on PVC base plate 11, gold mark compound draws 8 strokes, film line on nitrocellulose filter 9, sample pad 7 small part and edge are pasted in nitrocellulose filter 9 lower end, the edge of thieving paper 10 is pasted at nitrocellulose filter 9 epimere, the described corresponding circle of sample pad 7 or oval well 3, the corresponding strip watch window 4 of described nitrocellulose filter 9.
Described immunity test strip 6 is one.The top of chemistry test paper 20 loading section and the bottom of sample pad 7 got stuck by plastics on partition 22 separate, prevent the interference that chemical substance is possible.The position of well 3 is just in time positioned at above partition 22 place.
The oarse-grained collaurum preparation process of 60nm: 2000ml is through special processing (potassium dichromate: water: the concentrated sulphuric acid=1:2:18, by soaking glass flask milli-Q water 3 times after 8 hours after this mass ratio preparation treating fluid) flask accurately take 1500g ultrapure water, IKA Digital is set to 120 DEG C, and temperature probe inserts in flask liquid.After heating reaches design temperature, seethe with excitement and add 10% chlorauric acid solution 30g after 1 minute.Continue heating and fire 1 minute, adjusting knob, to maximum (top) speed, adds rapidly 1% citric acid three sodium solution 20g.Continue heating 5 minutes, stop heating stopping stirring, naturally cool to room temperature for subsequent use.Above-mentioned ultrapure water refers to that resistivity reaches the water of 18.2M Ω * cm (25 DEG C).
General colloid gold particle is the most stable at 20-40nm, and more than 40nm, the homogeneity of colloid gold particle and stability will sharply reduce, but product sensitivity and raw material are tired and also can be increased along with the increase of particle.Patent utilization gold chloride-trisodium citrate system of the present invention, improves temperature regulating device, and change and accurate reaction time put and chooses high purity raw material, finally prepare highly stable, homogeneous colloidal gold solution.
The 60nm bulky grain collaurum of preparation is used for the mark of antihuman hemoglobin monoclonal antibody, anti-human hoptoglobin monoclonal antibody, anti-human transferrin's monoclonal antibody, the advantages such as have low input, high production, sensitivity is high, and specificity is good.
60nm bulky grain colloid gold label antihuman hemoglobin monoclonal antibody: get the above-mentioned colloidal gold solution 100mL being cooled to room temperature, add the solution of potassium carbonate of 1mL0.1mol/L, stirs 30 minutes.Add 0.1mg antihuman hemoglobin monoclonal antibody, continue stirring 30 minutes.Add BSA (bovine serum albumin(BSA)) solution of 10% of 1mL, stir 30 minutes.4 DEG C, 12000rpm, after centrifugal 30 minutes, removes supernatant, by the resuspended precipitation of sodium phosphate buffer of the 0.01mol/L PH7.0 containing 1%BSA (bovine serum albumin(BSA)), forms the antihuman hemoglobin colloid gold label thing of 5ml high concentration multiple.
60nm bulky grain colloid gold label Hb-Hp monoclonal antibody: get the above-mentioned colloidal gold solution 100mL being cooled to room temperature, add the solution of potassium carbonate of 1mL0.1mol/L, stirs 30 minutes.Add 0.1mg Hb-Hp monoclonal antibody, continue stirring 30 minutes.Add BSA (bovine serum albumin(BSA)) solution of 10% of 1mL, stir 30 minutes.4 DEG C, 12000rpm, after centrifugal 30 minutes, removes supernatant, by the resuspended precipitation of sodium phosphate buffer of the 0.01mol/L PH7.0 containing 1%BSA (bovine serum albumin(BSA)), form the Hb-Hp monoclonal antibody colloid gold label thing of 5ml high concentration multiple.
60nm bulky grain colloid gold label transferrins monoclonal antibody: get the above-mentioned colloidal gold solution 100mL being cooled to room temperature, add the solution of potassium carbonate of 1mL0.1mol/L, stirs 30 minutes.Add 0.1mg transferrins monoclonal antibody, continue stirring 30 minutes.Add BSA (bovine serum albumin(BSA)) solution of 10% of 1mL, stir 30 minutes.4 DEG C, 12000rpm, after centrifugal 30 minutes, removes supernatant, by the resuspended precipitation of sodium phosphate buffer of the 0.01mol/L PH7.0 containing 1%BSA (bovine serum albumin(BSA)), forms the transferrins monoclonal antibody colloid gold label thing of 5ml high concentration multiple.
BioDotXYZ3060 is pneumatic, and colloidal gold composite 8 is rule on nitrocellulose filter by micro-quantitation nozzle: the colloid gold label thing marked three kinds is respectively in Homogeneous phase mixing after the ratio PG2 dilution of 1:1.5:1, form colloidal gold composite 8, line is on nitrocellulose filter 9, bag by good nitrocellulose filter 9 at 37 DEG C, the oven drying 8 hours that relative humidity is less than 20%.PG2 formula is as following table:
| Name of material | Content (%) |
| Bovine serum albumin(BSA) | 10％ |
| Tris | 5％ |
| Sucrose | 20％ |
| Trehalose | 5％ |
| Tween | 0.05％ |
| Sodium azide | 0.02％ |
On nitrocellulose filter 9, the process of antibody bag quilt is: with the PB of PH7.2, haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody are diluted to 1.0mg/ml, 0.5mg/ml, 1.5mg/ml respectively, sheep anti-mouse igg is diluted to 0.8mg/ml BioDotXYZ3060 and sprays film instrument difference parallel scribing in order on nitrocellulose filter 9, and discharge rate is set to 1.8ul/cm.Draw film complete after be placed in 37 DEG C of drying rooms immediately, dry 8 hours for subsequent use.Coating buffer formula is as following table:
| Name of material | Content |
| Bovine serum albumin(BSA) | 0.5％ |
| PB | 0.01M |
| Trehalose | 3％ |
| Sodium azide | 0.02％ |
Described nitrocellulose filter 9 is provided with parallel nature controlling line 15 and detection line, gold mark compound draws film line 8.Control line 15 place is coated with sheep anti-mouse igg, detection line place is coated with antihuman hemoglobin monoclonal antibody, anti-human hoptoglobin monoclonal antibody, anti-human transferrin's monoclonal antibody simultaneously, and gold mark compound draws the gold mark compound containing haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody 12 in film line 8 place.
Described thieving paper 10 is paper materials that water-intake capacity is stronger, PVC base plate 11 is the PVC base plates with sticky gum layer, described sample pad 7 be one deck densification have certain thickness glass fibre, it is that nitrocellulose filter is embedded with colored particle that described gold mark compound draws film line 8, can be a kind of colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle or fluorescent grain.
Sample pad 7 processes: according to the form below formula (percentagewising) is prepared, and uniform spreading is in sample pad 7 and maintain 37 DEG C, and relative humidity is less than 20% dry.Sample pad formula is as table:
Viscosity PVC base plate 11 pastes bag successively by good nitrocellulose filter 9, sample pad 7 and thieving paper 10.
Chemistry test paper 20 (chemical method is occult blood detection test paper just) containing 5% (W/W) cumyl hydroperoxide, 5% (W/W) ortho-aminotoluene, 10% (W/W) superoxol.After above-mentioned solution in mass ratio (cumyl hydroperoxide: ortho-aminotoluene: hydrogen peroxide=1:1:2) mixing, thieving paper is tailored growth 6mm, the slice of wide 4mm, take out dry after immersion treatment, the water suction scraps of paper are fixed in the groove of glass lower end, have between thieving paper and glass plastics get stuck upper plastics cut off separate, avoid chemical substance to interfere with each other.
During detection, in circular or oval well, drip 3-4 drip the sample liquid handled well by sample collecting device, 5 to 8 minutes interpretation immunity test strips.Meanwhile, drip the superoxol of 10% at chemical test paper watch window, observe color change.Result finally in conjunction with two kinds of detection methods judges final detection result.
, in strip watch window, all there is purple red zone at detection line and nature controlling line position in the full positive: for immunological method.Represent in sample to there is haemoglobin, Hb-Hp, transferrins.For chemical method, there is blue reaction, show that result is positive.
Full feminine gender: for immunological method, in strip watch window, there is purplish red colour band in nature controlling line position, and all do not occur purplish red colour band in detection line position, represent in sample there is not haemoglobin, Hb-Hp, transferrins.For chemical method, there is not blue reaction, show that result is negative.
Invalid: in strip watch window, do not occur purplish red colour band in nature controlling line position, no matter detection line position occurs or does not all occur purplish red colour band, shows invalidate the test, should re-start detection.
Kit of the present invention is quick and precisely, easy to operate, stable performance, without any need for special instrument equipment, is applicable to the visiting of hospital and the self-inspection of family.Because this kit has larger reference significance to the early diagnosis of the lower digestive tract such as the carcinoma of the rectum or colon cancer tumour with discriminating clinically, add that the generaI investigation local basic diagnosis that fall behind relative to medical condition that the feature that it is quick, easy, accuracy is high is this type of disease provides a kind of means.
Kit sensitivity is adjustable in clinical meaning scope.Example haemoglobin (Hb) 200ng/ml, Hb-Hp (Hb-Hp) 25ng/ml, transferrins 40ng/ml are the values having clinical meaning.
Although give detailed description and explanation to the specific embodiment of the present invention above, but what should indicate is, those skilled in the art too much need not test and can implement the present invention is required for protection, the similar substitute of example: colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle, or fluorescent grain replaces colloid gold particle or transforms, or with the reagent that some reagent similar on physics and chemistry replaces the present invention to describe, or carry out various equivalence change and amendment, its function produced do not exceed that instructions and accompanying drawing contain yet spiritual time, all should within protection scope of the present invention.
Claims (13)
1. a haemoglobin, Hb-Hp, transferrins joint inspection kit, comprise get stuck (1), under get stuck (2), be placed in get stuck and under get stuck between immunity test strip (6) and Test paper (20), on get stuck and be provided with well (3), watch window (4) and chemical test paper watch window (17), under get stuck (2) have first groove (5) corresponding with immunity test strip (6) position and second groove (21) corresponding with chemical method test paper position, it is characterized in that immunity test strip (6) comprises thieving paper (10), nitrocellulose filter (9), sample pad (7) and PVC base plate (11), sample pad edge pastes on nitrocellulose filter, thieving paper edge pastes on nitrocellulose filter, nitrocellulose filter is fixed on PVC base plate, containing haemoglobin monoclonal antibody, the gold mark compound of Hb-Hp monoclonal antibody and transferrins monoclonal antibody is drawn on nitrocellulose filter, nitrocellulose filter is arranged with 3 detection lines in parallel, the gold mark compound of nature controlling line (15) and close nitrocellulose filter (9) bottom draws film line (8), nature controlling line (15) place is coated with sheep anti-mouse igg, article 3, detection line place is coated with haemoglobin monoclonal antibody simultaneously, hapto-hemoglobin monoclonal antibody and transferrins monoclonal antibody, gold mark compound is drawn film line place and is contained haemoglobin monoclonal antibody, the gold mark compound of Hb-Hp monoclonal antibody and transferrins monoclonal antibody.
2. haemoglobin, Hb-Hp, transferrins joint inspection kit as claimed in claim 1, it is characterized in that being provided with protruding partition (22) in the middle of the first groove (5) and the second groove (21) corresponding with chemical method test paper position, the bottom of Test paper (20) loading section and sample pad (7) is separated by cutting off (22), and well (3) is positioned at directly over partition (22) place.
3. haemoglobin, Hb-Hp, transferrins joint inspection kit as claimed in claim 2, it is characterized in that being chemical method test paper and sample pad (7) below well, well spills chemical method test paper and sample pad for circle or ellipse are also simultaneously outer, described nitrocellulose filter correspondence watch window (4) placement.
4. haemoglobin, Hb-Hp, transferrins joint inspection kit as claimed in claim 3, it is characterized in that getting stuck is provided with ID enrollment (18) or/and Quick Response Code (19).
5. haemoglobin, Hb-Hp, transferrins joint inspection kit as claimed in claim 4, is characterized in that PVC base plate (11) is the PVC base plate with sticky gum layer.
6. as claimed in claim 5 haemoglobin, Hb-Hp, transferrins joint inspection kit, is characterized in that sample pad (7) to be a layer thickness is the 8964 type glass fibre of 355.6-508.0um.
7. the preparation method of haemoglobin as claimed in claim 1, Hb-Hp, transferrins joint inspection kit, it is characterized in that Test paper (20) is the preparation of following method: mass concentration is respectively 5% cumyl hydroperoxide, 5% ortho-aminotoluene, mix in proportion with 10% superoxol, ratio is in mass ratio: cumyl hydroperoxide: ortho-aminotoluene: hydrogen peroxide=1:1:2, thieving paper is tailored into slice, takes out after immersion treatment and be drying to obtain;
Described sample pad preparation method is: the PH9.0 sodium borate buffer liquid, 5% casein, 5%PVP-10,1% sodium taurocholate, 1.8% product type that are respectively 10% by mass concentration are surfactant, the 0.02% Sodium azide preparation of RHODASURF ON-870, uniform spreading is in sample pad and maintain 37 DEG C, and relative humidity is dry under being less than 20% condition.
8. preparation method as claimed in claim 7, is characterized in that gold mark compound is drawn the colored particle that has that film line (8) is selected from colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, dye granule, latex particle or fluorescent grain and lined on nitrocellulose filter (9).
9. preparation method as claimed in claim 8, is characterized in that gold mark compound is 60nm collaurum and obtains for the mark of antihuman hemoglobin monoclonal antibody, anti-human hoptoglobin monoclonal antibody and anti-human transferrin's monoclonal antibody.
10. preparation method as claimed in claim 9, it is characterized in that the preparation method of gold mark compound is: every 2000ml flask takes 1500g ultrapure water, magnetic force heating stirrer IKA Digital is set to 120 DEG C, temperature probe inserts in flask liquid, after instant heating reaches design temperature, seethe with excitement and add 10% chlorauric acid solution 30g after 1 minute; Continue heating and fire 1 minute, adjusting knob is to maximum (top) speed, add rapidly 1% citric acid three sodium solution 20g, continuing heating stops heating stopping stirring after 5 minutes, naturally cool to room temperature and obtain 60nm colloidal gold solution, through by potassium dichromate before described flask uses: water: the mass ratio of the concentrated sulphuric acid=1:2:18 is prepared treating fluid and to be soaked after 8 hours milli-Q water 3 times.
11. preparation methods as claimed in claim 10, is characterized in that the preparation process of the gold mark compound containing haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody is:
(1) get the colloidal gold solution 100mL being cooled to room temperature, add the solution of potassium carbonate of 1mL, 0.1mol/L, stir 30 minutes, add 0.1mg antihuman hemoglobin monoclonal antibody, continue stirring 30 minutes;
(2) in the solution of step (1), add the bovine serum albumin solution of 10% of 1mL, stir 30 minutes, condition is 4 DEG C, 12000rpm, after centrifugal 30 minutes, remove supernatant, by the resuspended precipitation of damping fluid containing 10% bovine serum albumin(BSA), 0.05% tween, 20% sucrose, 5% trehalose, 0.02% Sodium azide, 5%Tris, form the colloid gold label compound of the high concentration multiple of 5ml;
(3) Hb-Hp monoclonal antibody and transferrins monoclonal antibody, marks to step (2) according to step (1) equally, obtains corresponding colloid gold label compound;
After (4) three kinds of monoclonal antibodies have marked, and be evenly coated on nitrocellulose filter (9) in the ratio of 1:1.5:1 with 2ul/cm, bag by good nitrocellulose filter (9) at 37 DEG C, relative humidity be less than 20% oven drying 8 hours for subsequent use.
12. preparation methods as claimed in claim 11, is characterized in that the step of antibody bag quilt on nitrocellulose filter is: with the phosphate buffer (PH7.2) of the 0.01M containing 0.5% bovine serum albumin(BSA), 3% trehalose, 0.02% Sodium azide, haemoglobin monoclonal antibody, Hb-Hp monoclonal antibody and transferrins monoclonal antibody are diluted to 1.0mg/ml, 0.5mg/ml, 1.5mg/ml respectively; Sheep anti-mouse igg is diluted to 0.8mg/ml, and spray film instrument parallel scribing in order respectively on nitrocellulose filter with BioDotXYZ3060, discharge rate is set to 1.8ul/cm, draw a film complete after immediately as 37 DEG C of drying rooms, dry 8 hours for subsequent use.
The detection method of the haemoglobin of 13. uses as described in one of claim 1-6, Hb-Hp, transferrins joint inspection kit, when it is characterized in that detecting, in circular or oval well, drip 3-4 drip the sample liquid handled well by sample collecting device, 5 to 8 minutes interpretation immunity test strips; Meanwhile, drip the superoxol of 10% at chemical test paper watch window, observe color change, the result finally in conjunction with two kinds of detection methods judges final detection result:
The full positive: for immunological method, in strip watch window, all there is purple red zone at detection line and nature controlling line position, represent in sample to there is haemoglobin, Hb-Hp, transferrins, for chemical method, there is blue reaction, show that result is positive;
Full feminine gender: for immunological method, in strip watch window, there is purplish red colour band in nature controlling line position, and all do not occur purplish red colour band in detection line position, represent in sample there is not haemoglobin, Hb-Hp, transferrins.For chemical method, there is not blue reaction, show that result is negative;
Invalid: in strip watch window, do not occur purplish red colour band in nature controlling line position, no matter detection line position occurs or does not all occur purplish red colour band, shows invalidate the test, should re-start detection.
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