CN205539004U - Detect NGAL and glycated haemoglobin's test paper - Google Patents
Detect NGAL and glycated haemoglobin's test paper Download PDFInfo
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Abstract
The utility model relates to an immunity chromatography detects technical field, concretely relates to detect NGAL and glycated haemoglobin's test paper, including sample pad, combination pad, nitrocellulose membranes, the paper that absorbs water, detection line a, detection line b, matter accuse line and PVC bottom plate, the sample pad combine to fill up nitrocellulose membranes reaches absorb water paper in proper order the overlap joint be in on the PVC bottom plate, detection line a, detection line b matter accuse line is peridium in nitrocellulose membranes in proper order, it has NGAL, hbA1c monoclonal antibody - colloidal gold tag layer to combine to fill up the peridium, detection line a peridium has the NGAL monoclonal antibody, detection line b peridium has the hbA1c monoclonal antibody, matter accuse solenoid is had goat -anti mouse igG, a detect NGAL and glycated haemoglobin's test paper of this scheme of adoption have sensitivity height, specificity strong, easy and simple to handle, criticize between difference little, detect quick, accurate advantage.
Description
Technical field
This utility model relates to immuno-chromatographic assay technology field, is specifically related to a kind of use colloidal gold immunity chromatography to measure human blood neutrophil gelatinase-associated lipocalin NGAL and a kind of Test paper detecting NGAL and glycolated hemoglobin of saccharification hemoglobin content.
Background technology
NGAL molecular weight is 25kD, is No. 2 members of lipocalin protein lipocalin superfamily, and Kjeldsen found in human neutrophil in 1993.NGAL and Gelatinase B, i.e. GELB (MMP-9) covalent bond, form the heterodimeric protein of 1351kD, human neutrophils's granulocyte secrete.Research shows, body is exposed under extraneous harmful physics and chemistry and biological factor, and whole blood NGAL concentration is apparently higher than normal level.Modern study shows, under physiological status, NGAL not only expresses at neutrophilic granulocyte, also all has expression at some other tissue such as parietal cell, hepatic duct cell, small intestinal paneth's cell and renal proximal tubular cell etc..NGAL participates in different physiology and pathological process, relates to each stage that development occurs of inflammatory immune response, tumor, and inseparable with the generation of injury of kidney development.Renal cells NGAL high expressed is caused so that it is can be detected in blood and urine, and raise and raise early than serum creatinine in ischemia, poisoning equivalent damage factor.
NGAL, as new A KI early diagnosis marker, all can detect in the case of ischemic, septic, renal transplantation and early diabetes etc. in urine and blood, and highly sensitive, can be as diagnosis index earlier.
Glycolated hemoglobin (HbA1c) is the product that in blood of human body, endoerythrocytic hemoglobin is combined with blood glucose.It is irreversible reaction that the combination of blood glucose and hemoglobin generates glycolated hemoglobin, and is directly proportional to blood sugar concentration, and keeps about 120 days, it is possible to before observing 120 days.The changes of contents of glycolated hemoglobin can be as diabetes monitoring and the index of diagnosis.
China nephrotic has reached 1.5 hundred million people, and wherein diabetics has been up to 98,000,000 people, other detection methods due to time length, costly.Monitor for China's diabetes and nephropathy and diagnosis brings huge difficulty.
In consideration of it, this utility model research and devise and a kind of detect neutrophil gelatinase-associated lipocalin NGAL and a kind of Test paper detecting NGAL and glycolated hemoglobin of glycolated hemoglobin in human blood.
Utility model content
The purpose of this utility model is to provide a kind of highly sensitive, high specificity, easy and simple to handle, differences between batches are little, neutrophil gelatinase-associated lipocalin NGAL content and a kind of Test paper detecting NGAL and glycolated hemoglobin of glycolated hemoglobin (HbA1c) in the other detection human blood that detects quick, accurate and applicable bed, in order to monitor the early diagnosis and therapy of acute injury of kidney, diabetes.
For achieving the above object, this utility model provides a kind of Test paper detecting NGAL and glycolated hemoglobin, including sample pad, pad, nitrocellulose filter, absorbent paper, detection line a, detection line b, nature controlling line and PVC base plate, described sample pad, described pad, described nitrocellulose filter and described absorbent paper are overlapped on described PVC base plate successively, described detection line a, detection line b, described nature controlling line are coated in nitrocellulose filter successively, and described pad is coated with NGAL, HbA1c monoclonal antibody colloid gold label nitride layer；Described detection line a is coated with NGAL monoclonal antibody, and described detection line b is coated with HbA1c monoclonal antibody, and described nature controlling line is coated with sheep anti-mouse igg.
The preparation method of a kind of Test paper detecting NGAL and glycolated hemoglobin of this utility model, comprises the following steps:
Step one, the preparation of gold colloidal: take 1 500mL round-bottomed flask, add 100mL deionized water, round-bottomed flask terminate a reflux condensing tube, use temperature shelves 350 DEG C that it is heated in constant temperature blender with magnetic force, rotating speed 420r/min, is heated to 5min, adds 125 μ L 1% gold chlorides.Continue heating 5min to after fluidized state, boiling standard be water temperature be 100 DEG C, 250 μ L 10% citric acid three sodium solutions are rapidly joined in round-bottomed flask, in round-bottomed flask, solution is by red rear continuation reacting by heating 10min of purple stain, until after solution is bright, stopping heating, continue to stir 5min with 420r/min, being cooled to room temperature, 4 DEG C seal preservation, i.e. prepare gold colloidal.
Step 2, the preparation of gold labeling antibody: take 1mL gold colloidal in 1.5mL centrifuge tube, regulate pH to 8.5.The HbA1c monoclonal antibody of the NGAL monoclonal antibody of 10 μ g/mL, 8ug/mL is added in centrifuge tube, stand 10min after mixing, in centrifuge tube, add 100 μ L 10%BSA solution, after mixing, stand 5min, prepare gold labeling antibody, i.e. NGAL, HbA1c antibody colloidal gold label.
Prepared golden labeling antibody solution is centrifuged 10min in 4 DEG C of 13500r/min, removes supernatant, precipitation gold mark dilute solution is diluted to original volume 3/2, saves backup in 4 DEG C.
Step 3, NGAL monoclonal antibody, HbA1c monoclonal antibody, sheep anti-mouse igg are coated: take and be diluted to 0.5mg/mL NGAL monoclonal antibody, 0.5mg/mL HbA1c monoclonal antibody and 1.0mg/mL sheep anti-mouse igg, use dispent to draw film instrument and are coated the detection line a to nitrocellulose filter, detection line b and nature controlling line position respectively.Wherein containing 0.3% methanol in 0.5mg/mL NGAL monoclonal antibody, containing 2.5% sucrose in 0.5mg/mLHbA1c monoclonal antibody, detection line a is 14mm away from nature controlling line distance, and detection line b is 8mm away from nature controlling line distance, drawing film concentration and be 1 μ L/cm, platform translational speed is 40mm/s.
Step 4, the assembling of test strips: (1) by nitrocellulose filter, i.e. NC film, and absorbent paper is pasted on PVC base plate respectively, 2mm above NC film is pushed down in absorbent paper；(2) the PVC base plate pasting NC film and absorbent paper is cut on cutting cutter reagent paper (3) wide for 4mm and sample pad and pad are cut into the wide bar of 4mm, pad is soaked in gold labeling antibody solution simultaneously, and it is positioned in 37 degrees Celsius of constant temperature and humidity drying casees drying, prepare gold mark pad；(4) the gold mark pad after drying carries out cutting by length 1cm.Gold pad is pasted on PVC base plate, pushes down 2mm below NC film, push down 2mm below gold mark pad by sample pad simultaneously.
Compared with prior art, this utility model has the advantage that
1, neutrophil gelatinase-associated lipocalin NGAL and a kind of Test paper detecting NGAL and glycolated hemoglobin of glycolated hemoglobin in detection human blood of the present utility model, have the advantages such as high specificity, highly sensitive, the detection time is short, differences between batches are little, result is reliable and stable
2, the test strip of the present utility model detection time is short, and Turnaround Time is fast.
3, test strip of the present utility model is easy and simple to handle, it is not necessary to professional operates.
4, this utility model realizes programming operations to the preparation of gold colloidal, greatly reduces differences between batches.
5, the antibody labeling time of the present utility model is short, shortens more than 30min than Conventional temporal.
6, sample pad of the present utility model and gold pad have releasability and the water absorbing capacity of excellence, have more high accuracy.
7, in this utility model, antibody mode of being coated on NC film can improve stability and the hydrophilic of immuno-chromatographic test paper strip.
8, this utility model uses sodium chloride destruction method to determine final antibody labeling concentration, reduces antibody consumption, saved cost while improving test strips sensitivity.
Accompanying drawing explanation
Fig. 1 is the structural representation of a kind of Test paper detecting NGAL and glycolated hemoglobin of this utility model:
Fig. 2 is a kind of Test paper detection ideograph detecting NGAL and glycolated hemoglobin of this utility model；
Fig. 3 is the range of linearity dependency of the NGAL preparing a kind of Test paper detecting NGAL and glycolated hemoglobin of this utility model；
Fig. 4 is the range of linearity dependency of the HbA1c preparing a kind of Test paper detecting NGAL and glycolated hemoglobin of this utility model.
Wherein, 1 is sample pad, and 2 is pad, and 3 is nitrocellulose filter, and 4 is absorbent paper, and 5 is detection line a, and 6 is detection line b, and 7 is nature controlling line, and 8 is PVC base plate, and 9 is well, and T is chromatography direction.
Detailed description of the invention
With detailed description of the invention, this utility model is described in further detail below in conjunction with the accompanying drawings:
This utility model discloses a kind of Test paper detecting NGAL and glycolated hemoglobin as shown in Figure 1, 2, including sample pad 1, pad 2, nitrocellulose filter 3, absorbent paper 4, detection line a 5, detection line b 6 nature controlling line 7 and PVC base plate 8.Described sample pad 1, described pad 2, described nitrocellulose filter 3 and described absorbent paper 4 are bonded on described PVC base plate 8 successively；Described pad 2 is coated with NGAL, HbA1c clonal antibody colloid gold label thing；It is coated with neutrophil gelatinase-associated lipocalin NGAL monoclonal antibody on described detection line a 5, described detection line b 6 is coated with glycolated hemoglobin monoclonal antibody, described nature controlling line 7 is coated with sheep anti-mouse igg.
A kind of preparation method preparing a kind of Test paper detecting NGAL and glycolated hemoglobin, comprises the following steps:
Step one, the preparation of gold colloidal: take the round-bottomed flask of a 500mL, add 100mL deionized water, a reflux condensing tube is terminated on round-bottomed flask, use temperature to keep off 350 DEG C in constant temperature blender with magnetic force it is heated, rotating speed 420r/min, it is heated to 5min, adds 125uL 1% gold chloride.Continue heating 5min to after fluidized state, boiling standard be water temperature be 100 DEG C, 250 μ L 10% citric acid three sodium solutions are rapidly joined in round-bottomed flask, in round-bottomed flask, solution is by red rear continuation reacting by heating 10min of purple stain, until after solution is bright, stopping heating, continue to stir 5min with 420r/min, being cooled to room temperature, 4 DEG C seal preservation, i.e. prepare gold colloidal.
Step 2, the preparation of gold labeling antibody: take 1mL gold colloidal in 1.5mL centrifuge tube, regulate pH to 8.5.The HbA1c monoclonal antibody of the NGAL monoclonal antibody of 10 μ g/mL, 8ug/mL is added in centrifuge tube, stand 10min after mixing, in centrifuge tube, add 100 μ L 10%BSA solution, after mixing, stand 5min, prepare gold labeling antibody, i.e. NGAL, HbA1c antibody colloidal gold label.
Prepared golden labeling antibody solution is centrifuged 10min in 4 DEG C of 13500r/min, removes supernatant, precipitation gold mark dilute solution is diluted to original volume 3/2, saves backup in 4 DEG C.
Step 3, NGAL monoclonal antibody, HbA1c monoclonal antibody, sheep anti-mouse igg are coated: take and be diluted to 0.5mg/mL NGAL monoclonal antibody, 0.5mg/mL HbA1c monoclonal antibody and 1.0mg/mL sheep anti-mouse igg, use dispent to draw film instrument and are coated the detection line a to nitrocellulose filter, detection line b and nature controlling line position respectively.Wherein containing 0.3% methanol in 0.5mg/mL NGAL monoclonal antibody, containing 2.5% sucrose in 0.5mg/mLHbA1c monoclonal antibody, detection line a is 14mm away from nature controlling line distance, and detection line b is 8mm away from nature controlling line distance, drawing film concentration and be 1 μ L/cm, platform translational speed is 40mm/s.
Step 4, the assembling of test strips: (1) by nitrocellulose filter, i.e. NC film, and absorbent paper is pasted on PVC base plate respectively, 2mm above NC film is pushed down in absorbent paper；(2) the PVC base plate pasting NC film and absorbent paper is cut into the wide test strips of 4mm on cutting cutter；(3) sample pad and pad are cut into the wide bar of 4mm, pad are soaked in gold labeling antibody solution simultaneously, and are positioned in 37 degrees Celsius of constant temperature and humidity drying casees drying, prepare gold mark pad；(4) the gold mark pad after drying carries out cutting by length 1cm.Gold pad is pasted on PVC base plate, pushes down 2mm below NC film, push down 2mm below gold mark pad by sample pad simultaneously.
Optimal way as implementing: the cleaning of container used to experiment before described step one: first experiment glass container is soaked in the chloroformic solution of 5% dichlorodimethylsilane 1 minute, distilled water flushing after drying at room temperature, then drying for standby.
Optimal way as implementing: in described step one, deionized water resistivity used is 18.2 Ω.
Optimal way as implementing: in described step 2, gold mark diluent is to be 7.4 containing 3% beta-schardinger dextrin-, 0.01% casein, 0.5%BSA, the pH of 0.05% tween, the PBS of 0.01M.
Optimal way as implementing: in described step 4 (3), also include the step that sample pad is carried out pre-treatment, the strip of 4mm will be cut into respectively by sample pad cutting cutter, with containing 5% sucrose, 1%BSA, the PBS of the 0.01M that pH is 7.6 of 0.2% tween.
The determination of antibody optimum mark amount:
Sodium chloride destroys method: take 20 1.5mL centrifuge tubes, to it from 1 20 numberings, is separately added into 1mL colloidal gold solution, is subsequently adding 10 μ l 0.1M K2CO3Regulation pH, mixing.1 10 pipes add 0 μ g, 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g neutrophil gelatinase-associated lipocalin NGAL antibody, 11 20 pipes add 0 μ g, 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g glycolated hemoglobin antibody, concussion mixing.Last 1 to No. 20 pipe 100 μ L 10%NaCl solution respectively, stand the color change observing every centrifuge tube, illustrate that when there being centrifuge tube color to become purple this antibody concentration is not to be best suitable for label concentration, color keeps red constant label concentration for being suitable for label concentration, and keeping red constant minimum mark concentration is minimum mark amount.
Table one: the determination of neutrophil gelatinase-associated lipocalin NGAL antibody optimum protein labelled amount
Table two: glycolated hemoglobin antibody optimum mark amount determines
The determination of antibody optimum mark pH value
Sodium chloride destroys method: take 7 1.5mL centrifuge tubes, to it from 17 numberings, is separately added into 1mL colloidal gold solution, is then adjusted by colloidal gold solution pH value to 6,7,7.5,8,8.5,9,9.5 respectively by 17 sequence numbers, mixing.Add 10ng neutrophil gelatinase-associated lipocalin NGAL antibody and 8ng glycolated hemoglobin antibody, concussion mixing.Last 1 to No. 7 pipe 100 μ L 10%NaCl solution respectively, stand the color change observing every centrifuge tube, illustrate that when there being centrifuge tube color to become purple this pH value is not to be best suitable for labelling pH value, and color keeps red constant pH value for being best suitable for labelling pH value.
Table three: the determination of optimum mark pH value
Test strips performance test
The sensitivity of 1 test strips
0.5mg/mL neutrophil gelatinase-associated lipocalin NGAL standard solution negative whole blood is diluted to respectively the series of standards solution of 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1500ng/mL, each normal concentration takes 50 μ l, add in 200 μ L0.01M pH7.6PBS, mixing, take 75 μ L and add well, detect after timing 3min.Instrument can detect that the minimum OD value corresponding concentration producing change is 20ng/mL, and therefore the minimum detectability of neutrophil gelatinase-associated lipocalin NGAL is 20ng/mL.Taking patient whole blood's sample that HbAle egg is 16%, be diluted to the series of standards concentration of 0%, 1.5%, 3%, 6%, 12%, 16% respectively, each normal concentration takes 50 μ l, add in 200 μ L0.01M pH7.6PBS, mixing, takes 75 μ L and adds well, detect after timing 3min.Instrument can detect that the minimum OD value corresponding concentration producing change is 3%, and therefore the minimum detectability of glycolated hemoglobin is 3%.
The reperformance test of 2 test strips
Compound concentration is 0ng/mL (0%), NGAL and the HbA1c hybrid standard product solution of 50ng/mL (6%), 1000ng/mL (16%), is the concentration of HbA1c inside bracket.Take and detect with a batch of 90 test strips, each concentration is measured by 30 test strips, test OD value, concentration be the CV value of 0ng/mL (0%) be 7%, the CV value of 50ng/mL (6%) be 8%, the CV value of 1000ng/mL (16%) be 6.5%.According to testing result, this test strips has preferable repeatability.
The stability test of 3 test strips
Test strips is stored in equipped with in the aluminium foil bag of desiccant, is placed in 37 DEG C of thermostatic drying chambers, seal and preserve.Tested after 1,2,3,4 weeks respectively.Compound concentration is 0ng/mL (0%), NGAL and the HbA1c hybrid standard product solution of 50ng/mL (6%), 1000ng/mL (16%), is the concentration of HbA1c, tests inside bracket.At the 4th week, each concentration standards detection OD value did not reduce.Illustrate that this test strips can stably be deposited 2 years.
The drafting of standard curve
1.NGAl Specification Curve of Increasing
The NGAl standard substance negative whole blood taking 0.5mg/mL is diluted to the series of standards concentration of 0ng/mL, 20ng/mL, 50ng/mL, 125ng/mL, 312ng/mL, 782ng/mL, 1500ng/mL respectively.Each concentration retest 3 times, averages, and with concentration as X, draws standard working curve with OD value for Y, and listing equation through the expression formula of statistical fit standard working curve is Y=3.28638/ [1+ (X/457.86033)-2.32345]+0.11853, fitting coefficient R2=0.99618. result is shown in accompanying drawing 3, and Fig. 3 is linearity test standard working curve, and NGAL measurement range is 20ng/mL～1500ng/mL.
2.HbA1c Specification Curve of Increasing
Take patient's anticoagulated whole blood specimen that HbA1c is 16%, it is configured to the whole blood sample that HbA1c is 1.5%, 3%, 6%, 8%, 16%, each concentration retest 3 times, record OD value, with concentration as X, measuring OD value is Y drafting standard working curve, and listing equation through the expression formula of fit standard working curve is: Y=2.81517/ [1+ (X/6.39003)-2.69146]-0.09493, fitting coefficient R2=0.99342. result is shown in accompanying drawing 4, and Fig. 4 is linearity test standard working curve.HbA1c measurement range is 3%～16%.
Interference test
One, a fresh anticoagulated whole blood specimen is divided into 6 parts, every part of 1mL, 1st, 2 parts add glucose and are respectively the 100 μ L height sugar substances of 30mmol/L, 50mmol/L, 3rd, 4 parts add total bilirubin and are respectively the serum 100 μ L of 0.2mmol/L, 0.4mmol/L, 5th, 6 parts add triglyceride and are respectively the serum 100 μ l of 7mmol/L, 10mmol/L, measure HbA1c and NGAL value after mixing.
Two, gather patient whole blood's specimen, be injected separately into containing EDTA, heparin, structure 3 kinds of anticoagulant of rafter acid sodium, after mixing, measure HbA1c and NGAL value.
Three, result shows, is below 30mmol/L high sugar specimen at glucose, and total bilirubin is the jaundice specimen of below 0.2mmol/L and blood fat specimen that triglyceride is below 10mmol/L measures HbA1c and NGAL without substantially interfering with to this method.Use EDTA, anticoagulant heparin agent to experimental result also without substantially interfering with.
All deformation that the scientific research personnel of this area directly derives from this utility model disclosure or associates, are all considered as protection domain of the present utility model.
Claims (1)
1. the Test paper detecting NGAL and glycolated hemoglobin, it is characterized in that: include sample pad, pad, nitrocellulose filter, absorbent paper, detection line a, detection line b, nature controlling line and PVC base plate, described sample pad, described pad, described nitrocellulose filter and described absorbent paper are overlapped on described PVC base plate successively, described detection line a, detection line b, described nature controlling line are coated in nitrocellulose filter successively, and described pad is coated with NGAL, HbA1c monoclonal antibody colloid gold label nitride layer；Described detection line a is coated with NGAL monoclonal antibody, and described detection line b is coated with HbA1c monoclonal antibody, and described nature controlling line is coated with sheep anti-mouse igg.
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