CN106443012B - A kind of test strips and preparation method thereof and the application in microdose urine protein and β2-microglobulin joint-detection - Google Patents
A kind of test strips and preparation method thereof and the application in microdose urine protein and β2-microglobulin joint-detection Download PDFInfo
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- 238000012360 testing method Methods 0.000 title claims abstract description 64
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- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 29
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- 108010088751 Albumins Proteins 0.000 claims abstract description 48
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 44
- 239000007788 liquid Substances 0.000 claims description 37
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- 235000021240 caseins Nutrition 0.000 claims description 13
- 241001494479 Pecora Species 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 11
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 11
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 11
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 10
- 239000005018 casein Substances 0.000 claims description 9
- 239000003365 glass fiber Substances 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000000020 Nitrocellulose Substances 0.000 claims description 6
- 229920001220 nitrocellulos Polymers 0.000 claims description 6
- 239000003381 stabilizer Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims 1
- 102000036639 antigens Human genes 0.000 abstract description 22
- 108091007433 antigens Proteins 0.000 abstract description 22
- 239000000427 antigen Substances 0.000 abstract description 15
- 238000002203 pretreatment Methods 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
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- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 12
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- 238000002372 labelling Methods 0.000 description 5
- 206010020772 Hypertension Diseases 0.000 description 4
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 4
- LUVOJBWJNHWVNG-UHFFFAOYSA-N [Na].[Na].[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O Chemical compound [Na].[Na].[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O LUVOJBWJNHWVNG-UHFFFAOYSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 229940021722 caseins Drugs 0.000 description 4
- 235000019628 coolness Nutrition 0.000 description 4
- 230000003907 kidney function Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 150000003625 trehaloses Chemical class 0.000 description 4
- 238000010790 dilution Methods 0.000 description 3
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- 235000013601 eggs Nutrition 0.000 description 3
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- 238000002965 ELISA Methods 0.000 description 2
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- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
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- 231100001027 nephrosis Toxicity 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
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- 208000014674 injury Diseases 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
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- 210000003296 saliva Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to technical field of biological chemistry detection more particularly to a kind of test strips and preparation method thereof and the application in microdose urine protein and β2-microglobulin joint-detection.Test strips provided by the invention, the sample pad being disposed on bottom plate, gold-labelled pad, chromatographic film, blotting paper；Wherein, gold-labelled pad, the antibody or antigen of certain content are coated in chromatographic film so that the detection limit of good control test strips is just the cut off values of albumin and β2-microglobulin.Also, the antibody concentration that test strips provided by the invention use enables the detection of albumin and β2-microglobulin to be carried out at the same time without interfering with each other.Therefore, test strips using the present invention can realize the direct detection to urine specimen, the step of saving pre-treatment, and its detection more quickly, it is reliable.Experiment shows that test strips accuracy rate provided by the invention is higher, and the preparation method very simple of the test strips.
Description
Technical field
The present invention relates to technical field of biological chemistry detection more particularly to a kind of test strips and preparation method thereof with urinate it is micro-
Measure the application in albumin and β2-microglobulin joint-detection.
Background technology
Microdose urine protein (MAU) refers to occurring micro albumin in urine.Albumin is normal in a kind of blood
Protein, under normal circumstances, the albumin in urine is few for body metabolism, and albumin is no more than 20 μ g in every liter of urine（<20μg/
mL）.When lesion occurs for renal blood vessels, the function of kidney filtration protein (especially albumin) is changed, albumin in urine
Amount will be increased to 20 μ g/mL or more.Therefore, clinically commonly used microdose urine protein monitors the generation of nephrosis, and
As detection diabetes, the important indicator of hypertension, angiocardiopathy, nephrosis injury of blood vessel.
β2-microglobulin（β2-MG）It is a kind of small molecule ball egg generated by lymphocyte, blood platelet, polymorphonuclear leukocyte
In vain, molecular mass 11800, the single chain polypeptide being made of 99 amino acid.β 2-MG are widely present in blood plasma, urine, brain ridge
In liquid, saliva and colostrum.The synthetic ratio of normal person β 2-MG and fairly constant from the burst size on cell membrane, β 2-MG can be from
Glomerulus freely filters, and 99.9% absorbs in proximal tubular, and decomposes and destroy in renal cells；So positive reason
Under condition, the discharge of β 2-MG is very micro, usually not more than 0.2 μ g/mL.When proximal convoluted tubule is slightly damaged, β 2-MG are urinated
It obviously increases.Diabetes, hypertensive patient's early stage urine β 2-MG and its kidney function damage degree are significantly correlated.
As it can be seen that accurately early diagnosis of the content of albumin and β 2-MG to a variety of diseases, early treatment in detection urine
There are important reference value and clinical meaning.And it as can realizes to microdose urine protein and urinal beta2 microglobulin joint-detection, then
The recall rate of diabetes Renal function in early period damage can be improved, caused by effectively being clinical diagnosis hypertension and diabetes
Renal function in early period damage provides important reference frame.
Currently, the testing principle of the Test paper of commercially available β 2-MG mostly uses double antibody sandwich method.To other quality testings to be measured
When survey, the high sensitivity of double antibody sandwich method can play testing result more positive effect.But experiment shows using double
Antibody sandwich detects, and to the minimum detection limit of albumin up to 200ng/mL, 2ng/ml is limited to the detection of β 2-MG.It should
Detection limits hundreds of times also lower than contents of the MAU and β 2-MG in normal human.Test strips differentiate determinand content whether be more than
Normal value is realized by whether generating band, and it is more than threshold value to generate band and then prompt determinand content.However, double antibody presss from both sides
The too low detection limit of heart method but brings puzzlement to the detection of MAU and β 2-MG.Detection limit it is too low, cause the test paper before use,
It needs to be diluted urine sample using specific dilution, otherwise the testing result of Healthy People also will present the positive.And it is dilute
Releasing must carry out according to specific multiple, not only increase the complexity of operation, and operating error is also easy pair in dilution
Testing result has an impact.
Can more effectively be that clinical diagnosis hypertension and diabetes are drawn if be capable of providing suitable for the test strips of detection limit
The Renal function in early period damage risen provides important reference frame.
Invention content
In view of this, the technical problem to be solved in the present invention be to provide a kind of test strips and preparation method thereof with urinate it is micro-
Measure the application in albumin and β2-microglobulin joint-detection.ELISA test strip limit provided by the invention is reasonable.
Test strips provided by the invention, the sample pad being disposed on bottom plate, gold-labelled pad, chromatographic film, blotting paper；
Gold-labelled pad supports the albumin antibody of colloid gold label and the β2-microglobulin antibody of colloid gold label；
The amount of the albumin antibody of colloid gold label is calculated as 0.33 μ g/cm with the amount of albumin antibody2；
The amount of the β2-microglobulin antibody of colloid gold label is calculated as 0.11 μ g/cm with the amount of β2-microglobulin antibody2；
T1 lines and T2 lines is arranged in chromatographic film, and T1 lines, which are drawn, albumin；T2 lines, which are drawn, β2-microglobulin；
The amount of albumin is with albuminometer for 3.5 μ g/cm；
The amount of β2-microglobulin is calculated as 1.17 μ g/cm with β2-microglobulin.
C lines are nature controlling line, and C lines are also set up in the chromatographic film of test strips provided by the invention；The C lines, which are drawn, sheep anti mouse.
Prolong chromatography direction in chromatographic film and sets gradually T2 lines, T1 lines, C lines.
In the present invention, the material of bottom plate is PVC；The material of sample pad is glass fibre；The material of gold-labelled pad is glass fibers
Dimension；The material of chromatographic film is nitrocellulose.
The width of test strips provided by the invention is 3.5mm.
The principle of test strips provided by the invention be by competition law to albumin in urine and β2-microglobulin simultaneously into
Row detection.By adjusting the concentration of sample diluting liquid on the antibody, antigen and sample pad loaded in test strips, to good control
The detection limit of test strips processed is just the cut off values of albumin and β2-microglobulin.And then it realizes and urine sample is directly detected
Without carrying out special pre-treatment（Dilution or concentration）.Also, the antibody concentration that test strips provided by the invention use makes
The detection of albumin and β2-microglobulin can be carried out at the same time without interfering with each other.Therefore, test strips provided by the invention can
The accuracy detected to albumin and β2-microglobulin is improved, more effectively judges the 2 microballoon egg of albumin and β in urine specimen
Whether white be more than normal contents.Its easy to operate, rapid reaction, high specificity, be suitble to Site Detection and it is economical and practical the advantages that.
The preparation method of test strips of the present invention, including：
Step 1：With the PB solution treatment sample pads containing casein；
Step 2：The β2-microglobulin antibody of the albumin antibody of colloid gold label and colloid gold label is spread to gold-labelled pad；
Step 3：Albumin and β2-microglobulin are crossed respectively to chromatographic film；
Step 4：The sample pad that step 1 ~ 3 obtains, gold-labelled pad, chromatographic film are laid with to bottom plate successively, re-lay suction
After water paper, test strips are made after cutting；
The amount of the albumin antibody of colloid gold label is calculated as 0.33 μ g/cm with the amount of albumin antibody2；
The amount of the β2-microglobulin antibody of colloid gold label is calculated as 0.11 μ g/cm with the amount of β2-microglobulin antibody2；
The amount of albumin is 3.5 μ g/cm；
The amount of β2-microglobulin is 1.17 μ g/cm.
In the present invention, step 1 includes：Sample diluting liquid is spread to sample pad；
Sample diluting liquid is the PB solution comprising casein, and wherein the mass fraction of casein is a concentration of of 0.5%, PB
0.02mol/L；
The dosage of sample diluting liquid is 5mL/90cm2。
The pH value of the sample diluting liquid is 7.4.
PB solution is phosphate buffer, is formulated by sodium dihydrogen phosphate, disodium hydrogen phosphate and water.
The paving is to sample pad specifically, make sample pad and sample diluting liquid haptoreaction 5min, and after draining, 37 DEG C are reacted
16h。
In the present invention, step 2 includes：
Step A：Albumin is dissolved to a concentration of 3mg/ml of albumin with the PB solution containing trehalose；A liquid is made；
Step B：β2-microglobulin is dissolved to a concentration of 1mg/ml of β2-microglobulin with the PB solution containing trehalose；
B liquid is made；
Step C：The A liquid, B liquid are crossed to chromatographic film；
The usage amount of the A liquid is 35 μ L/30cm；The usage amount of the B liquid is 35 μ L/30cm.
A concentration of 0.01mol/L of PB in the PB solution containing trehalose；The mass fraction of trehalose is 3%.
A described stroke of most use stroke film instrument carries out 42 DEG C of reaction 2h after stroke film.
Step 2 further includes the steps that drawing C lines, specially：Sheep anti mouse is dissolved to goat-anti with the PB solution containing trehalose
A concentration of 1mg/ml of mouse；It is crossed to chromatographic film with the dosage of 35 μ L/30cm；PB's is dense in the PB solution containing trehalose
Degree is 0.01mol/L；The mass fraction of trehalose is 3%.
In the present invention, step 3 includes：
Step a：Prepare colloidal gold solution；
Step b：It is quiet after the colloidal gold solution, albumin antibody, β2-microglobulin antibody are mixed with solution of potassium carbonate
Set 20min；Obtained solution a；
Step c：The solution a is mixed with stabilizer, after standing 10min, after centrifugation, reject supernatant, with suspension
Mixing, obtained solution b；
Step d：The solution b is spread to gold mark film, 37 DEG C of reaction 4h.
A concentration of 6 μ g/mL of albumin antibody in the solution a；A concentration of 2 μ g/mL of β2-microglobulin antibody；
The volume ratio of the suspension and solution a are 1:1；
It is 5mL/90cm that the solution b, which spreads to gold and marks the dosage of film,2。
The preparation of colloidal gold solution uses trisodium citrate reduction method.
Specifically, the preparation method of colloidal gold solution is：The trisodium citrate for being 1% by 15 mL ~ 20mL mass fractions
Aqueous solution is mixed with 1L water boil 2min after, be added 10mL mass fractions be 2% ~ 4% aqueous solution of chloraurate, ebuillition of heated reaction
10min is settled to 1L after cooling.
A concentration of 0.2mol/L of potassium carbonate in the solution of potassium carbonate.
The volume ratio of the colloidal gold solution and solution of potassium carbonate is 5:0.03.
Stabilizer described in step c is PEG20000 solution, and wherein the mass fraction of PEG20000 is 1%.Stabilizer with it is molten
The volume ratio of liquid a is 5:0.1.
The rotating speed centrifuged described in step c is 12000 turns, and temperature is 4 DEG C, time 25min.
The suspension is the PB solution containing sucrose, PEG20000 and casein.
The pH value of suspension is 7.4；The mass fraction of sucrose is 5%；The mass fraction of PEG20000 is 1%；Casein
Mass fraction is 1%.A concentration of 0.02mol/L of PB solution.
Two detection lines, respectively T1 and T2 is arranged in test strips provided by the invention, is respectively used to detection albumin and β 2
Microglobulin realizes the synchronous detection of the two.The preparation method of test strips provided by the invention is easy to operate, in preparation process
In, the concentration and dosage of each solution are strictly controlled, so that the test strips prepared can have suitable detection to limit, so as to
It is enough to realize to whether albumin in sample and β2-microglobulin are fast and accurately detected more than normal value.
Application of the test strips provided by the invention in preparing microdose urine protein and β2-microglobulin joint-detection product.
Test strips provided by the invention are that urine sample is directly added dropwise in sample to the testing principle of microdose urine protein
After on pad, sample enters gold mark film along chromatography direction, and albumin and β2-microglobulin in sample and the antigen on gold mark film are competing
It strives and combines gold labeling antibody, if nonantigenic presence in measuring samples, gold labeling antibody first and T lines during swimming（T1 lines and T2
Line）Envelope antigen combines, and forms the visible red line of naked eyes, and extra gold labeling antibody continues swimming and nature controlling line（C lines）Sheep anti mouse two
Anti-binding, also forms the visible red line of a naked eyes, this result is judged to feminine gender.If with the presence of antigen, sample in measuring samples
In antigen, envelope antigen competition and gold labeling antibody combine, envelope antigen is suppressed, and antigen in sample and gold labeling antibody combine
After continue swimming, recombined until with the secondary antibody of C lines, at this point, T lines（T1 lines or T2 lines）It does not develop the color, and the aobvious red of C lines, knot
Fruit is judged to the positive（T1 lines or T2 lines are colourless, and C lines are red, for the positive）；If C lines do not develop the color in result, test strips have failed,
It cannot reuse.
The present invention also provides a kind of microdose urine proteins and β2-microglobulin combined detection kit, including the present invention to carry
The test strips of confession.
The present invention also provides a kind of joint-detection microdose urine proteins and the united method of β2-microglobulin with the present invention
The test strips of offer are detected sample to be tested；The sample to be tested is not diluted urine.
Test strips provided by the invention, the sample pad being disposed on bottom plate, gold-labelled pad, chromatographic film, blotting paper；
Wherein, gold-labelled pad, the antibody or antigen of certain content are coated in chromatographic film so that the detection of good control test strips
Limit is just the cut off values of albumin and β2-microglobulin.Also, the antibody concentration that test strips provided by the invention use makes
The detection of albumin and β2-microglobulin can be carried out at the same time without interfering with each other.Therefore, test strips using the present invention can
The step of realizing the direct detection to urine specimen, saving pre-treatment, and its detection is more quickly, reliably.Experiment shows this
It is higher to invent the test strips accuracy rate provided, and the preparation method very simple of the test strips.
Description of the drawings
Fig. 1 shows positive test symbol；
Fig. 2 shows negative result.
Specific implementation mode
The present invention provides a kind of test strips and preparation method thereof to combine inspection in microdose urine protein and β2-microglobulin
Application in survey, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular
It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this
Invention.The method of the present invention and application are described by preferred embodiment, and related personnel can obviously not depart from this
Methods herein and application are modified or are suitably changed and combined in invention content, spirit and scope, to realize and apply
The technology of the present invention.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is further explained：
Embodiment 1
It is prepared by colloid gold particle：It takes 1L water to be added in round-bottomed flask, places and be heated to boiling in electric jacket, take 1% citric acid
Trisodium 15mL ~ 20mL additions are boiled 2 minutes, and 2% ~ 4% chlorauric acid solution 10mL is added, and make the reaction 10min coolings of its ebuillition of heated
After to be settled to 1L etc. to be used.
Draw film：Take sheep anti mouse, microdose urine protein antigen（Purchased from HYTEST）, β2-microglobulin antigen（Purchased from Guilin English
U.S. special biology）, sheep anti mouse, microdose urine protein antigen and β2-microglobulin are resisted with the PB+3% trehaloses of 0.01mol/L respectively
It is 35 μ L/30 cm that original is diluted to 1 mg/mL, 3mg/mL and 1mg/mL usage amount successively, and film is drawn to nitrocellulose with film instrument is drawn
Film（2 cm of Sai Duolisi, 140 films）On, 42 DEG C of reaction 2h.
Label：It takes 30 μ L of 5mL colloidal gold solution 0.2mol/L solution of potassium carbonate to be uniformly mixed, takes microdose urine protein anti-
Body（HYTEST 6B11）30 μ g, β2-microglobulin antibody（Great Britain and America spy 1E9）10 μ g are uniformly mixed static 20 minutes, and 100 μ are added
The 1%PEG20000 of L, static 10 minutes, 12000 turns 4 DEG C centrifuged 25 minutes, abandon supernatant, and colloidal gold suspension is added（PH value 7.4
0.02mol/L PB+5% sucrose+1%PEG20000+1% caseins）5mL is spread to the glass fibre of 3cm × 30cm, 37 DEG C
React 4h.
Fill item：By sample pad, gold-labelled pad, the chromatographic film pulled and blotting paper are attached to successively on PVC bottom plates, are uniformly cut
At the film item of 3.5 mm wide.
Comparative example 1
It is prepared by colloid gold particle：It takes 1L water to be added in round-bottomed flask, places and be heated to boiling in electric jacket, take 1% citric acid
Trisodium 15mL ~ 20mL additions are boiled 2 minutes, and 2% ~ 4% chlorauric acid solution 10mL is added, and make the reaction 10min coolings of its ebuillition of heated
After to be settled to 1L etc. to be used.
Draw film：Take sheep anti mouse, microdose urine protein antigen（Purchased from HYTEST）, β2-microglobulin antigen（Purchased from Guilin English
U.S. special biology）, sheep anti mouse, microdose urine protein antigen and β2-microglobulin are resisted with the PB+3% trehaloses of 0.01mol/L respectively
It is 35 μ L/30 cm that original is diluted to 1mg/mL, 3mg/mL and 1mg/mL usage amount successively, and film is drawn to nitrocellulose with film instrument is drawn
Film（2 cm of Sai Duolisi, 140 films）On, 42 DEG C of reaction 2h.
Label：It takes 30 μ L of 5mL colloidal gold solution 0.2mol/L solution of potassium carbonate to be uniformly mixed, takes microdose urine protein anti-
Body（HYTEST 6B11）50 μ g, β2-microglobulin antibody（Great Britain and America spy 1E9）20 μ g are uniformly mixed static 20 minutes, and 100 μ are added
The 1%PEG20000 of L, static 10 minutes, 12000 turns 4 DEG C centrifuged 25 minutes, abandon supernatant, and colloidal gold suspension is added（PH value 7.4
0.02mol/L PB+5% sucrose+1%PEG20000+1% caseins）5mL is spread to the glass fibre of 3cm × 30cm, 37 DEG C
React 4h.
Fill item：By sample pad, gold-labelled pad, the chromatographic film pulled and blotting paper are attached to successively on PVC bottom plates, are uniformly cut
At the film item of 3.5 mm wide.
Comparative example 2
It is prepared by colloid gold particle：It takes 1L water to be added in round-bottomed flask, places and be heated to boiling in electric jacket, take 1% citric acid
Trisodium 15mL ~ 20mL additions are boiled 2 minutes, and 2% ~ 4% chlorauric acid solution 10mL is added, and make the reaction 10min coolings of its ebuillition of heated
After to be settled to 1L etc. to be used.
Draw film：Take sheep anti mouse, microdose urine protein antigen（Purchased from HYTEST）, β2-microglobulin antigen（Purchased from Guilin English
U.S. special biology）, sheep anti mouse, microdose urine protein antigen and β2-microglobulin are resisted with the PB+3% trehaloses of 0.01mol/L respectively
It is 35 μ L/30 cm that original is diluted to 1 mg/mL, 3mg/mL and 1mg/mL usage amount successively, and film is drawn to nitrocellulose with film instrument is drawn
Film（2 cm of Sai Duolisi, 140 films）On, 42 DEG C of reaction 2h.
Label：It takes 30 μ L of 5mL colloidal gold solution 0.2mol/L solution of potassium carbonate to be uniformly mixed, takes microdose urine protein anti-
Body（HYTEST 6B11）15 μ g, β2-microglobulin antibody（Great Britain and America spy 1E9）5 μ g are uniformly mixed static 20 minutes, and 100 μ L are added
1%PEG20000, static 10 minutes, 12000 turns 4 DEG C centrifuged 25 minutes, abandon supernatant, and colloidal gold suspension is added（PH value 7.4
0.02mol/L PB+5% sucrose+1%PEG20000+1% caseins）5mL is spread to the glass fibre of 3cm × 30cm, 37 DEG C
React 4h.
Fill item：By sample pad, gold-labelled pad, the chromatographic film pulled and blotting paper are attached to successively on PVC bottom plates, are uniformly cut
At the film item of 3.5 mm wide.
Comparative example 3
It is prepared by colloid gold particle：It takes 1L water to be added in round-bottomed flask, places and be heated to boiling in electric jacket, take 1% citric acid
Trisodium 15mL ~ 20mL additions are boiled 2 minutes, and 2% ~ 4% chlorauric acid solution 10mL is added, and make the reaction 10min coolings of its ebuillition of heated
After to be settled to 1L etc. to be used.
Draw film：Take sheep anti mouse, microdose urine protein antibody（HYTEST 1C8）, β2-microglobulin antibody（The special biology of Great Britain and America
6D11）, respectively use 0.01mol/L PB+3% trehaloses by sheep anti mouse, microdose urine protein antibody and β2-microglobulin antibody according to
Secondary 1 mg/mL, 1mg/mL and 1mg/mL usage amount that is diluted to is 35 μ L/30 cm, and film is drawn to nitrocellulose filter with film instrument is drawn
（2 cm of Sai Duolisi, 140 films）On, 42 DEG C of reaction 2h.
Label：It takes 30 μ L of 5mL colloidal gold solution 0.2mol/L solution of potassium carbonate to be uniformly mixed, takes microdose urine protein anti-
Body（HYTEST 6B11）25 μ g, β2-microglobulin antibody（Great Britain and America spy 1E9）15 μ g are uniformly mixed static 20 minutes, and 100 μ are added
The 1%PEG20000 of L, static 10 minutes, 12000 turns 4 DEG C centrifuged 25 minutes, abandon supernatant, and colloidal gold suspension is added（PH value 7.4
0.02mol/L PB+5% sucrose+1%PEG20000+1% caseins）5mL is spread to the glass fibre of 3cm × 30cm, 37 DEG C
React 4h.
Fill item：By sample pad, gold-labelled pad, the chromatographic film pulled and blotting paper are attached to successively on PVC bottom plates, are uniformly cut
At the film item of 3.5 mm wide.
Embodiment 2
By microdose urine protein antigen and β2-microglobulin antigen diluent at 0.1ng/ml, 0.5ng/ml, 2ng/ml,
20ng/ml, 200ng/ml, 1 μ g/ml, 2 μ g/ml, 10 μ g/ml, 20 μ g/ml, 50 μ g/ml take 30 respectively, and each condition is with 3
Item, which is detected embodiment 1 and the test strips of comparative example 1 ~ 3, directly to be reacted after antigen addition sample pad, sees result after five minutes.
As a result such as table 1：
1 ELISA test strip of table limits
And the cut off values of microdose urine protein (MAU) and β2-microglobulin (β 2-MG) are respectively 20 μ g/ml and 0.2 μ
g/ml。
As shown in Table 1：
1 test strips of embodiment are respectively 20 μ g/ml and 200ng/ml to microdose urine protein and β2-microglobulin detection limit,
It is consistent with cut off values.
The detection limit of 1 microdose urine protein of comparative example and β2-microglobulin is respectively 50 μ g/ml or more and 1 μ g/ml,
The detection limit of 2 microdose urine protein of comparative example and β2-microglobulin is respectively 10ug/ml and 200ng/ml,
Comparative example 3（Double antibody sandwich method, the prior art）The detection of microdose urine protein and β2-microglobulin limits
200ng/ml and 2ng/ml is less than 100 times of cut off values.
The detection limit of test strips made from embodiment 1 is most appropriate from sensitivity, and sample can be straight without pre-treatment
It connects for detecting.
Embodiment 3
Microdose urine protein and each 10 parts of β2-microglobulin positive sample are taken, 10 parts of normal person's sample is detected, with reality
The test strips for applying example 1 are detected.See result within 5 minutes.Testing result such as table 2：
2 testing result of table
Table 2 shows, microdose urine protein be in positive sample beta-2 microglobulin some be positive, and 2 microballoon eggs of β
White is that equally also some is positive to positive sample microdose urine protein.And in ' negative ' specimens, then testing result both
It is all negative.The result is consistent completely with expection, illustrates that test strips accuracy rate provided by the invention is very high.
Wherein, positive findings such as Fig. 1, negative findings such as Fig. 2.
It the above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (6)
1. a kind of test strips, which is characterized in that including bottom plate and the sample pad being successively set on the bottom plate, gold-labelled pad, layer
Analyse film, blotting paper；
The gold-labelled pad is supported with the albumin antibody of colloid gold label and the β2-microglobulin antibody of colloid gold label；
The amount of the albumin antibody of the colloid gold label is calculated as 0.33 μ g/cm with the amount of albumin antibody2；
The amount of the β2-microglobulin antibody of the colloid gold label is calculated as 0.11 μ g/cm with the amount of β2-microglobulin antibody2；
T1 lines and T2 lines are provided in the chromatographic film, the T1 lines, which are drawn, albumin；The T2 lines, which are drawn, β2-microglobulin；
The amount of the albumin is 3.5 μ g/cm；
The amount of the β2-microglobulin is 1.17 μ g/cm；
Preparation method includes：
Step 1：Sample diluting liquid is spread to sample pad；The sample diluting liquid is the phosphate buffer comprising casein,
The mass fraction of middle casein is 0.5%, phosphatic a concentration of 0.02mol/L；The dosage of the sample diluting liquid is 5mL/
90cm2；
Step 2：Prepare colloidal gold solution；The colloidal gold solution, albumin antibody, β2-microglobulin antibody and potassium carbonate is molten
20min, obtained solution a are stood after liquid mixing；The solution a is mixed with stabilizer, after standing 10min, through centrifugation, reject supernatant
It after liquid, is mixed with suspension, obtained solution b；The solution b is spread to gold-labelled pad, 37 DEG C of reaction 4h；
A concentration of 6 μ g/mL of albumin antibody in the solution a；A concentration of 2 μ g/mL of β2-microglobulin antibody；The suspension
The volume ratio of liquid and solution a are 1:1；It is 5mL/90cm that the solution b, which is spread to the dosage of gold-labelled pad,2；
Step 3：Albumin is dissolved to a concentration of 3mg/ml of albumin with the phosphate buffer containing trehalose；A is made
Liquid；β2-microglobulin is dissolved to a concentration of 1mg/ml of β2-microglobulin with the phosphate buffer containing trehalose；B is made
Liquid；The A liquid, B liquid are crossed to chromatographic film；
The usage amount of the A liquid is 35 μ L/30cm；The usage amount of the B liquid is 35 μ L/30cm；
Step 4：The sample pad that step 1 ~ 3 obtains, gold-labelled pad, chromatographic film are laid with to bottom plate successively, re-lay blotting paper
Afterwards, test strips are made after cutting.
2. test strips according to claim 1, which is characterized in that be additionally provided with C lines in the chromatographic film；The C lines are drawn
There is sheep anti mouse.
3. test strips according to claim 1, which is characterized in that
The material of the bottom plate is PVC；
The material of the sample pad is glass fibre；
The material of the gold-labelled pad is glass fibre；
The material of the chromatographic film is nitrocellulose.
4. the preparation method of claim 1 ~ 3 any one of them test strips, which is characterized in that including：
Step 1：Sample diluting liquid is spread to sample pad；The sample diluting liquid is the phosphate buffer comprising casein,
The mass fraction of middle casein is 0.5%, phosphatic a concentration of 0.02mol/L；The dosage of the sample diluting liquid is 5mL/
90cm2；
Step 2：Prepare colloidal gold solution；The colloidal gold solution, albumin antibody, β2-microglobulin antibody and potassium carbonate is molten
20min is stood after liquid mixing；Obtained solution a；The solution a is mixed with stabilizer, after standing 10min, through centrifugation, reject supernatant
It after liquid, is mixed with suspension, obtained solution b；The solution b is spread to gold mark film, 37 DEG C of reaction 4h；It is white in the solution a
A concentration of 6 μ g/mL of protein antibodies；A concentration of 2 μ g/mL of β2-microglobulin antibody；The volume ratio of the suspension and solution a
It is 1:1；It is 5mL/90cm that the solution b, which spreads to gold and marks the dosage of film,2；
Step 3：Albumin is dissolved to a concentration of 3mg/ml of albumin with the phosphate buffer containing trehalose；A is made
Liquid；β2-microglobulin is dissolved to a concentration of 1mg/ml of β2-microglobulin with the phosphate buffer containing trehalose；B is made
Liquid；The A liquid, B liquid are crossed to chromatographic film；The usage amount of the A liquid is 35 μ L/30cm；The usage amount of the B liquid is 35 μ
L/30cm；
Step 4：The sample pad that step 1 ~ 3 obtains, gold-labelled pad, chromatographic film are laid with to bottom plate successively, re-lay blotting paper
Afterwards, test strips are made after cutting.
5. claim 1 ~ 3 any one of them test strips are preparing microdose urine protein and β2-microglobulin joint-detection product
In application.
6. a kind of microdose urine protein and β2-microglobulin combined detection kit, which is characterized in that including claim 1 ~ 3 times
Test strips described in one.
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| CN109725159B (en) * | 2018-12-28 | 2021-10-08 | 江苏众红生物工程创药研究院有限公司 | Human beta2Quantitative detection test paper card of microglobulin and clinical application |
| CN117288966B (en) * | 2023-11-27 | 2024-02-27 | 山东康华生物医疗科技股份有限公司 | Kit for combined detection of IGF-1 and IGFBP-3 and lysate and buffer solution used by kit |
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