CN103983792B - A kind of immunoglobulin (Ig) lateral chromatography detection system - Google Patents
A kind of immunoglobulin (Ig) lateral chromatography detection system Download PDFInfo
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- CN103983792B CN103983792B CN201410234215.XA CN201410234215A CN103983792B CN 103983792 B CN103983792 B CN 103983792B CN 201410234215 A CN201410234215 A CN 201410234215A CN 103983792 B CN103983792 B CN 103983792B
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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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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Abstract
The present invention relates to field of medicaments, be specifically related to based on lateral chromatography isolation technics for determination of immunoglobulin test strips and application thereof, the method is highly sensitive, high specificity.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to based on lateral chromatography isolation technics for determination of immunoglobulin lateral chromatography detection system.
Background technology
Immunoglobulin (Ig) (immunoglobulin, Ig) be one group of protein with antibody activity, Ig is produced by thick liquid cell, is mainly present in biosome blood and other body fluid (comprising tissue fluid and exocrine secretion), accounts for 20% of plasma proteins total amount; Also can be distributed in b cell surface.Immunoglobulin (Ig) great majority in human plasma are present in gamma globulin (gamma globulin).Immunoglobulin (Ig) can be divided into IgG, IgA, IgM, IgD, IgE five class, and wherein IgG is the main antibody component of serum, accounts for 75% of serum I g.
IgA (immunoglobulin A) content in normal human serum is only second to IgG, accounts for 10 ~ 20% of Immunoglobulins in Serum.From structure, IgA have monomer, binary, three bodies and polymer point.Serotype and secreting type two kinds is divided into again by its immunologic function.Serotype IgA is present in serum, and its content accounts for about 85% of total IgA.Though serotype IgA has some function of IgG and IgM, in serum, do not show important immunologic function.Secretory IgA is present in juice, as saliva, tear, colostrum, nose and bronchial secretion liquid, gastro-intestinal Fluid, urine, sweat etc.Secretory IgA is the main antibody of body mucous membrane local anti-infective immunity.Therefore also known as mucous membrane local antibody.Therefore, special IgA change is relevant to some membrane disease, as Epstein-Barr virus VCA antigentic specificity IgA raises with nasal cavity disease closely related.
IgM accounts for the 5%-10% of serum immune globulin total amount, and serum-concentration is about 1mg/ml.Monomer I gM is expressed in cell surface with film mating type (mIgM), forms B cell antigen receptor (BCR).Secreting type IgM is pentamer, and be the maximum Ig of molecular weight, sedimentation coefficient is 19S, is called macroglobulin, generally not by vascular wall, is mainly present in blood.Pentamer IgM, containing 10 Fab section, has very strong antigen binding capacity; Containing 5 Fc sections, activating complement easier in IgG.Natural blood group antibody is IgM, and the blood transfusion that blood group is not inconsistent can cause significant hemolysis reaction.IgM is the antibody synthesizing the earliest in ontogenetic process and secrete, and can produce IgM the fetus in embryonic development late period, therefore Cord blood IgM raises prompting fetus intrauterine infection (as the infection such as rubella virus or cytomegalovirus).IgM is also the antibody occurred the earliest in Primary humoral immune response, is body anti-infectious " vanguard "; Detect IgM prompting in serum recently to infect, can be used for the early diagnosis infected.Surface Ig M is the principal ingredient of B cell antigen receptor.Only express the mark that mIgM is immature B cells.In course of infection, first IgM occurs, but the duration is not long, is the mark of recent infection.Therefore, specific IgM measures the Early judgement to infectious diseases, has important diagnostic value to carry out early treatment.
Immunoglobulin E (IgE) is the homocytotropic antibody that a class has δ chain, is the main antibody participating in the pathogenesis adjustments such as allergic rhinitis, allergic asthma and eczema.Since Japanese scholars Ishizaka in 1966 finds IgE, about the research of IgE makes substantial progress, and successively found IgE acceptor at mast cell, basocyte, acidophil and Macrophage Surface, also comprise Serum of Sufferers from Allergic Asthma from various anaphylactia patient the specific IgE isolated for multiple pollen, dirt mite, mould and animal skin respectively, in recent years confirm that many cell factors such as IL-4, gamma interferon all take part in the adjustment of IgE synthesis.IgE antibody can start speed and send out phase allergic reaction, also can bring out tardy phase allergic reaction.
Immunoglobulin content assay method special in blood has immunoturbidimetry, lateral chromatography method, ELISA etc., and wherein, immunoturbidimetry simple economy, can robotization, takes short; ELISA method detection sensitivity is high, special good, but two kinds of methods all need corresponding instrument and equipment, and lateral chromatography rule directly can carry out result judgement, uses more convenient, flexible.
Lateral chromatography technology, can be divided into two classes by its principle, and a class is based on enzymatic reaction colour developing, highly comes quantitatively to develop the color; The another kind of colored marker that then uses is as emulsion particle, electroselenium, collaurum and liposome etc., during chromatography, label and determinand react that the compound formed is caught by the corresponding part be fixed on chromatographic material and enrichment develops the color, according to the presence or absence of band that chromatographic material develops the color or how many come qualitative or quantitative.Immunochromatography technique based on enzymatic reaction is displayed one's abilities due to determinand be quantitatively with in test strips with enzymatic activity for foundation, therefore its measurement result is by the impact of the environmental factor such as enzyme stability and sample substrate, pH value, incubative time and temperature.
Immune colloidal gold technique is the solid phase labelling immunoassay grown up after three large labelling techniques (fluorescein, radioactive isotope and enzyme).Colloidal gold immunochromatographimethod (colloidal gold immunochromatography assay, the GICA) fast diagnose test paper bar appearing at clinical diagnosis field the eighties is based upon a kind of immunoassay technology on gold-marking immunity diafiltration basis.It has, and simple and quick, result is clear and definite, without the need to advantages such as complex operations skill and specific installation, susceptibility are high, easy to carry, oneself becomes a new direction of clinical trial diagnostic field development.But, in IgM, IgA, IgD, IgE test process, due to the IgG competition binding antigen of blood middle high concentration, affect the Accurate Determining of IgM, IgA, IgD, IgE test, therefore for realizing the interference all needing to remove or reduce IgG to arbitrary mensuration of IgM, IgA, IgD, IgE.
Such as, mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) size is 200nm, in variform, bacterium colony starts, in " mulberries shape ", can become " decocting poached egg " shape after repeatedly going down to posterity, i.e. surface elevation, central dense, the irregular small colony in edge.Mp can containing tissue or cell, and in containing the agar medium of yeast leachate and 20% horse serum well-grown.Poor growth under anaerobic environment.This bacterium energy glucose fermentation, can grow and produce acid in the nutrient culture media containing glucose.In addition, Mp can carry out DNA/RNA simultaneously copies, in double division growth.Mp membranous antigen can stimulate human body to produce complement fixation antibody and growth inhibition antibody.Some composition on Mp cell membrane same Wa Saier (Wasserman) reacting positive serum, streptococcus m G and human erythrocyte I type antigen antiserum can play cross reaction.
Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) is the pathogen of mankind's Eaton agent pneumonia (or claim primary atypical pneumonia), mainly through droplet infection, 2-3 week in latent period, the incidence of disease with teenager and children higher.Clinical symptoms is lighter or asymptomatic, if there is the respiratory symptom that also just headache, pharyngalgia, heating, cough etc. are general, how to occur in time autumn and winter.
The diagnostic method of mycoplasma pneumoniae mainly relies on to be separated and cultivates and serological test, and wherein complement fixation test (CFT), enzyme linked immunosorbent assay (ELISA) etc. are commonly used in serological test.
After human infection mycoplasma pneumoniae, specific IgM, IgG, IgA antibody can be produced.IgM antibody occurs early, and generally within 1 week, occur after infection, 3-4 week reaches peak, reduces gradually later.Latent period due to mycoplasma pneumoniae infection is 2-3 week, and when patient occurs symptom and goes to a doctor, IgM antibody has reached quite high level, and therefore the IgM antibody positive can be used as the auxiliary diagnosis of acute HIV infection.In traditional lateral chromatography system, by by specific IgG blocking agent (as high concentration anti-igg antibody) reason place sample pad, sample pad IgG blocking agent can close IgG antigen binding site, reduce specific antibody to be combined with Mp, but in such a system due to the swimming together with serum target IgM of IgG blocking agent, do not close the IgG that Mp is special completely in serum and can mark bond (GC-Mp) by competition binding gold, the mensuration of interference IgM.In addition, because IgG blocking agent is usually very low to variable region binding ability in conjunction with human IgG conserved region, therefore its efficiency of blockading also can have a strong impact on the mensuration of IgM.Therefore, develop mycoplasma pneumoniae IgM test paper bar and have important value in the auxiliary diagnosis of mycoplasma pneumoniae infection.
In sum.The method that can adapt to clinical rapid and accurate determination specific immune globulin is extremely urgent.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of special lateral chromatography detection system, its principle has the emulsion particle that cancellated sample pad can fix specified particle diameter, and then the anti-IgG be combined with emulsion particle by covalence key is fixed on sample pad.To utilize in sample pad fixing anti-IgG to catch IgG in sample, make IgG and IgM, IgA, IgE be separated, and then utilize gold mark lateral chromatography system measurement specific IgM, IgA, IgE, effectively reduce the interference of IgG.The method is easy and simple to handle, quick, susceptibility is high, specificity is good.
The present invention is achieved by the following technical solutions:
The present invention's one is used for immunoglobulin (Ig) lateral chromatography detection system, comprises sample pad, pad, the NC film of emulsion particle being fixed with the activation of interfering material scavenger reagent, described immunoglobulin (Ig) be in IgM, IgA, IgE one or more.
One of the present invention is used for determination of immunoglobulin lateral chromatography detection system and also comprises adsorptive pads, PVC liner plate and cartridge.
A kind of lateral chromatography detection system for determination of immunoglobulin of the present invention, includes but not limited to lateral chromatography detector, lateral chromatography test paper, lateral chromatography check-out console, measures test strips etc.Preferably measure test strips.
The emulsion particle of interfering material scavenger reagent activation of the present invention refers to the emulsion particle (LP-anti-IgG) that anti-human IgG antibodies activates.
Immobilized LP-anti-IgG of the present invention is anti-human IgG antibodies (anti-IgG).
Immobilized LP-anti-IgG of the present invention is obtained by covalent coupling for anti-human IgG antibodies anti-IgG and active emulsion particle LP, is combined in order to IgG in the sample of people source.Preferred described immobilized LP-anti-IgG is that anti-human IgG antibodies anti-IgG and active emulsion particle LP is obtained by the coupling of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) method valency.
Sample pad of the present invention is netted tunica fibrosa (Fusion5, GE), catches particle diameter 2.0-2.6um emulsion particle, is fixed.
Described fixing emulsion particle can catch IgG in sample, and by itself and IgM, IgA, IgE, any one is separated, and effectively reduces IgG interference in measuring, improves the susceptibility detected.
The anti-IgG that sample pad of the present invention is fixed is the antibody anti-Fc IgG in goat anti-human igg's holoprotein antibody anti-whole IgG or goat anti-human igg Fc region.The anti-IgG that preferred described sample pad is fixed is goat anti-human igg's holoprotein antibody anti-whole IgG.
Preferably described sample pad fixed L P-anti-IgG concentration is 0.02-2.0mg/cm
2, preferred concentration is 0.2-2.0mg/cm
2, optium concentration is 0.2mg/cm
2.
Immunoglobulin (Ig) of the present invention to be preferably in IgM, IgA, IgE one or more.
Pad of the present invention refers to the pad of the Mp antigen being coated with colloid gold label, specifically refers to there is colloid gold particle Colloidal Gold, the pad of the Mp antigen of CG mark, and the IgM that wherein CG-Mp can be special in sample is combined and forms compound.Pad of the present invention may be used for the judgement of Mp specific IgM result.
The NC film that NC film of the present invention refers to the C line of T line and the anti-Mp antibody being coated with anti-human IgM (anti-IgM) antibody mark bond respectively and is goldenly marked bond and be combined with IgM-gold.NC film of the present invention may be used for the judgement of Mp specific IgM result.
Pad of the present invention, NC film, when measuring in other specific IgM, IgA, IgE, only need replace with corresponding antigen, specific antibody and anti-human IgM or IgA or IgE by antigen, specific antibody and anti-human IgM antibodies.
Decision method
Because mycoplasma pneumoniae IgG and IgM coexists in blood, in order to reduce the interference of IgG in IgM mensuration process, improve susceptibility, therefore test strips selects anti-human IgG antibodies and emulsion particle 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) method covalent coupling, form stable covalent chemical bond, obtain active emulsion particle.By a certain amount of active emulsion particle process sample pad, the sample pad of certain material can fix the emulsion particle of specified particle diameter, and the emulsion particle of activation can be fixed on sample pad.In the lateral chromatography of pattern detection, due to the affine combination of IgG and the anti-IgG in sample, and then be separated with IgM, then IgM by being combined with gold mark bond, the catching of T line and C line, finally carry out result of determination according to the colour developing degree of T line and C line.
Beneficial effect
In order to beneficial effect of the present invention is better described, be illustrated by following test example.The present invention is selected by anti-IgG kind and concentration, the gold mark aspect such as bond and T linear protein kind sample pad bag.
Test example 1 is by the test strips of the sample pad assembling of coating anti-IgG and measure clinical sample (100 example) contrast test by the test strips that the sample pad being fixed with LP-anti-IgG is assembled
1.IgM measures
Respectively with coating anti-IgG sample pad assembling test strips and be fixed with LP-anti-IgG sample pad assembling test strips measure clinical sample each 100 example, the anti-mycoplasma pneumoniae antibody IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3404814th, tool (entering) word 2013) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 1.
Screening technique is as follows:
1) select anti-IgG to be coated with sample pad respectively, LP-anti-IgG bag is by sample pad;
2) chromatograph test strip preparation method is prepared according to embodiment 1;
3) 100 μ L diluted sample are added respectively, room temperature reaction 15min;
4) according to colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different sample pad encrusting substance of table 1 and specificity
Visible by table 1, the test strips mensuration clinical sample assembled by the sample pad being fixed with LP-anti-IgG is in susceptibility, specificity and always have coincidence rate all higher than the test strips of the sample pad assembling of coating anti-IgG.
2.IgA measures:
Respectively by the test strips of the sample pad assembling of coating anti-IgG with measure clinical sample each 100 examples (adopting Epstein-Barr virus capsid antigen (VCA) IgA antibody detection kit (euzymelinked immunosorbent assay (ELISA)) of same sunrise biological production (state's food medicine prison No. 3400467th, tool (standard) word 2013) to carry out yin and yang attribute judgement) by the test strips of the sample pad assembling being fixed with LP-anti-IgG, calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 2.
Screening technique is as follows:
1) select anti-IgG to be coated with sample pad respectively, LP-anti-IgG bag is by sample pad;
2) chromatograph test strip preparation method is prepared according to embodiment 3;
3) 100 μ L diluted sample are added respectively, room temperature reaction 15min;
4) according to colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different sample pad encrusting substance of table 2 and specificity
Visible by table 2, the test strips mensuration clinical sample assembled by the sample pad being fixed with LP-anti-IgG is in susceptibility, specificity and always have coincidence rate all higher than the test strips of the sample pad assembling of coating anti-IgG.
3.IgE measures:
Respectively by the test strips of the sample pad assembling of coating anti-IgG with measure clinical sample each 100 examples (adopting the dermatophagoides pteronyssinus allergen specificity antibody IgE detection kit (euzymelinked immunosorbent assay (ELISA)) of enlightening news biological production (state's food medicine prison No. 3400432nd, tool (standard) word 2011) to carry out yin and yang attribute judgement) by the test strips of the sample pad assembling being fixed with LP-anti-IgG, calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 3.
Screening technique is as follows:
1) select anti-IgG to be coated with sample pad respectively, LP-anti-IgG bag is by sample pad;
2) chromatograph test strip preparation method is prepared according to embodiment 4;
3) 100 μ L diluted sample are added respectively, room temperature reaction 15min;
4) according to colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different sample pad encrusting substance of table 3 and specificity
Visible by table 3, the test strips mensuration clinical sample assembled by the sample pad being fixed with LP-anti-IgG is in susceptibility, specificity and always have coincidence rate all higher than the test strips of the sample pad assembling of coating anti-IgG.
The susceptibility of the different anti-IgG kind of test example 2 and specificity
1.IgM measures
With be fixed with goat anti-human igg-Fc antibody (anti-Fc IgG) sample pad assembling test strips and be fixed with goat anti-human igg's holoprotein antibody (anti-whole IgG) measure clinical sample each 100 example, (the anti-mycoplasma pneumoniae antibody IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3404814th, tool (entering) word 2013) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 4.
Screening technique is as follows:
1) LP-anti-Fc IgG, LP-anti-whole IgG is selected to wrap by sample pad respectively;
2) chromatograph test strip preparation method is with embodiment 1;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different anti-IgG kind of table 4 and specificity
From table 4, measure test strips with being fixed with the sample pad that test strips that goat anti-human igg's holoprotein antibody (anti-whole IgG) assembles all is better than with being fixed with goat anti-human igg-Fc antibody (anti-Fc IgG) at susceptibility, specificity, total coincidence rate.
2.IgA measures
Be fixed with goat anti-human igg-Fc antibody (anti-Fc IgG) sample pad assembling test strips and be fixed with goat anti-human igg's holoprotein antibody (anti-whole IgG) measure clinical sample each 100 example, (adopting Epstein-Barr virus capsid antigen (VCA) IgA antibody detection kit (euzymelinked immunosorbent assay (ELISA)) of same sunrise biological production (state's food medicine prison No. 3400467th, tool (standard) word 2013) to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 5.
Screening technique is as follows:
1) LP-anti-Fc IgG, LP-anti-whole IgG is selected to wrap by sample pad respectively;
2) chromatograph test strip preparation method is with embodiment 3;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different anti-IgG kind of table 5 and specificity
From table 5, measure test strips with being fixed with the sample pad that test strips that goat anti-human igg's holoprotein antibody (anti-whole IgG) assembles all is better than with being fixed with goat anti-human igg-Fc antibody (anti-Fc IgG) at susceptibility, specificity, total coincidence rate.
3.IgE measures
Be fixed with goat anti-human igg-Fc antibody (anti-Fc IgG) sample pad assembling test strips and be fixed with goat anti-human igg's holoprotein antibody (anti-whole IgG) measure clinical sample each 100 example, (adopting the dermatophagoides pteronyssinus allergen specificity antibody IgE detection kit (euzymelinked immunosorbent assay (ELISA)) of enlightening news biological production (state's food medicine prison No. 3400432nd, tool (standard) word 2011) to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 6.
Screening technique is as follows:
1) LP-anti-Fc IgG, LP-anti-whole IgG is selected to wrap by sample pad respectively;
2) chromatograph test strip preparation method is with embodiment 4;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
The susceptibility of the different anti-IgG kind of table 6 and specificity
From table 6, measure test strips with being fixed with the sample pad that test strips that goat anti-human igg-Fc antibody (anti-Fc IgG) assembles all is better than with being fixed with goat anti-human igg's holoprotein antibody (anti-whole IgG) at susceptibility, specificity, total coincidence rate.
Test the selection of 3 sample pad LP-anti-IgG concentration
(1) mycoplasma pneumoniae IgM measures
1, clinical sample (100 examples are measured by the test strips only prepared by 1%BSA pre-service sample pad, the anti-mycoplasma pneumoniae antibody IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3404814th, tool (entering) word 2013) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 3.
Screening technique is as follows:
1) select and only use 1%BSA pre-service sample pad;
2) chromatograph test strip preparation method embodiment 1;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
2, clinical sample (100 examples are measured by the test strips of the sample pad assembling being fixed with LP-anti-IgG, the anti-mycoplasma pneumoniae antibody IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3404814th, tool (entering) word 2013) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 7.
Screening technique is as follows:
1) concentration is selected to be 2.0ug/cm respectively
2, 20ug/cm
2, 200ug/cm
2, 2000ug/cm
2lP-anti-IgG (representing with anti-IgG concentration) bag by sample pad;
2) chromatograph test strip preparation method is with embodiment 1;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
3, result: as seen from Table 7, it is 0.02-2.0mg/cm that sample pad fixes anti-IgG concentration (representing with anti-IgG concentration)
2between, no matter sensitivity, specificity or coincidence rate are all relatively good, and especially concentration is 0.2mg/cm
2sensitivity, specificity are best.
Table 7 sample pad anti-human IgG antibodies concentration is selected
(2) Chlamydia pneumoniae IgM measures
1, clinical sample (100 examples are measured by the test strips only prepared by 1%BSA pre-service sample pad, the anti-chlamydia pneumoniae (cp) IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3402559th, tool (entering) word 2010) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 8.
Screening technique is as follows:
1) select and only use 1%BSA pre-service sample pad;
2) chromatograph test strip preparation method is with embodiment 2;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
2, clinical sample (100 examples are measured by the test strips of the sample pad assembling being fixed with LP-anti-IgG, the anti-chlamydia pneumoniae (cp) IgM detection kit (enzyme linked immunosorbent assay) (state's food medicine prison No. 3402559th, tool (entering) word 2010) adopting EUROIMMUN Medizinische Labordiagnostika AG to produce carries out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 8.
Screening technique is as follows:
1) concentration is selected to be 2.0ug/cm respectively
2, 20ug/cm
2, 200ug/cm
2, 2000ug/cm
2lP-anti-IgG (representing with anti-IgG concentration) bag by sample pad;
2) chromatograph test strip preparation method is with embodiment 2;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
3, result: as seen from Table 8, sample pad fixed L P-anti-IgG concentration (representing with anti-IgG concentration) is 0.02-2.0mg/cm
2between, no matter sensitivity, specificity or coincidence rate are all relatively good, and especially concentration is 0.2mg/cm
2sensitivity, specificity are best.
Table 8 sample pad anti-human IgG antibodies concentration is selected
(3) Epstein-Barr virus capsid antigen (VCA) IgA measures
1, clinical sample (100 examples are measured by the test strips only prepared by 1%BSA pre-service sample pad, Epstein-Barr virus capsid antigen (VCA) IgA antibody detection kit (euzymelinked immunosorbent assay (ELISA)) (state's food medicine prison No. 3400467th, tool (standard) word 2013) with sunrise biological production is adopted to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 9.
Screening technique is as follows:
1) select and only use 1%BSA pre-service sample pad;
2) chromatograph test strip preparation method is with embodiment 3;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
2, clinical sample (100 examples are measured by the test strips of the sample pad assembling being fixed with LP-anti-IgG, Epstein-Barr virus capsid antigen (VCA) IgA antibody detection kit (euzymelinked immunosorbent assay (ELISA)) (state's food medicine prison No. 3400467th, tool (standard) word 2013) with sunrise biological production is adopted to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 9.
Screening technique is as follows:
1) concentration is selected to be 2.0ug/cm respectively
2, 20ug/cm
2, 200ug/cm
2, 2000ug/cm
2lP-anti-IgG (representing with anti-IgG concentration) bag by sample pad;
2) chromatograph test strip preparation method is with embodiment 3;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
3, result: as seen from Table 9, sample pad fixed L P-anti-IgG concentration (representing with anti-IgG concentration) is 0.02-2.0mg/cm
2between, no matter sensitivity, specificity or coincidence rate are all relatively good, and especially concentration is 0.2mg/cm
2sensitivity, specificity are best.
Table 9 sample pad anti-human IgG antibodies concentration is selected
(4) dermatophagoides pteronyssinus allergen specificity antibody IgE measures:
1, clinical sample (100 examples are measured by the test strips only prepared by 1%BSA pre-service sample pad, the dermatophagoides pteronyssinus allergen specificity antibody IgE detection kit (euzymelinked immunosorbent assay (ELISA)) of enlightening news biological production (state's food medicine prison No. 3400432nd, tool (standard) word 2011) is adopted to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 10.
Screening technique is as follows:
1) select and only use 1%BSA pre-service sample pad;
2) chromatograph test strip preparation method is with embodiment 4;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
2, clinical sample (100 examples are measured by the test strips of the sample pad assembling being fixed with LP-anti-IgG, the dermatophagoides pteronyssinus allergen specificity antibody IgE detection kit (euzymelinked immunosorbent assay (ELISA)) of enlightening news biological production (state's food medicine prison No. 3400432nd, tool (standard) word 2011) is adopted to carry out yin and yang attribute judgement), calculate the susceptibility of detection system, specificity, positive coincidence rate, negative match-rate and total coincidence rate, the results are shown in Table 10.
Screening technique is as follows:
1) concentration is selected to be 2.0ug/cm respectively
2, 20ug/cm
2, 200ug/cm
2, 2000ug/cm
2lP-anti-IgG (representing with anti-IgG concentration) bag by sample pad;
2) chromatograph test strip preparation method is with embodiment 4;
3) 100 μ L diluted sample are added, room temperature reaction 15min;
4) according to T line colour developing deciding degree result, namely colourless is negative, and redness is positive.
3, result: as seen from Table 10, sample pad fixed L P-anti-IgG concentration (representing with anti-IgG concentration) is 0.02-2.0mg/cm
2between, no matter sensitivity, specificity or coincidence rate are all relatively good, and especially concentration is 0.2mg/cm
2sensitivity, specificity are best.
Table 10 sample pad anti-human IgG antibodies concentration is selected
Accompanying drawing explanation
Fig. 1 lateral chromatography system and composition
Fig. 2 lateral chromatography system is explained in detail
Fig. 3 NC film T/C line bag quilt
Following embodiment is only better understand the present invention, does not have restriction to protection scope of the present invention.
Embodiment
Embodiment 1 mycoplasma pneumoniae IgM measures test strips preparation
Agents useful for same:
Active Latex Particle (Latex Particle): Merck, 39510001
(2-(N-morpholinyl) ethyl sulfonic acid (sigma, M3671) damping fluid: take 4.88g MES in 900mL ultrapure water regulates pH5.0 after dissolving completely, is settled to 1L 25mM MES.
EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) solution: sigma, 39391
PH7.510mM PBS: take 0.27g potassium dihydrogen phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g sodium hydrogen phosphate (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g potassium chloride (KCl) (traditional Chinese medicines, 10016308) in 900mL ultrapure water, regulate pH to 7.5 after dissolving completely, be then settled to 1L with volumetric flask.
10mM Tris-HCl: take 1.576g (traditional Chinese medicines, xw020034) in 900mL ultrapure water, is adjusted to required pH value after dissolving completely, is then settled to 1L.
1% citrate buffer solution: take 1.0g trisodium citrate (traditional Chinese medicines, 10019428) in 100mL ultrapure water, to dissolving completely.
5%BSA: take 5g BSA (amresco, 0332) in 100ml PBS solution, to dissolving completely.
Terminological interpretation:
Mp: i.e. mycoplasma pneumoniae antigen, is purchased from mybiosource, article No. MBS230193.
LP: namely Latex Particle abridges, emulsion particle, its particle surface have reactive group can with protein covalent bond, be purchased from Merck company, article No. 39510001.
Anti-IgM: i.e. anti-human IgM antibodies, a kind of can with the immunoglobulin (Ig) of the affine combination of people IgM, be purchased from Jackson company, article No. 109-005-043.
Anti-IgG: i.e. anti-human IgG antibodies, a kind of can with the immunoglobulin (Ig) of the affine combination of human IgG.
Anti-whole: the i.e. one of anti-human IgG antibodies, can with the immunoglobulin (Ig) of the affine combination of human IgG holoprotein, be purchased from Jackson company, article No. 109-005-003.
Anti-Fc: the i.e. one of anti-human IgG antibodies, can with the immunoglobulin (Ig) of human IgG Fc fragment specific bond, be purchased from Jackson company, article No. 109-005-003.
Anti-Mp: i.e. anti-Mp antibody, the immunoglobulin (Ig) that can be combined with Mp, is purchased from Calbioreagents company, article No. P056.
(1) emulsion particle (Latex Particle, Merck) preparation process
1, Latex Particle (LP) activation.
Get 1mg Latex Particle (Merck, 39510001), with 1mL25mM MES (pH5.0) (sigma, M3671) buffer solution 3 times (mixing 10min) at every turn;
1). preparation 10mg/mL EDC (M=191.7g/mol, C=52mM) solution (EDC is dissolved in cold 25mM MES, pH5.0); (EDC, sigma, 39391)
2). Latex Particle is resuspended with 440 μ L25mM MES (pH5.0);
3). add 10 μ L EDC solution, fully mix, slow jolting, 30min, room temperature;
4). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant, and use 1mL25mM MES, pH5.0 washs 3 times;
2, Latex Particle bag quilt
1). with the resuspended Latex Particle of 300 μ L25mM MES, pH5.0;
2). add 10ul10mg/ml anti-human IgG antibodies (anti-IgG, 25mM MES, pH5.0) solution;
3). use 25mM MES, pH5.0 makes reaction system constant volume be 500 μ L, fully mixes;
4). slowly jolting, room temperature 30-120min or 4 DEG C 2 hours;
5). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant;
3, Latex Particle washs and preserves
1). wash 4 times with 500 μ L25mM MES (pH5.0);
2). add 100 μ L0.1-0.5%BSA (amresco, 0332), 30-60min, room temperature;
3). with 100 μ L50mM Tris-HCl (pH6.8, containing 1%Tween-20) (sigma, 44112) washing 4 times, obtain the emulsion particle (LP-anti-IgG) of anti-IgG activation;
4). add 1mL5%BSA and preserve.
(2) gold mark bond preparation
1) get 100mL0.01%HAuCl4 (traditional Chinese medicines, 10010711) solution, be heated to boiling;
2) add 2.0mL1% citric acid three sodium solution, add thermal agitation 15min;
3) turn off heating turn-knob, suitable speed is stirred to room temperature, obtains colloidal gold solution (ColloidalGold, CG);
4) collaurum measuring 20.0mL is placed in the small beaker of 100.0mL, under the state stirred, slowly add 0.4mg mycoplasma pneumoniae antigen (Mp), stirs 30min;
5) adding 5%BSA to final concentration is 1%, continues to stir 30min;
6) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST (PBS is containing 0.05%Tween-20, v/v);
7) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST;
8) with the centrifugal 30min of 12000r/min, supernatant discarded night, precipitation 1%BSA is resuspended, obtains 1.0mL concentrate (i.e. gold mark bond), puts 4 DEG C of refrigerators for subsequent use.
(3) sample pad, pad pre-service
1) sample pad (Fusion5, GE) and pad (Shanghai Jinbiao Bio-Tech Co., Ltd.) are soaked 30min with 1%BSA, 37 DEG C of oven dry;
2) emulsion particle (LP-anti-IgG) anti-human IgG antibodies activated or anti-human IgG antibodies (anti-IgG) coat sample pad (0.2mg/cm
2), 37 DEG C of oven dry;
3) get gold mark bond and be added drop-wise to (10uL/cm on pad
2), 37 DEG C of oven dry.
(4) T/C line (detection line/nature controlling line) wraps quilt
C line (nature controlling line): by anti-for 1.0mg/mL rabbit Mp antibody (anti-Mp) bag by NC film (135s, Millipore) C line (see Fig. 3 a, Fig. 3 b), every bar 1.0 μ L, 37 DEG C of dryings.
T line (detection line): by 1.0mg/mL goat-anti people IgM (anti-IgM) bag by NC film (135s, Millipore) (see Fig. 3 c, Fig. 3 d), every bar 1.0 μ g, 37 DEG C of dryings.
(5) assemble
By sample pad (Fusion5, GE), pad (NJ25, Shanghai Jinbiao Bio-Tech Co., Ltd.), NC film (135s, Millipore), adsorptive pads (CH37M, Shanghai Jinbiao Bio-Tech Co., Ltd.), PVC liner plate (Shanghai Jinbiao Bio-Tech Co., Ltd.), cartridge (Jin Canhua), from down to up, carry out from inside to outside assembling (see Fig. 1,2).
Embodiment 2 Chlamydia pneumoniae IgM measures test strips preparation
Agents useful for same:
Active Latex Particle (Latex Particle): Merck, 39510001
(2-(N-morpholinyl) ethyl sulfonic acid (sigma, M3671) damping fluid: take 4.88g MES in 900mL ultrapure water regulates pH5.0 after dissolving completely, is settled to 1L 25mM MES.
EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) solution: sigma, 39391
PH7.510mM PBS: take 0.27g potassium dihydrogen phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g sodium hydrogen phosphate (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g potassium chloride (KCl) (traditional Chinese medicines, 10016308) in 900mL ultrapure water, regulate pH to 7.5 after dissolving completely, be then settled to 1L with volumetric flask.
10mM Tris-HCl: take 1.576g (traditional Chinese medicines, xw020034) in 900mL ultrapure water, is adjusted to required pH value after dissolving completely, is then settled to 1L.
1% citrate buffer solution: take 1.0g trisodium citrate (traditional Chinese medicines, 10019428) in 100mL ultrapure water, to dissolving completely.
5%BSA: take 5g BSA (amresco, 0332) in 100ml PBS solution, to dissolving completely.
Terminological interpretation:
Cpn: i.e. Chlamydia pneumoniae (Chlamydia pneumoniae, Cpn) antigen, is purchased from luxuriant and rich with fragrance roc biological.Anti-Cpn: i.e. anti-Cpn antibody, the immunoglobulin (Ig) that can be combined with Cpn, is purchased from
Calbioreagents company, article No. M002.
(1) emulsion particle (Latex Particle, Merck) preparation process
1, Latex Particle (LP) activation.
Get 1mg Latex Particle (Merck, 39510001), with 1mL25mM MES (pH5.0) (sigma, M3671) buffer solution 3 times (mixing 10min) at every turn;
5). preparation 10mg/mL EDC (M=191.7g/mol, C=52mM) solution (EDC is dissolved in cold 25mM MES, pH5.0); (EDC, sigma, 39391)
6). Latex Particle is resuspended with 440 μ L25mM MES (pH5.0);
7). add 10 μ L EDC solution, fully mix, slow jolting, 30min, room temperature;
8). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant, and use 1mL25mM MES, pH5.0 washs 3 times;
2, Latex Particle bag quilt
6). with the resuspended Latex Particle of 300 μ L25mM MES, pH5.0;
7). add 10ul10mg/ml anti-human IgG antibodies (anti-whole, 25mM MES, pH5.0) solution;
8). use 25mM MES, pH5.0 makes reaction system constant volume be 500 μ L, fully mixes;
9). slowly jolting, room temperature 30-120min or 4 DEG C 2 hours;
10). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant;
3, Latex Particle washs and preserves
5). wash 4 times with 500 μ L25mM MES (pH5.0);
6). add 100 μ L0.1-0.5%BSA (amresco, 0332), 30-60min, room temperature;
7). with 100 μ L50mM Tris-HCl (pH6.8, containing 1%Tween-20) (sigma, 44112) washing 4 times, obtain the emulsion particle (LP-anti-IgG) of anti-IgG activation;
8). add 1mL5%BSA and preserve.
(2) gold mark bond preparation
9) get 100mL0.01%HAuCl4 (traditional Chinese medicines, 10010711) solution, be heated to boiling;
10) add 2.0mL1% citric acid three sodium solution, add thermal agitation 15min;
11) turn off heating turn-knob, suitable speed is stirred to room temperature, obtains colloidal gold solution (ColloidalGold, CG);
12) collaurum measuring 20.0mL is placed in the small beaker of 100.0mL, under the state stirred, slowly add 0.4mg Chlamydia pneumoniae antigen (Cpn), stirs 30min;
13) adding 5%BSA to final concentration is 1%, continues to stir 30min;
14) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST (PBS is containing 0.05%Tween-20, v/v);
15) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST;
16) with the centrifugal 30min of 12000r/min, supernatant discarded night, precipitation 1%BSA is resuspended, obtains 1.0mL concentrate (i.e. gold mark bond), puts 4 DEG C of refrigerators for subsequent use.
(3) sample pad, pad pre-service
1) sample pad (Fusion5, GE) and pad (Shanghai Jinbiao Bio-Tech Co., Ltd.) are soaked 30min with 1%BSA, 37 DEG C of oven dry;
2) emulsion particle (LP-anti-whole) that anti-human IgG antibodies activates is coated sample pad (0.2mg/cm
2), 37 DEG C of oven dry;
3) get gold mark bond and be added drop-wise to (10uL/cm on pad
2), 37 DEG C of oven dry.
(4) T/C line (detection line/nature controlling line) wraps quilt
C line (nature controlling line): by anti-for 1.0mg/mL Cpn monoclonal antibody (anti-Cpn) bag by NC film (135s, Millipore) C line (see Fig. 3 a, Fig. 3 b), every bar 1.0 μ L, 37 DEG C of dryings.
T line (detection line): by 1.0mg/mL goat-anti people IgM (anti-IgM) bag by NC film (135s, Millipore) (see Fig. 3 c, Fig. 3 d), every bar 1.0 μ g, 37 DEG C of dryings.
(5) assemble
By sample pad (Fusion5, GE), pad (NJ25, Shanghai Jinbiao Bio-Tech Co., Ltd.), NC film (135s, Millipore), adsorptive pads (CH37M, Shanghai Jinbiao Bio-Tech Co., Ltd.), PVC liner plate (Shanghai Jinbiao Bio-Tech Co., Ltd.), cartridge (Jin Canhua), from down to up, carry out from inside to outside assembling (see Fig. 1,2).
Embodiment 3EB viral capsid antigen (VCA) IgA measures test strips preparation
Agents useful for same:
Active Latex Particle (Latex Particle): Merck, 39510001
(2-(N-morpholinyl) ethyl sulfonic acid (sigma, M3671) damping fluid: take 4.88g MES in 900mL ultrapure water regulates pH5.0 after dissolving completely, is settled to 1L 25mM MES.
EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) solution: sigma, 39391
PH7.510mM PBS: take 0.27g potassium dihydrogen phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g sodium hydrogen phosphate (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g potassium chloride (KCl) (traditional Chinese medicines, 10016308) in 900mL ultrapure water, regulate pH to 7.5 after dissolving completely, be then settled to 1L with volumetric flask.
10mM Tris-HCl: take 1.576g (traditional Chinese medicines, xw020034) in 900mL ultrapure water, is adjusted to required pH value after dissolving completely, is then settled to 1L.
1% citrate buffer solution: take 1.0g trisodium citrate (traditional Chinese medicines, 10019428) in 100mL ultrapure water, to dissolving completely.
5%BSA: take 5g BSA (amresco, 0332) in 100ml PBS solution, to dissolving completely.
Terminological interpretation:
Anti-IgA: i.e. anti-human IgA antibody, a kind of can with the immunoglobulin (Ig) of the affine combination of people IgA, be purchased from Jackson company, article No. 309-005-011.
VCA: i.e. Epstein-Barr virus (Epstein Barr Virus) capsid antigen, is purchased from Calbioreagents company, article No. A023.
Anti-VCA: i.e. anti-VCA antibody, the immunoglobulin (Ig) that can be combined with VCA, is purchased from
Calbioreagents company, article No. M032.
(1) emulsion particle (Latex Particle, Merck) preparation process
1, Latex Particle (LP) activation.
Get 1mg Latex Particle (Merck, 39510001), with 1mL25mM MES (pH5.0) (sigma, M3671) buffer solution 3 times (mixing 10min) at every turn;
9). preparation 10mg/mL EDC (M=191.7g/mol, C=52mM) solution (EDC is dissolved in cold 25mM MES, pH5.0); (EDC, sigma, 39391)
10). Latex Particle is resuspended with 440 μ L25mM MES (pH5.0);
11). add 10 μ L EDC solution, fully mix, slow jolting, 30min, room temperature;
12). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant, and use 1mL25mM MES, pH5.0 washs 3 times;
2, Latex Particle bag quilt
11). with the resuspended Latex Particle of 300 μ L25mM MES, pH5.0;
12). add 10ul10mg/ml anti-human IgG antibodies (anti-whole, 25mM MES, pH5.0) solution;
13). use 25mM MES, pH5.0 makes reaction system constant volume be 500 μ L, fully mixes;
14). slowly jolting, room temperature 30-120min or 4 DEG C 2 hours;
15). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant;
3, Latex Particle washs and preserves
9). wash 4 times with 500 μ L25mM MES (pH5.0);
10). add 100 μ L0.1-0.5%BSA (amresco, 0332), 30-60min, room temperature;
11). with 100 μ L50mM Tris-HCl (pH6.8, containing 1%Tween-20) (sigma, 44112)
Wash 4 times, obtain the emulsion particle (LP-anti-whole) of anti-whole activation;
12). add 1mL5%BSA and preserve.
(2) gold mark bond preparation
17) get 100mL0.01%HAuCl4 (traditional Chinese medicines, 10010711) solution, be heated to boiling;
18) add 2.0mL1% citric acid three sodium solution, add thermal agitation 15min;
19) turn off heating turn-knob, suitable speed is stirred to room temperature, obtains colloidal gold solution (ColloidalGold, CG);
20) collaurum measuring 20.0mL is placed in the small beaker of 100.0mL, under the state stirred, slowly add 0.4mg VCA, stirs 30min;
21) adding 5%BSA to final concentration is 1%, continues to stir 30min;
22) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST (PBS is containing 0.05%Tween-20, v/v);
23) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST;
24) with the centrifugal 30min of 12000r/min, supernatant discarded night, precipitation 1%BSA is resuspended, obtains 1.0mL concentrate (i.e. gold mark bond), puts 4 DEG C of refrigerators for subsequent use.
(3) sample pad, pad pre-service
1) sample pad (Fusion5, GE) and pad (Shanghai Jinbiao Bio-Tech Co., Ltd.) are soaked 30min with 1%BSA, 37 DEG C of oven dry;
2) emulsion particle (LP-anti-whole) that anti-human IgG antibodies activates is coated sample pad (0.2mg/cm
2), 37 DEG C of oven dry;
3) get gold mark bond and be added drop-wise to (10uL/cm on pad
2), 37 DEG C of oven dry.
(4) T/C line (detection line/nature controlling line) wraps quilt
C line (nature controlling line): by anti-for 1.0mg/mL VCA monoclonal antibody (anti-VCA) bag by NC film (135s, Millipore) C line (see Fig. 3 a, Fig. 3 b), every bar 1.0 μ L, 37 DEG C of dryings.
T line (detection line): by anti-human for 1.0mg/mL rabbit IgA (anti-IgA) bag by NC film (135s, Millipore) (see Fig. 3 c, Fig. 3 d), every bar 1.0 μ g, 37 DEG C of dryings.
(5) assemble
By sample pad (Fusion5, GE), pad (NJ25, Shanghai Jinbiao Bio-Tech Co., Ltd.), NC film (135s, Millipore), adsorptive pads (CH37M, Shanghai Jinbiao Bio-Tech Co., Ltd.), PVC liner plate (Shanghai Jinbiao Bio-Tech Co., Ltd.), cartridge (Jin Canhua), from down to up, carry out from inside to outside assembling (see Fig. 1,2).
Embodiment 4 dermatophagoides pteronyssinus specific IgE measures test strips preparation
Agents useful for same:
Active Latex Particle (Latex Particle): Merck, 39510001
(2-(N-morpholinyl) ethyl sulfonic acid (sigma, M3671) damping fluid: take 4.88g MES in 900mL ultrapure water regulates pH5.0 after dissolving completely, is settled to 1L 25mM MES.
EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) solution: sigma, 39391
PH7.510mM PBS: take 0.27g potassium dihydrogen phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g sodium hydrogen phosphate (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g potassium chloride (KCl) (traditional Chinese medicines, 10016308) in 900mL ultrapure water, regulate pH to 7.5 after dissolving completely, be then settled to 1L with volumetric flask.
10mM Tris-HCl: take 1.576g (traditional Chinese medicines, xw020034) in 900mL ultrapure water, is adjusted to required pH value after dissolving completely, is then settled to 1L.
1% citrate buffer solution: take 1.0g trisodium citrate (traditional Chinese medicines, 10019428) in 100mL ultrapure water, to dissolving completely.
5%BSA: take 5g BSA (amresco, 0332) in 100ml PBS solution, to dissolving completely.
Terminological interpretation:
Anti-IgE: i.e. antihuman IgE antibody, a kind of can with the immunoglobulin (Ig) of the affine combination of people IgE, be purchased from Calbioreagents company, article No. M348.
Df: i.e. dermatophagoides pteronyssinus (Dermatophagoides farinae, Df) antigen, is purchased from INDOORBiotechnologies company, article No. RP-DF2-1.
Anti-Df: i.e. anti-Df antibody, the immunoglobulin (Ig) that can be combined with Df, is purchased from Hytest company, article No. 3K15.
(1) emulsion particle (Latex Particle, Merck) preparation process
1, Latex Particle (LP) activation.
Get 1mg Latex Particle (Merck, 39510001), with 1mL25mM MES (pH5.0) (sigma, M3671) buffer solution 3 times (mixing 10min) at every turn;
13). preparation 10mg/mL EDC (M=191.7g/mol, C=52mM) solution (EDC is dissolved in cold 25mM MES, pH5.0); (EDC, sigma, 39391)
14). Latex Particle is resuspended with 440 μ L25mM MES (pH5.0);
15). add 10 μ L EDC solution, fully mix, slow jolting, 30min, room temperature;
16). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant, and use 1mL25mM MES, pH5.0 washs 3 times;
2, Latex Particle bag quilt
16). with the resuspended Latex Particle of 300 μ L25mM MES, pH5.0;
17). add 10ul10mg/ml anti-Fc (anti-Fc, 25mM MES, pH5.0) solution;
18). use 25mM MES, pH5.0 makes reaction system constant volume be 500 μ L, fully mixes;
19). slowly jolting, room temperature 30-120min or 4 DEG C 2 hours;
20). by the centrifuge tube centrifugal (3000rmp, 5min) reacted, remove supernatant;
3, Latex Particle washs and preserves
13). wash 4 times with 500 μ L25mM MES (pH5.0);
14). add 100 μ L0.1-0.5%BSA (amresco, 0332), 30-60min, room temperature;
15). with 100 μ L50mM Tris-HCl (pH6.8, containing 1%Tween-20) (sigma, 44112) washing 4 times, obtain the emulsion particle (LP-anti-Fc) of anti-IgG activation;
16). add 1mL5%BSA and preserve.
(2) gold mark bond preparation
25) get 100mL0.01%HAuCl4 (traditional Chinese medicines, 10010711) solution, be heated to boiling;
26) add 2.0mL1% citric acid three sodium solution, add thermal agitation 15min;
27) turn off heating turn-knob, suitable speed is stirred to room temperature, obtains colloidal gold solution (ColloidalGold, CG);
28) collaurum measuring 20.0mL is placed in the small beaker of 100.0mL, under the state stirred, slowly add 0.4mg DMS former (Df), stirs 30min;
29) adding 5%BSA to final concentration is 1%, continues to stir 30min;
30) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST (PBS is containing 0.05%Tween-20, v/v);
31) with the centrifugal 30min of 12000r/min, supernatant discarded night, resuspended with PBST;
32) with the centrifugal 30min of 12000r/min, supernatant discarded night, precipitation 1%BSA is resuspended, obtains 1.0mL concentrate (i.e. gold mark bond), puts 4 DEG C of refrigerators for subsequent use.
(3) sample pad, pad pre-service
1) sample pad (Fusion5, GE) and pad (Shanghai Jinbiao Bio-Tech Co., Ltd.) are soaked 30min with 1%BSA, 37 DEG C of oven dry;
2) emulsion particle (LP-anti-Fc) that anti-human IgG antibodies activates is coated sample pad (0.2mg/cm
2), 37 DEG C of oven dry;
3) get gold mark bond and be added drop-wise to (10uL/cm on pad
2), 37 DEG C of oven dry.
(4) T/C line (detection line/nature controlling line) wraps quilt
C line (nature controlling line): by anti-for 1.0mg/mL Df monoclonal antibody (anti-Df) bag by NC film (135s, Millipore) C line (see Fig. 3 a, Fig. 3 b), every bar 1.0 μ L, 37 DEG C of dryings.
T line (detection line): by 1.0mg/mL anti human IgE monoclonal antibody (anti-IgE) bag by NC film (135s, Millipore) (see Fig. 3 c, Fig. 3 d), every bar 1.0 μ g, 37 DEG C of dryings.
(5) assemble
By sample pad (Fusion5, GE), pad (NJ25, Shanghai Jinbiao Bio-Tech Co., Ltd.), NC film (135s, Millipore), adsorptive pads (CH37M, Shanghai Jinbiao Bio-Tech Co., Ltd.), PVC liner plate (Shanghai Jinbiao Bio-Tech Co., Ltd.), cartridge (Jin Canhua), from down to up, carry out from inside to outside assembling (see Fig. 1,2).
Claims (10)
1. for an immunoglobulin (Ig) lateral chromatography detection system, it is characterized in that, comprise sample pad, pad, the NC film of emulsion particle being fixed with the activation of interfering material scavenger reagent, described immunoglobulin (Ig) be in IgM, IgA, IgE more than one.
2. lateral chromatography detection system as claimed in claim 1, is characterized in that, the emulsion particle of described interfering material scavenger reagent activation refers to the emulsion particle LP-anti-IgG that anti-human IgG antibodies activates.
3. lateral chromatography detection system as claimed in claim 2, is characterized in that, described immobilized LP-anti-IgG is obtained by covalent coupling for anti-human IgG antibodies anti-IgG and active emulsion particle LP, is combined in order to IgG in the sample of people source.
4. lateral chromatography detection system as claimed in claim 3, it is characterized in that, described immobilized LP-anti-IgG is that anti-human IgG antibodies anti-IgG and LP is obtained by 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) method covalent coupling.
5. lateral chromatography detection system as claimed in claim 1, it is characterized in that, described sample pad is netted tunica fibrosa, catches particle diameter 2.0-2.6 μm emulsion particle, is fixed.
6. the lateral chromatography detection system as described in claim 3 or 4, is characterized in that: described anti-IgG is the antibody anti-Fc IgG in goat anti-human igg's holoprotein antibody anti-whole IgG or goat anti-human igg Fc region.
7. the lateral chromatography detection system as described in claim 2-4 any one, is characterized in that, described sample pad fixed L P-anti-IgG concentration is 0.02-2.0mg/cm
2.
8. lateral chromatography detection system as claimed in claim 7, it is characterized in that, described sample pad fixed L P-anti-IgG concentration is 0.2mg/cm
2.
9. lateral chromatography detection system as claimed in claim 1, it is characterized in that, described pad refers to the antigen being coated with colloid gold label.
10. lateral chromatography detection system as claimed in claim 1, it is characterized in that, described NC film is the NC film being coated with the T line of specific antibody and the C line of anti-human IgM or IgA or IgE antibody.
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| CN108348913A (en) * | 2015-10-26 | 2018-07-31 | 幸福科技公司 | Lateral flow assay device and method |
| CN105403562A (en) * | 2015-12-16 | 2016-03-16 | 合肥泰特信息科技有限责任公司 | Mycoplasma detector |
| CN105891509B (en) * | 2016-05-03 | 2018-05-11 | 同昕生物技术（北京）有限公司 | For quantitatively detecting the lateral chromatography system of alpha-fetoprotein variant |
| CN109142743B (en) * | 2018-07-25 | 2022-02-08 | 宙斯生命科技(常州)有限公司 | Multiple detection kit for self-immune liver antibody |
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