CN110462383A - Immunoassays and its application method based on magnetic-particle - Google Patents
Immunoassays and its application method based on magnetic-particle Download PDFInfo
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- CN110462383A CN110462383A CN201880014031.9A CN201880014031A CN110462383A CN 110462383 A CN110462383 A CN 110462383A CN 201880014031 A CN201880014031 A CN 201880014031A CN 110462383 A CN110462383 A CN 110462383A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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Abstract
The present invention partially describes for using magnetic-particle the improved method for detecting the analyte in biological sample, measurement and kit.It the described method comprises the following steps: contacting sample with the magnetic conjugate comprising magnetic-particle and capture portion, the capture portion is configured as in conjunction with the analytes of interest analytes in the sample;Contact the sample with the report conjugate comprising reporter and report bound fraction, the report bound fraction is configured as in conjunction with the analytes of interest analytes in the sample;Make the analytes of interest analytes in conjunction with the capture portion and the report bound fraction;The analytes of interest analytes is separated from the sample by applying magnetic field to analysis chamber;And the presence of the analytes of interest analytes is detected by detecting the reporter, is not present or horizontal.
Description
Cross reference to related applications
The U.S. Provisional Application No.62/450,623 and in August, 2017 that the International Application claim is submitted on January 26th, 2017
The U.S. Provisional Application No.62/544 submitted for 11st, 393 equity, the full content of the provisional application is by reference simultaneously
Enter herein.
Invention field
Invention as described herein relates generally to improved method, measurement and the examination of the analyte in detection biological sample
Agent box.
Background of invention
Analyte detection is analyzed in the various industries such as medicine and biological study and environmental science with various clinical and non-
Clinical application.Conventional method for analyzing analyte detection includes such as enzyme linked immunosorbent assay (ELISA) (ELISA), mass spectrum and high pressure liquid
The measurement of phase chromatography (HPLC).HPLC and mass spectrum can be used for testing and analyzing object based on charge and/or size, and ELISA is available
Object can be tested and analyzed by capturing agent and the antigen of detection agent (for example, antibody, aptamer etc.) identification on based on analyte.It is special
Not, ELISA detection has become detection method more commonly used in life science.However, traditional ELISA may be time-consuming
, because it is related to various incubations and washing step, and possibly enough sensitivity can not be provided for various applications.In addition,
Parameter for carrying out ELISA measurement is alterable height, so that the measurement is difficult to develop into general-purpose platform, especially
It is difficult to develop into the home diagnostic method of personal use person.
In fact, health care will be benefited a great deal if easily and biomarker continually can be monitored at home.Citing
For, compared with medical professional, the health status that individual is more suitable for monitoring oneself and guides nursing, because of medical professionalism people
Member's time and resource in terms for the treatment of patient is limited.In addition, obtaining health and fitness information, individual can more accurately plan life,
Including for example planning the reproduction time.
Therefore, there is still a need for the improved method for the analyte in test sample, these improved methods and conventional method
Compared to the time needed for executing and input the sensitivity for reducing, while maintaining or improving detection.
Summary of the invention
This document describes immunoassays;Kit including the measurement;And using feeling in the measurement test sample
The presence of interest analysis object is not present or horizontal method.
In some respects, the present invention relates to system and the sides for testing and analyzing object (such as the antigen for carrying out biological sample)
Method, the separation that the system and method utilize magnetic to mediate have higher sensitivity than existing method, such as dependent on emerging to sense
The detection based on antibody of interesting antigen (such as can be used for antigen associated with the health status of individual).Compared to exempting from for this field
Epidemic disease measurement, immunoassays, kit and method as described herein provide sizable advantage.In fact, in some embodiment party
In case, invention as described herein includes saving sample preparation, this has no precedent in known immunoassays.This field shows
There is method to need a large amount of sample preparation.It here, in some embodiments, can be by sample and required reagent (including magnetism
Conjugate and reporter or report conjugate) it is mixed in analysis chamber, and apply magnetic field as " traction (pull downwards
Down then) " step is visualized and/or is quantified to reporter.
In one embodiment, the present invention includes a kind of for the presence of analytes of interest analytes in test sample, do not deposit
Or horizontal method.In some embodiments, sample can be body fluid as herein defined.
In one embodiment, method of the invention may include the step being added to sample in analysis chamber.One
In a little embodiments, it may include the sample being delivered to sample with analysis chamber in fluid communication in analysis chamber that sample, which is added to,
Collector (for example, absorbent or wick material).In some embodiments, then sample can be fed by sample divider
It analyzes in chamber.In some embodiments, method of the invention may include making sample and comprising magnetic-particle and capture portion
Magnetic conjugate contact, the capture portion be configured as combine sample in analytes of interest analytes;In some embodiments
In, magnetic conjugate can be placed at sample divider, and the step of contacting sample and magnetic conjugate is in sample
Occur at product collector.In some embodiments, before sample is added in sample divider, magnetism can be conjugated
Object is embedded in a part of sample divider.In some embodiments, method of the invention may include making to analyze in chamber
Sample is contacted with magnetic conjugate.In some embodiments, capture portion be antibody, antigen-binding fragment, antigen, receptor,
Ligand, aptamer, aptamer receptor, nucleic acid or small molecule.In some embodiments, capture portion is capture antibody.In some realities
It applies in scheme, magnetic conjugate includes magnetic-particle and capture antibody.
In some embodiments, method described herein, which can comprise the following steps that, makes sample and reporter or comprising report
It accuses object and reports the report conjugate contact of bound fraction, the report bound fraction is configured as combining interested in sample
Analyte.In some embodiments, can by reporter or report conjugate be placed at sample divider, and make sample with
The step of reporter or report conjugate contact, can occur at sample divider.In some embodiments, by sample
Before being added in sample divider, can will reporter or report conjugate insertion sample divider a part in.One
In a little embodiments, method described herein may include that the sample made analyze in chamber is contacted with reporter or report conjugate
Step.In some embodiments, report bound fraction is antibody, antigen-binding fragment, antigen, receptor, ligand, aptamer, fits
Receptor body, nucleic acid or small molecule.In some embodiments, report bound fraction is reporter antibody.In some embodiments,
Report that conjugate includes reporter and reporter antibody.In some embodiments, method described herein may include make it is interested
The step of analyte is in conjunction with capture antibody and reporter antibody.In some embodiments, method described herein may include leading to
It crosses and applies the step of magnetic field separates analytes of interest analytes from sample to analysis chamber.In some embodiments, this paper institute
The method stated may include detecting the presence of analytes of interest analytes by examining report object, being not present or horizontal step.
In some embodiments, reporter may include metal core (for example, metal particle or metal nanoparticle) and
It may include silica shell.In some embodiments, reporter includes metal core and has silica shell.In some implementations
In scheme, reporter includes multiple quantum dots.In some embodiments, reporter includes the metal with silica shell
Core, and silica shell is impregnated with multiple quantum dots.In some embodiments, reporter includes metal core, and metal
Core may include or another metal as described herein.
In some embodiments, reporter as described herein can be fluorescence report object, phosphorescence reporter or colorimetric report
It accuses object (such as coloured particle), the absorption or scattering that the reporter can be configured to measurement light are (alternatively, for example by colorimetric point
Analysis measure certain color in the presence/absence of).
In some embodiments, method described herein, which can comprise the further steps of:, makes sample and magnetic conjugate
After contact, by applying magnetic field to analysis chamber come the analytes of interest analytes in concentrating sample;Then it reduces in analysis chamber
The volume of sample.In some embodiments, method described herein, which may additionally include, makes sample contact it with report conjugate
Before the step of deactivating magnetic field.
In some embodiments, method described herein, which can comprise the further steps of:, makes sample and magnetic conjugate
After contact, by applying magnetic field to analysis chamber come the analytes of interest analytes in concentrating sample;One is removed from analysis chamber
Determine the sample of volume;And the sample of the buffer of certain volume and/or other volume is added in analysis chamber.Some
In embodiment, method described herein further includes in the step for making sample with reporting that conjugate deactivates magnetic field before contacting
Suddenly.
In some embodiments, reporter antibody biotin labeling as described herein.In some embodiments, herein
Reporter streptavidin functionalization.In some embodiments, reporter antibody chain as described herein
Mould avidin protein tag.In some embodiments, reporter as described herein is with biotin functionalized.
In some embodiments, analytes of interest analytes as described herein can be any analyte as described herein and/
Or biomarker.In some embodiments, analytes of interest analytes can be selected from human chorionic gonadotrophin (hCG), rush
Corpus luteum hormone (LH)/lutropin (Lutropin), prostate-specific antigen (PSA), herpes simplex virus (HSV) are anti-
Body, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation biomarker, troponin, Lay
Nurse disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol biomarker, thyroid swash
Element, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biomarker, liver function biomarker
Object, estrogen, progesterone, lactic acid and their combination.In some embodiments, bacterium can be A group of streptococcus
(Streptococcus-A), Chlamydia and/or micrococcus gonococcus (Gonorrhea).In some embodiments, inflammation biology mark
Note object can be CRP, SAA and/or MP8.In some embodiments, liver function biomarker can be ALT and/or AST.
In some embodiments, analytes of interest analytes can be selected from the group being made up of: ovulation biomarker, Aschheim-Zondek mark
Remember object, Strep throat biomarker, prostate cancer biomarker, bleb biomarker, diabetes biomarker
Object, inflammation biomarker, heart attack biomarker, Chlamydia biomarker, bacterial biomarkers object, Lyme disease
Biomarker, cholesterol biomarker, hypothyroidism biomarker, hepatitis C biomarker, rhinopathy
Malicious biomarker, influenza biomarker, liver function biomarker, fertility biomarker, muscular fatigue biomarker
Object and their combination.
In some embodiments, ovulation biomarker can be originated from the sample based on urine, blood or serum.One
In a little embodiments, Aschheim-Zondek marker can be originated from the sample based on urine or blood.In some embodiments, hammer
Bacterium property laryngitis biomarker can be originated from the sample based on saliva.In some embodiments, the sample based on saliva can be with
It is the aliquot of saliva, cheek swab or brush,throat.In some embodiments, prostate cancer biomarker can be with source
From the sample based on blood, serum or urine.In some embodiments, bleb biomarker can be originated from derived from blood or
The sample of saliva.
In one embodiment, method described herein can detecte the presence of analytes of interest analytes in sample, not deposit
Or it is horizontal.
In some embodiments, method described herein can comprise the following steps that make sample with comprising magnetic-particle and
The magnetic conjugate of capture portion contacts, and the capture portion is configured as combining the analytes of interest analytes in sample;Make sample
It is contacted with the report conjugate comprising reporter and report bound fraction, the report bound fraction is configured as combining in sample
Analytes of interest analytes;Make analytes of interest analytes in conjunction with capture portion and report bound fraction;By applying to analysis chamber
Magnetic field separates analytes of interest analytes from sample;And/or depositing for analytes of interest analytes is detected by examining report object
, be not present or horizontal.
In some embodiments, methods herein, which can comprise the following steps that, ties sample with comprising reporter and report
The report conjugate contact of part is closed, the report bound fraction is configured as combining the analytes of interest analytes in sample;Make sample
Product are contacted with the magnetic conjugate comprising magnetic-particle and capture antibody, and the capture portion is configured as combining the sense in sample
Interest analysis object;Make analytes of interest analytes in conjunction with capture portion and report bound fraction;By applying magnetic field to analysis chamber
Analytes of interest analytes is separated from sample;And/or the presence, no of analytes of interest analytes is detected by examining report object
In the presence of or it is horizontal.
In some embodiments, method described herein can comprise the following steps that make sample with comprising magnetic-particle and
The magnetic conjugate of capture portion contacts, and the capture portion is configured as combining the analytes of interest analytes in sample;Make to feel emerging
Interesting analyte is in conjunction with capture portion;By to analysis chamber apply magnetic field with by magnetic conjugate and with the magnetic conjugate
The analytes of interest analytes of association is drawn downwards, so that analytes of interest analytes be separated from sample;Make sample and comprising reporter
With the report conjugate contact of report bound fraction, the report bound fraction is configured as combining the analysis interested in sample
Object;Make analytes of interest analytes in conjunction with report bound fraction;By to analysis chamber apply magnetic field by analytes of interest analytes and with
The report bound fraction that the analytes of interest analytes combines is separated from sample;And/or by with light source and Photoelectric Detection
Device examining report object detects the presence of analytes of interest analytes, is not present or horizontal.
In some embodiments, method described herein, which can comprise the following steps that, makes sample and comprising reporter and report
The report conjugate contact of bound fraction is accused, the report bound fraction is configured as combining the analytes of interest analytes in sample;
Make analytes of interest analytes in conjunction with report bound fraction;Contact sample with the analyte of magnetic particle labels, described magnetic
The analyte of grain label is configured as combining report conjugate in the case where analytes of interest analytes is not present in the sample;Pass through to
Sample applies magnetic field and separates the analyte of magnetic particle labels from sample;And/or it is detected by examining report object
The presence of analytes of interest analytes is not present or horizontal.
In some embodiments, method described herein can comprise the following steps that make sample with comprising magnetic-particle and
The magnetic conjugate of capture portion contacts, and the capture portion is configured as combining the analytes of interest analytes in sample;Make to feel emerging
Interesting analyte is in conjunction with capture portion;Contact sample with the report bound fraction comprising biotin labeling, the report combines
It is partially configured as combining the analytes of interest analytes in sample;Make sample and the reporter comprising streptavidin label
Contact, the streptavidin label are configured as combining biotin labeling;It will feel emerging by applying magnetic field to sample
Interesting analyte is separated from sample;And/or the presence of analytes of interest analytes is detected by examining report object, be not present or
It is horizontal.
In some embodiments, method described herein can comprise the following steps that make sample with comprising magnetic-particle and
The magnetic conjugate of capture portion contacts, and the capture portion is configured as combining the analytes of interest analytes in sample;Make to feel emerging
Interesting analyte is in conjunction with capture portion;Contact sample with the report bound fraction marked comprising streptavidin, institute
Report bound fraction is stated to be configured as combining the analytes of interest analytes in sample;Make sample and the reporter comprising biotin labeling
Contact, the biotin labeling are configured as combining strepto- avidin protein tag;It will feel emerging by applying magnetic field to sample
Interesting analyte is separated from sample;And/or the presence of analytes of interest analytes is detected by examining report object, be not present or
It is horizontal.
In one embodiment, method described herein, which can comprise the following steps that, is added to sample in analysis chamber;
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as combining in sample
Analytes of interest analytes;Make analytes of interest analytes in conjunction with capture antibody;By applying magnetic field to analysis chamber to sew magnetism
It closes object and is drawn downwards with the analytes of interest analytes of the magnetic conjugate association, so that analytes of interest analytes be divided from sample
From;Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as combining sample
In analytes of interest analytes;Make analytes of interest analytes in conjunction with reporter antibody;And by being detected with light source and photoelectric detector
Reporter detects the presence of analytes of interest analytes, is not present or horizontal.
In some embodiments, method described herein can be to negative sample (that is, not including analytes of interest analytes
Sample) it executes, so that it is determined that analytes of interest analytes is not present in sample.
In one embodiment, method described herein, which can comprise the following steps that, is added to sample in analysis chamber;
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as combining in sample
Analytes of interest analytes;Make analytes of interest analytes in conjunction with capture antibody;Contact sample with the analyte for reporting substance markers, institute
The analyte for stating report substance markers is configured as combining magnetic conjugate in the case where analytes of interest analytes is not present in the sample;
Analytes of interest analytes is separated from sample by applying magnetic field to analysis chamber;And it is detected by examining report object and feels emerging
The presence of interesting analyte is not present or horizontal.
In one embodiment, method described herein, which can comprise the following steps that, is added to sample in analysis chamber;
Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as combining in sample
Analytes of interest analytes;Make analytes of interest analytes in conjunction with reporter antibody;Contact sample with the analyte of magnetic particle labels, institute
The analyte for stating magnetic particle labels is configured as combining report conjugation in the case where analytes of interest analytes is not present in the sample
Object;The analyte of magnetic particle labels is separated from sample by applying magnetic field to sample;And by examining report object come
It detects the presence of analytes of interest analytes, be not present or horizontal.
In one embodiment, method described herein, which can comprise the following steps that, is added to sample in analysis chamber;
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as combining in sample
Analytes of interest analytes;Make analytes of interest analytes in conjunction with capture antibody;Make sample and the reporter antibody comprising biotin labeling
Contact, the reporter antibody are configured as combining the analytes of interest analytes in sample;Make sample and comprising streptavidin egg
The reporter of white marker contacts, and the streptavidin label is configured as combining biotin labeling;By to sample
Apply magnetic field to separate analytes of interest analytes from sample;And depositing for analytes of interest analytes is detected by examining report object
, be not present or horizontal.
It in some embodiments, include contact procedure (for example, making sample and magnetic conjugation in method described herein
Object, reporter antibody, the conjugate for reporting substance markers and/or the contact of report conjugate) in the case where, this contact procedure may include
By sample (it may contain analyte) and corresponding magnetic conjugate, reporter antibody, the conjugate and/or report for reporting substance markers
Conjugate is accused to be incubated within the selected period and at a temperature of selected.
Detailed description of the invention
When read in conjunction with the accompanying drawings, foregoing summary and following specific embodiments are better understood with.
When Figure 1A to Fig. 1 D is shown presence or absence of antigen, the example of the sandwich mode of immunoassays as described herein
Property feature.Figure 1A shows the exemplary parameter of sandwich mode: (a) analytes of interest analytes (such as hCG);(b) to interested point
Analyse the special antibody #1 (such as the anti-hCG 4F9 of MoLogic) of object;(c) the antibody #2 special to analytes of interest analytes (such as
The anti-hCG 5011 of Medix);(d) as shown here, antibody 1 and 2 can be simultaneously in conjunction with analyte;(e) fluorescence report object;With
And (f) magnetic-particle.There are analyte, compound will be formed, be not only labeled but also magnet can be attracted to.Figure
1B shows magnetic conjugate and reports the addition of conjugate.Fig. 1 C shows magnetic and draws downwards.Fig. 1 D shows analyte
Detection.
When Fig. 2A to Fig. 2 F is shown presence or absence of antigen, the independent addition mode of immunoassays as described herein
Example feature.Fig. 2A shows the exemplary parameter of individually addition mode: (a) analytes of interest analytes (hCG);(b) emerging to sense
The special antibody #1 of interesting analyte (such as the anti-hCG 4F9 of MoLogic);(c) the antibody #2 special to analytes of interest analytes (such as
The anti-hCG 5011 of Medix);(d) as shown here, antibody 1 and 2 can be simultaneously in conjunction with analyte;(e) fluorescence report object;With
And (f) magnetic-particle.Fig. 2 B shows the addition of magnetic conjugate.Fig. 2 C shows magnetic and draws downwards.Fig. 2 D shows report
The addition of conjugate.Fig. 2 E shows second of magnetic and draws downwards.Fig. 2 F shows the detection of analyte.
When Fig. 3 A to Fig. 3 E is shown presence or absence of antigen, the competition sexual norm of immunoassays as described herein shows
Example property feature.Fig. 3 A shows the exemplary parameter of competition sexual norm: (a) analytes of interest analytes;(b) to analytes of interest analytes spy
Different antibody;(c) fluorescence report object;And (d) magnetic-particle.In the absence of the analyte, the bound site on antibody
Point is still available, so that compound can be made between magnetic-particle and reporter.There are analyte, trip
From antigen can binding site on blocking antibody, thus prevent to form compound with magnetic particles and reporter.Fig. 3 B is shown
The addition of magnetic conjugate.Fig. 3 C shows the addition of the analyte of report substance markers.Fig. 3 D shows magnetic and draws downwards.Fig. 3 E
Show the detection of analyte.
When Fig. 4 A to Fig. 4 G is shown presence or absence of antigen, the example of the three-level schema of immunoassays as described herein
Property feature.Fig. 4 A shows the exemplary parameter of three-level schema: (a) analytes of interest analytes;(b) special to analytes of interest analytes and
With the antibody #1 of biotin labeling;(c) the antibody #2 special to analytes of interest analytes;(d) as shown here, antibody 1 and 2 can
Simultaneously in conjunction with analyte;(e) the fluorescence report object of streptavidin functionalization is used;And (f) magnetic-particle.It is depositing
In the case where analyte, compound will be formed with the biotin of magnetic particle labels.It is marked when by streptavidin
Reporter when being added in the compound, reporter in conjunction with the compound and will will generate fluorescent composition, and the fluorescence is multiple
Closing object will be attracted to magnet.Fig. 4 B shows the addition of magnetic conjugate.Fig. 4 C shows magnetic and draws downwards.Fig. 4 D is shown
The addition of the antibody of biotin labeling.Fig. 4 E shows the addition of the reporter of streptavidin label.Fig. 4 F is shown
Second magnetic is drawn downwards.Fig. 4 G shows the detection of analyte.
Fig. 5 shows the sensitivity of the measurement for detecting human chorionic gonadotrophin (hCG) of the invention.*
Elecsys electrochemiluminescent immunoassay HCG STAT (601 module of cobas e, 602 module of cobas e).1fM=10-15
Mol/L.
Fig. 6 shows the sensitivity of the measurement for detecting metakentrin (LH) of the invention.* Elecsys electrochemistry
Luminescent immunoassay LH (cobas).1fM=10-15Mol/L.
Fig. 7 shows the sensitivity of the measurement for detecting prostate-specific antigen (PSA) of the invention.*Elecsys
Electrochemiluminescent immunoassay Free PSA (cobas).1fM=10-15Mol/L.
When Fig. 8 A to Fig. 8 D is shown presence or absence of bacterium, the Bacteria Detection mode of immunoassays as described herein
Example feature.Fig. 8 A shows the exemplary parameter of Bacteria Detection mode: (a) interested bacterium;(b) on bacterium surface
The special antibody #1 of analyte;(c) the antibody #2 special to the analyte on bacterium surface (may be identical as antibody #1);
(d) fluorescence report object;And (e) magnetic-particle.The antibody of many labels can be immediately in conjunction with single bacterium, and generation can be inhaled
The bacterium for leading to magnet and being fluorescently labeled.Fig. 8 B shows magnetic conjugate and reports the addition of conjugate.Fig. 8 C is shown
Magnetic is drawn downwards.Fig. 8 D shows the detection of analyte.
Fig. 9 shows the sensitivity of the measurement for detecting A group of streptococcus of the invention.
Figure 10, which is shown, detects E3G in exemplary competition sexual norm immunoassays of the invention.
Figure 11 shows the PSA measured value of immunoassays as described herein and the PSA realized in clinical labororatory is measured
Correlation between value.
Figure 12 shows the type testing to hCG in urine sample as measured by immunoassays as described herein.
Figure 13 shows the curve graph of result of the quantization from Figure 12.
Figure 14 shows staircase curve, and this graph illustrate the fraction positives as the function of number of days after ovulation.As herein
The immunoassays (i.e. Confer Magneto) are shown with purple.First Response is with pink display.With black
With white as reference, at the 10th day, Confer Magneto was top curve, and First Response is bottom curve.
Figure 15 shows the summary of gestation data, shows and gives number of days reading before First Response as the positive
Sample percentage.
Figure 16 shows the frontlighting exciting method arranged using the dichroscope with 405nm laser-excitation source.Dichroic
Mirror: Thor Labs, DMLP490R, 25mm x 36mm long leads to dichroscope, 490nm cutoff wavelength.Filter: Thor Labs
FGL610 filter.
Figure 17 A and Figure 17 B show the result that the scheme of c reactive protein is detected for analyzing blood serum sample.Figure 17 A shows
Go out the CRP concentration series in buffer, and Figure 17 B shows the CRP concentration series in mark-on serum.
Specific embodiment
The present invention is based in part on following discovery: for example, application relevant for health, can be used Magneto separate with sensitive
And effective mode detects analytes of interest analytes.
In some respects, the present invention provides a kind of for detecting the presence of analyte in solution, being not present or horizontal
Method, under conditions of the bonding agent of label is placed in permission bonding agent in conjunction with analyte;It will be comprising combining
Under conditions of the particle of agent is placed in permission bonding agent in conjunction with analyte;Applying has the magnetic field of sufficient intensity will include analysis
The gained compound of object and label is separated from the solution;And the label in detection compound.
In various embodiments, the amount of the label detected is compared presence or absence of magnetic field.For example,
In some embodiments, the presence of label (for example, using any technology as described herein) instruction analyte is detected.This
Outside, in some embodiments, amount (for example, using any technology as described herein) the instruction analyte of the label detected
Amount.In various embodiments, the amount of the label detected and the amount of analyte are proportional.In some embodiments, it does not examine
Measuring (or detecting few) label (for example, using any technology as described herein) instruction analyte, there is no (or basic
There is no).
In various embodiments, about the presence of analytes of interest analytes in sample, be not present or horizontal information guiding
Health care or life style relevant to health determine.
Immunoassays
Invention section as described herein it is related to for analyte in test sample for detecting changing for analytes of interest analytes
Into immunoassays, the sample may include humoral sample.
In one embodiment, the present invention includes that can be used for the presence of analytes of interest analytes in test sample, be not present
Or horizontal immunoassays.
In some embodiments, immunoassays as described herein may include analysis chamber, and the analysis chamber may include
Sample, magnetic conjugate, reporter or report conjugate, magnet, light source and/or photoelectric detector.
In some embodiments, immunoassays as described herein may include with analysis chamber be connected or otherwise with
Analyze the sample divider of chamber in fluid communication.In some embodiments, sample divider may include the suction of absorbable sample
Receipts and/or wick material, then sample is fed into analysis chamber by sample divider.In some embodiments, it can incite somebody to action
One or more of magnetic conjugate, reporter and report conjugate are placed in sample divider, so that when adding sample
When into sample divider, sample can contact magnetic conjugate, reporter, report conjugate or their combination.Some
It, can be by one or more of magnetic conjugate, reporter and report conjugate insertion sample divider in embodiment
In a part.
In some embodiments, sample divider includes sponge, foam or film (such as polyurethane sponge or foam), or
Another adsorbent and/or absorbing material.In some embodiments, sample divider may include cellulose, NC Nitroncellulose
And/or polyvinylidene fluoride (PVDF) film, sponge or foam.In some embodiments, sample divider can be Porex suction
It attached dose, can be based on PE/PET's.In some embodiments, Porex adsorbent may include the removing of Porex conjugate
Layer, can be the sinter based on PE.
In some embodiments, magnet can be permanent magnet, can with analysis chamber separate, so as to analyze chamber
Apply magnetic field.In some embodiments, magnet can be electromagnet, can be activated or deactivate, so as to analysis
Chamber applies magnetic field.
In some embodiments, light source is connected to analysis chamber and can be configured to that light transmission is made to pass through analysis chamber
A part.
In some embodiments, analysis chamber can be a chamber or two chambers or three chambers or four
Chamber.In some embodiments, analysis chamber can be one or more chambers or two or more chambers or three
Or more chamber or four or more chambers.In some embodiments, analysis chamber may include multiple chambers.One
In a little embodiments, multiple chambers can be in fluid communication.In some embodiments, sample, reporter or report can be sewed
It closes object and magnetic conjugate mixes in the first chamber.In certain embodiments, magnetic field can be applied in second chamber,
And the light source for being connected to analysis chamber can be configured to that light transmission is made to pass through second chamber.In some embodiments, herein
The method and step can execute in the independent chamber of each comfortable analysis cavity room.In some embodiments, analysis chamber can be with
It is a chamber, and all method and steps can execute in the same chamber.
In some embodiments, photoelectric detector can connect to analysis chamber (for example, towards light source and light source pair
It is neat or opposite with light source), and can be configured to detect the light by lit transmissive by analysis chamber, to measure the transmission of light
And/or it absorbs.In some embodiments, photoelectric detector can connect to analysis chamber ((orthogonal photograph orthogonal with light source
It is bright)), and can be configured to test and analyze the reporter in a part of chamber or report the fluorescence and/or phosphorescence of conjugate.
In some embodiments, photoelectric detector can connect to analysis chamber ((transillumination) opposite with light source), and can quilt
The fluorescence and/or phosphorescence of the reporter or report conjugate that are configured to test and analyze in a part of chamber.In some embodiment party
In case, photoelectric detector can connect to analysis chamber and (be aligned with light source, such as (suitable by dichroscope as shown in figure 16
Optical illumination)), and can be configured to test and analyze chamber a part in reporter or report conjugate fluorescence and/or
Phosphorescence.In some embodiments, photoelectric detector may include one or more photomultiplier detectors and photodiode
Detector.As used herein, term " photoelectric multiplier " or " photomultiplier tube " refer to through photoelectric effect and secondary electron hair
Penetrate the optical detection component that incident photon is converted into electronics.Term photomultiplier tube is intended to include containing for current multiplication
The device of independent dynode and contain those of one or more channel electron multipliers device.As used herein, term
" fluorescence detector " or " photoelectric detector " refers to the device that output signal is generated when being irradiated with luminous energy.Therefore, term optics
Detector system is considered as pointing out energy in physical quantity or transactional purpose from one in the broadest sense
Kind form is converted to another form of device.Fluorescence detector includes but is not limited to photomultiplier tube and photodiode.Such as
Used herein, term " photodiode " refers to solid state photodetector type, including but not limited to PN, PIN, APD, CMOS and
CCD.In some embodiments, photoelectric detector may include the detector based on PN, PIN-based detector, based on APD's
One or more of detector, the detector based on CMOS and detector based on CCD.In some embodiments, it analyzes
Chamber includes photoelectric detector as described herein.In some embodiments, analyzing chamber to include includes the detection based on PN
One of device, PIN-based detector, the detector based on APD, the detector based on CMOS and detector based on CCD
Or more persons.
In some embodiments, magnetic conjugate as described herein may include magnetic-particle and form with the magnetic-particle
The capture antibody of conjunction.In some embodiments, magnetic-particle can be in conjunction with capture antibody.
In some embodiments, report conjugate as described herein may include reporter and reporter antibody.In some realities
It applies in scheme, reporter can be in conjunction with reporter antibody.
Binding partners and antibody
In some embodiments, capture portion and report bound fraction can be identical or different.In some embodiments
In, capture portion and/or report bound fraction can be antibody, antigen-binding fragment, antigen, receptor, ligand, aptamer, aptamer
Receptor, nucleic acid or small molecule.In some embodiments, capture portion can be capture antibody.In some embodiments, it reports
Accusing bound fraction can be reporter antibody.
In some embodiments, it captures antibody and/or reporter antibody can be selected from by for as described herein interested
The group of the antibody composition of analyte.In some embodiments, capturing antibody and/or reporter antibody can be selected from and be made up of
Group: Anti-hCG antisera, anti-LH antibody, anti-psa antibody, anti-HSV antibody, anti-E3G antibody, antibacterium cell surface protein antibody,
Anti- HbA1 C antibody, anti-c reactive protein antibody, anti-inflammatory biomarker antibody, anti-troponin antibodies, anti-lime
Sick antibody, anti-LDL biomarker antibody, anti-HDL biomarker antibody, anti-total cholesterol biomarker antibody, resist it is female
Hormone antibody, antiprogestin antibody, antithyrotropic hormone antibody, anti-hepatitis c virus biomarker antibody, rhinovirus
Biomarker antibody, anti influenza biomarker antibody, anti-liver function biomarker antibody, antifertility biomarker are anti-
Body, muscular fatigue biomarker antibody and their combination.
In some embodiments, capturing antibody and/or reporter antibody can be in combination with selected from the group being made up of
Analytes of interest analytes antibody: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate
Specific antigen (PSA), herpes simplex virus (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, C
Reactive protein, inflammation biomarker, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biology
Marker, total cholesterol biomarker, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker
Object, influenza virus biomarker, liver function biomarker, estrogen, progesterone, lactic acid and their combination.Some
In embodiment, bacterium can be A group of streptococcus, Chlamydia and/or micrococcus gonococcus.In some embodiments, inflammation biology
Marker can be CRP, SAA and/or MP8.In some embodiments, liver function biomarker can be ALT and/or
AST.In some embodiments, analytes of interest analytes can be selected from the group being made up of: ovulation biomarker, gestation are raw
Substance markers object, Strep throat biomarker, prostate cancer biomarker, bleb biomarker, diabetes biology
Marker, inflammation biomarker, heart attack biomarker, Chlamydia biomarker, bacterial biomarkers object, Lay
Nurse disease biomarker, cholesterol biomarker, hypothyroidism biomarker, hepatitis C biomarker,
Rhinovirus biomarker, influenza biomarker, liver function biomarker, fertility biomarker, muscular fatigue biology
Marker and their combination.
In various embodiments, binding partners as described herein may include but be not limited to antibody, including but not limited to
Single-chain antibody, antigen binding antibody fragment, antigen (such as combining its antibody), receptor, ligand, aptamer, aptamer receptor, core
Acid, small molecule etc..
In some embodiments, it captures antibody and/or reporter antibody can be selected from the group being made up of: INN-hCG-
2, INN-hCG-2,5008-SP5,5008-SP5 and 5011SPRN-1 or its functional variety.In some embodiments, it captures
Antibody and/or reporter antibody can be one or more antibody described in table 1.
Listed in table 1 be can be used for detecting the exemplary antibodies of hCG to (for example, select one of following object or
The two come generate to):
Table 1
Have determined that selection can be used successfully to several key factors of the antibody pair of urine.For example, in combination with energy
Power is prerequisite.In some embodiments, antibody allows for combining intact hCG and β subunit.With high parent
Antibody with power but low combination rate is effective but needs to be incubated for for a long time.Following antibody is successfully used in detection human urine
Endogenous hCG:INN-hCG-2, INN-hCG-22,5008-SP5,5014-SPTN5 and 5011SPRN-1.Therefore, of the invention
Embodiment be related to using or INN-hCG-2, INN-hCG-22,5008-SP5,5014-SPTN5 and 5011 SPRN-1 or its function
One or both of energy segment.
In some embodiments, it captures antibody and/or reporter antibody can be Fitzgerald 10-L15A and 10-
L15B or its functional variety.
In some embodiments, it captures antibody and/or reporter antibody can be anti-psa 5001 (Medix), anti-PsA
5012 (Medix) or its functional variety.
In some embodiments, it captures antibody and/or reporter antibody can be the targeting A manufactured by Biospacific
The polyclonal antibody or 2601 SPTN-5 of monoclonal A group of streptococcus of group of streptococcus antigen or 2603 SPTN-5 antibody or its function
Variant.
In various embodiments, analyte to be detected can actually be any analyte, and condition is available pair
The special binding partners of the analyte.In various embodiments, analyte can be simultaneously by least two binding partners
In conjunction with.In various embodiments, analyte is combined gametophyte in same epitope or different epitopes and combines.In various embodiment party
In case, analyte can be or may include nucleic acid, peptide or protein matter, carbohydrate, lipid or any combination of them.
In various embodiments, invention as described herein is covered such as detection human chorionic gonadotrophin (hCG) and is made
For a part of pregnancy tests.Intact hCG includes the dimerization formed between two hCG subunits i.e. α-hCG and β-hCG
Body.In some embodiments, using a pair of of antibody of one or more epitopes on antibody, such as identification α-hCG and β-hCG
Detect hCG.In one embodiment, using the antibody test hCG of identification β-hCG.In various embodiments, in this paper institute
Using any known antibody for α-hCG or β-hCG in the invention stated.In some embodiments, antibody includes INN-
HCG-2, INN-hCG-2,5008-SP5,5008-SP5 and 5011 SPRN-1 or its functional variety.In some embodiments,
Compared to conventional pregnancy tests in the market, such as (as used herein, by those of First Response exploitation pregnancy tests
" First Response " refers to the over the counter chromatographic immunoassay measurement for qualitative detection human chorionic gonadotrophin (hCG)),
Method described herein can detect hCG earlier and more accurately.
In various embodiments, such as detection metakentrin (LH)/lutropin is covered in invention as described herein
As a part for identifying the test of ovulation.The exemplary antibodies that can be used for the identification LH of methods described herein include but not
It is limited to Fitzgerald 10-L15A and 10-L15B or its functional variety.
In some embodiments, invention as described herein also covers detection oestrone -3- glucosiduronic acid (E3G) as another
Kind is for identifying the biomarker of ovulation.In some embodiments, it is all compared to conventional ovulation test in the market by
(the non-place (ClearBlue Digital Ovulation Test) is tested in ovulating such as ClearBlue number for ClearBlue exploitation
Square LH test) or other ovulation tests, method described herein can detect LH or E3G earlier and more accurately.In some realities
It applies in scheme, method described herein suffers from Stein-Leventhal syndrome (PCOS) particularly suitable for prediction, because high LH baseline cannot
Use the ovulation of the women of currently marketed ovulation test.
In various embodiments, the present invention covers detection prostate-specific antigen (PSA).It can be used for side described herein
The exemplary antibodies of the identification PSA of method include but is not limited to anti-psa 5001 (Medix), anti-PsA 5012 (Medix) or its function
Variant.
In various embodiments, the present invention covers present in detection herpes simplex virus (HSV) or blood or serum
For the antibody of HSV.In some embodiments, method described herein be related to detecting HSV-1 (herpes of mouth) or blood or
The antibody of HSV-1 is directed to present in serum.In other embodiments, method described herein is related to detecting HSV-2 (reproduction
Device bleb) or for HSV-2 antibody.For example, HSV-1 or HSV-2 antigen can be used as binding partners to detect blood or blood
For the presence of the antibody of HSV-1 or HSV-2 in clear.
In various embodiments, the present invention covers detection A group of streptococcus (Streptococcus-A/Strep-A).It can
Exemplary antibodies for detecting A group of streptococcus include but is not limited to the targeting A group of streptococcus antigen manufactured by Biospacific
Polyclonal antibody or monoclonal A group of streptococcus 2601SPTN-5 or 2603SPTN-5 antibody or its functional variety.In some realities
It applies in scheme, (has patient with sympotoms by oneself for detecting compared to routine test such as QuickVue Dipstick Strep A test
Brush,throat A group of streptococcus antigen immunofluorescent test), method described herein can detect earlier and more accurately
A group of streptococcus antigen.
In one embodiment, such as QuickVue Dipstick is quickly tested compared to currently marketed other
The sensitivity of Strep A test, the sensitivity of method described herein is at least about 10 times, about 20 times, about 30 times, about 40 times,
About 50 times, about 60 times, about 70 times, about 80 times, about 90 times or at least about 100 times.In various embodiments, with other streptococcus
Bacterium such as B group of streptococcus, C group of streptococcus or G group of streptococcus are compared, and method described herein has enhancing to A group of streptococcus
Specificity.
In various embodiments, the present invention covers the various infection of detection, including stranguria syndrome and choamydiae infection.It can be used
The Exemplary antigens of antibody test in methods described herein include but is not limited to that Chlamydia LPS KDO- trisaccharide, Chlamydia are main
All antigens (including any major outer membrane protein) of outer membrane protein, Neisseria gonorrhoeae.
In various embodiments, the present invention covers the various diseases of detection or illness, including diabetes and inflammation.It can make
Exemplary antigens with the antibody test in methods described herein include but is not limited to HbA1 C and c reactive protein.It can be with
Including by other antigens that method described herein detects can be immune by currently marketed ELISA or sandwich ELISA
Measure any known antigen of detection.
In certain embodiments, method described herein can cover using a kind of antibody (for example, capture antibody or report
Antibody).In various embodiments, invention as described herein is covered using Multiple Antibodies, such as one or more captures
Antibody and one or more reporter antibodies.In some embodiments, invention as described herein is using at least about 2 kinds, about 3
Kind, about 4 kinds, about 5 kinds, about 6 kinds, about 7 kinds, about 8 kinds, about 9 kinds, about 10 kinds, about 11 kinds, about 12 kinds, about 13 kinds, about 14 kinds, about
15 kinds, about 16 kinds, about 17 kinds, about 18 kinds, about 19 kinds, about 20 kinds, about 21 kinds, about 22 kinds, about 23 kinds, about 24 kinds, about 25 kinds, about
26 kinds, about 27 kinds, about 28 kinds, about 29 kinds, about 30 kinds, about 31 kinds, about 32 kinds, about 33 kinds, about 34 kinds, about 35 kinds, about 36 kinds, about
37 kinds, about 38 kinds, about 39 kinds, about 40 kinds, about 45 kinds, about 50 kinds, about 55 kinds, about 60 kinds, about 65 kinds, about 70 kinds, about 75 kinds, about
80 kinds, about 85 kinds, about 90 kinds, about 95 kinds, about 100 kinds, about 110 kinds, about 120 kinds, about 130 kinds, about 140 kinds, about 150 kinds, about
160 kinds, about 170 kinds, about 180 kinds, about 190 kinds, about 200 kinds, about 250 kinds, about 300 kinds, about 350 kinds, about 400 kinds, about 450
Kind or about 500 kinds, about 750 kinds, about 1000 kinds, about 1250 kinds, about 1500 kinds, about 1750 kinds, about 2000 kinds, about 3000 kinds, about
4000 kinds, about 5000 kinds, about 6000 kinds, about 7000 kinds, about 8000 kinds, about 9000 kinds, about 10000 kinds of antibody.In some implementations
In scheme, invention as described herein is using at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9
A, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20
A, about 25, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80
A, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160
It is a, about 170, about 180, about 190, about 200 or about 250 or about 500 or about 750 or about 1000,
Or about 1250 or about 1500 or about 1750 or about 2000 or about 3000 or about 4000 or about 5000,
Or about 6000 or about 7000 or about 8000 or about 9000 or about 10000 antibody pair.
In some embodiments, invention as described herein can utilize multivalent antibody.For example, the present invention can be related to divalent or
Trivalent single chain variable fragment antibody (for example, each can be respectively containing about 4 or about 6 analytes or more binding site)
Coupling.In other embodiments, method described herein can be related to the aggregation for chemically forming Multiple Antibodies.This
A variety of multifunctional connectors can be used to execute.
It is expected that the speed and sensitivity of analysis analyte detection can be improved using a variety of binding partners.
In various embodiments, method described herein minimizes false positive signal.In some embodiments, this hair
Bright method reduces signal by control pH value of solution.In some embodiments, it is controlled by using buffer appropriate molten
Liquid pH, the buffer can have specificity to antibody used.In some embodiments, using Tris/ boric acid/EDTA
Buffer and/or buffer containing EDTA.Various buffers can be used in the present invention.It can be used for running in the present invention solidifying
The exemplary buffer of glue includes but is not limited to single buffer system, such as Boratex, sodium acetate, sodium citrate, lithium borate,
Tris/ acetic acid/EDTA, Tris/ acetic acid, Tris- acetic acid, Tris acetic acid EDTA, Tris/TAPS/EDTA buffer, Bis-
Tris/HCl buffer, Tris- acetic acid SDS, MOPS, MOPS/Tris/SDS/EDTA, MOPS/Tris/EDTA, MOPS/Tris/
SDS、MOPS/Tris、MES、MES/Tris/SDS/EDTA、MES/Tris/EDTA、MES/Tris/SDS、MES/Tris、Tris-
Glycine;Or double buffering system, such as gel side is Tris EDTA, and the other side is boric acid.It can be used for stablizing the other of pH
Exemplary buffer includes but is not limited to Boratex, sodium acetate, sodium citrate, lithium borate, Tris-HCl, TAPS, Tris/ second
Acid/EDTA, Tris- acetic acid, Tris acetic acid EDTA, Tris/TAPS/EDTA buffer, ammonium hydrogen carbonate, sodium bicarbonate, phosphate
Buffer, guanidine hydrochloride, guanidine thiocyanate, Bis-Tris/HCl buffer, Tris- acetic acid SDS, MOPS, MOPS/Tris/EDTA,
MOPS/Tris/SDS, MOPS/Tris, MES, MES/Tris/SDS/EDTA, MES/Tris/SDS, MES/Tris and Tris- are sweet
Propylhomoserin.
In some embodiments, using passivator such as Tween, BSA, polyethylene glycol or casein.It can be used for this hair
Bright other exemplary passivator include but is not limited to glycerol, sucrose, glucose, TritonX, SDS, LDS, Sigmacoat,
DNA oligonucleotides, isinglass, whole serum, polyvinyl alcohol, polyvinylpyrrolidone, salmon sperm dna, silane and silica.
Use the method for immunoassays
Method described herein includes for the presence of analytes of interest analytes in test sample, is not present or horizontal side
Method.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as
In conjunction with the analytes of interest analytes in sample;
Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as tying
Close the analytes of interest analytes in sample;
C. make analytes of interest analytes in conjunction with capture antibody and reporter antibody;
D. interested to associate by magnetic conjugate and with the magnetic conjugate by applying magnetic field to analysis chamber
Analyte is drawn downwards, so that analytes of interest analytes be separated from sample;And
E. by detecting the presence of analytes of interest analytes with light source and photoelectric detector examining report object, being not present or water
It is flat.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as tying
Close the analytes of interest analytes in sample;
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as
In conjunction with the analytes of interest analytes in sample;
C. make analytes of interest analytes in conjunction with capture antibody and reporter antibody;
D. interested to associate by magnetic conjugate and with the magnetic conjugate by applying magnetic field to analysis chamber
Analyte is drawn downwards, so that analytes of interest analytes be separated from sample;And
E. by detecting the presence of analytes of interest analytes with light source and photoelectric detector examining report object, being not present or water
It is flat.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as
In conjunction with the analytes of interest analytes in sample;
B. make analytes of interest analytes in conjunction with capture antibody;
C. interested to associate by magnetic conjugate and with the magnetic conjugate by applying magnetic field to analysis chamber
Analyte is drawn downwards, so that analytes of interest analytes be separated from sample;
Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as tying
Close the analytes of interest analytes in sample;
E. make analytes of interest analytes in conjunction with reporter antibody;
F. interested to associate by magnetic conjugate and with the magnetic conjugate by applying magnetic field to analysis chamber
Analyte and report conjugate are drawn downwards, so that analytes of interest analytes be separated from sample;And
G. by detecting the presence of analytes of interest analytes with light source and photoelectric detector examining report object, being not present or water
It is flat.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as
In conjunction with the analytes of interest analytes in sample;
B. make analytes of interest analytes in conjunction with capture antibody;
Contact sample with the analyte for reporting substance markers, the analyte of the report substance markers is configured as in sample
In there is no magnetic conjugate is combined in the case where analytes of interest analytes;
D. interested to associate by magnetic conjugate and with the magnetic conjugate by applying magnetic field to analysis chamber
Analyte is drawn downwards, so that analytes of interest analytes be separated from sample;And
E. by detecting the presence of analytes of interest analytes with light source and photoelectric detector examining report object, being not present or water
It is flat.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the report conjugate comprising reporter and reporter antibody, the reporter antibody is configured as tying
Close the analytes of interest analytes in sample;
B. make analytes of interest analytes in conjunction with reporter antibody;
Contact sample with the analyte of magnetic particle labels, the analyte of the magnetic particle labels is configured as
There is no report conjugate is combined in the case where analytes of interest analytes in sample;
D. the analyte of magnetic particle labels is separated from sample by applying magnetic field to sample;And
E. by detecting the presence of analytes of interest analytes with light source and photoelectric detector examining report object, being not present or water
It is flat.
In some embodiments, method described herein can comprise the following steps that
Contact sample with the magnetic conjugate comprising magnetic-particle and capture antibody, the capture antibody is configured as
In conjunction with the analytes of interest analytes in sample;
B. make analytes of interest analytes in conjunction with capture antibody;
Contact sample with the reporter antibody comprising biotin labeling, the reporter antibody is configured as combining in sample
Analytes of interest analytes;
Contact sample with the reporter marked comprising streptavidin, the streptavidin mark
Note is configured as combining biotin labeling;
E. analytes of interest analytes is separated from sample by applying magnetic field to sample;And
F. the presence of analytes of interest analytes is detected by examining report object, is not present or horizontal.
In some embodiments of methods described herein, magnetic conjugate (including capture antibody) can with reporter or
Report conjugate adds simultaneously.
In some embodiments of methods described herein, magnetic conjugate (including capture antibody) and reporter or report
Conjugate can be added separately.
In various embodiments, method of the invention may include the step being added to sample in analysis chamber.One
In a little embodiments, it may include the sample being delivered to sample with analysis chamber in fluid communication in analysis chamber that sample, which is added to,
Collector (for example, absorbent and/or wick material).In some embodiments, sample divider then can by sample into
It is given in analysis chamber.
In some embodiments, one or more of magnetic conjugate, reporter and report conjugate can be set
At sample divider, and the step of contacting sample and magnetic conjugate, reporter or report conjugate is in sample
Occur at collector.In some embodiments, before sample is added in sample divider, magnetism can be conjugated
In one or more of object, reporter and report conjugate a part of insertion sample divider.In some embodiments,
Method described herein may include that the sample made analyze in chamber is contacted with magnetic conjugate, reporter and/or report conjugate.
In some embodiments of methods described herein, reporter can be fluorescence report object, phosphorescence reporter or ratio
Color reporter (such as coloured particle), the absorption or scattering that the reporter can be configured to measurement light are (alternatively, for example pass through ratio
Colour analysis measure certain color in the presence/absence of).
In some embodiments, method described herein, which can comprise the further steps of:, makes sample and magnetic conjugate
After contact, by applying magnetic field to analysis chamber come the analytes of interest analytes in concentrating sample;Then it reduces in analysis chamber
The volume of sample.In some embodiments, method described herein, which may additionally include, makes sample contact it with report conjugate
Before the step of deactivating magnetic field.
In some embodiments, the volume for reducing sample in analysis chamber can be for example, by carrying out a part of volume
Siphon is carried out by removing whole samples and being resuspended with new smaller size smaller.
In some embodiments, method described herein, which can comprise the further steps of:, makes sample and magnetic conjugate
After contact, by applying magnetic field to analysis chamber come the analytes of interest analytes in concentrating sample;One is removed from analysis chamber
Determine the sample of volume;And the sample of the buffer of certain volume and/or other volume is added in analysis chamber.Some
In embodiment, method described herein may include in the step for making sample with reporting that conjugate deactivates magnetic field before contacting
Suddenly.
In some embodiments, method described herein may include by the buffer of certain volume and/or other volume
Sample be added to analysis chamber in step.
In some embodiments, method described herein can comprise the following steps that draws downwards by magnetic conjugate
After (i.e. application magnetic field) and before or after making sample and reporter or report conjugate contact, removed from analysis chamber
Remove the sample of certain volume.
In some embodiments of methods described herein, reporter antibody biotin labeling, and reporter strepto-
Avidin functionalization.In some embodiments of methods described herein, reporter antibody streptavidin
Functionalization, and reporter biotin labeling.
It in some embodiments, include contact procedure (for example, making sample and magnetic conjugation in method described herein
Object, reporter antibody, the conjugate for reporting substance markers and/or the contact of report conjugate) in the case where, this contact procedure may include
By sample (it may contain analyte) and corresponding magnetic conjugate, reporter antibody, the conjugate and/or report for reporting substance markers
Conjugate is accused to be incubated within the selected period and at a temperature of selected.
In some embodiments of methods described herein, analytes of interest analytes can be as described herein any interested
Analyte.In some embodiments, analytes of interest analytes can be selected from the group being made up of: human chorionic gonadotrophin
(hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA), herpes simplex virus (HSV) antibody,
Oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation biomarker, troponin, lime
Sick antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol biomarker, thyroid swash
Element, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biomarker, liver function biomarker
Object, estrogen, progesterone, lactic acid and their combination.In some embodiments, bacterium can be A group of streptococcus, Chlamydia
And/or micrococcus gonococcus.In some embodiments, inflammation biomarker can be CRP, SAA and/or MP8.In some implementations
In scheme, liver function biomarker can be ALT and/or AST.In some embodiments, analytes of interest analytes can be selected
Free group consisting of: ovulation biomarker, Aschheim-Zondek marker, Strep throat biomarker, prostate
Cancer biomarker, bleb biomarker, diabetes biomarker, inflammation biomarker, heart attack biology mark
Remember object, Chlamydia biomarker, bacterial biomarkers object, Lyme disease biomarker, cholesterol biomarker, thyroid gland
Hypofunction biomarker, hepatitis C biomarker, rhinovirus biomarker, influenza biomarker, liver function
Biomarker, fertility biomarker, muscular fatigue biomarker and their combination.
In some embodiments, ovulation biomarker can be originated from the sample based on urine, blood or serum.One
In a little embodiments, Aschheim-Zondek marker can be originated from the sample based on urine or blood.In some embodiments, hammer
Bacterium property laryngitis biomarker can be originated from the sample based on saliva.In some embodiments, prostate cancer biomarker
The sample based on blood, serum or urine can be originated from.In some embodiments, bleb biomarker can be derived from
Blood or the sample of saliva.
In certain embodiments, analytes of interest analytes can be selected from the group that is made up of: hCG, c reactive protein, LH,
PSA, HSV, E3G, bacterium (such as A group of streptococcus) and their combination.
In some embodiments of methods described herein, sample can be body fluid as described herein.In some implementations
In scheme, method described herein may include obtaining humoral sample from patient.
In certain embodiments, method described herein covers sandwich method, independent adding method, competing method and three
Grade method.
For example, sandwich method as described herein can be very suitable for handling small fluid sample volumes with immunoassay format.This
Independent adding method described in text may make can handle bigger fluid volume with the sensitivity of raising.Competitive assay method
Can be used for the measurement of following situations: user cannot find can in combination with the capture antibody and reporter antibody of analyte, such as
When analyte is small molecule.Three-level measuring method can provide three binding events to enhance the dynamics of system.It can lead to
Cross sandwich method, independent adding method or competition assay method form application three-level binding motif.
In various embodiments, immunoassays as described herein, method and kit allow to analytes of interest analytes into
Row individual's baseline alignment (base lining).In such embodiment, immunoassays, method and kit as described herein
Allow to determine the normal assay object range of each individual user.In some embodiments, if there is the individual with individual
Any deviation of normal assay object range, then alert user.
In various embodiments, the analyte (such as antigen) that can detecte is any biomarker of biological event
Object.In some embodiments, biological event may include illness events (i.e. disease biomarker), inflammatory episode (i.e. inflammation
Biomarker), reproduction event (i.e. reproduction biomarker) and/or aging event (i.e. aging biomarker).
In various embodiments, the present invention relates to the biologies for using system and method detection biological event as described herein
Marker.In various embodiments, the method for carrying out pregnant detection using system and method as described herein is provided.Each
In kind embodiment, the method for carrying out ovulation tests using system and method as described herein is provided.In various embodiments
In, it provides and carries out the method for prostate health detection (for example, the presence of detection cancer using system and method as described herein
Or a possibility that developing cancer).In various embodiments, it provides and carries out bleb inspection using system and method as described herein
The method of survey.In various embodiments, it provides and carries out streptococcal infection detection using system and method as described herein
Method.
In various embodiments, method described herein includes the various detection techniques for example for report signal.This
Class detection technique can be related to microscope, spectrophotometer, fluorimeter, pipe luminometer or plate luminometer, x-ray film, magnetic field, sudden strain of a muscle
Bright body, fluorescence-activated cell sorting (FACS) device, microfluidic device, device based on bead etc..
In some embodiments, magnetic-particle is paramagnetic particle.In some embodiments, paramagnetic particle is to receive
Rice grain or particle.In some embodiments, paramagnetic particle is bead, such as nanometer bead or microballon.In various implementations
In scheme, paramagnetic particle is magnetic Nano bead or microballon, and particle is allowed to be held and/or manipulated by magnet.In some realities
It applies in scheme, paramagnetic particle is the metal nanoparticle coated with thin (about 2nm) graphene carbon-like layer.In some embodiments
In, paramagnetic particle is coated, such as coated with streptavidin or PEG.The exemplary magnetic that can be used
Particle is DYNABEAD (THER MOFISHER), MACS bead (MILTENYI BIOTEC), TURBOBEADS
(TURBOBEADS)、ABSOLUTE MAG STREPTAVIDIN MAGNETIC PARTICLES(CREATIVE
) and GOLD NANOPARTICLES (SIGMAALDRICH) DIAGNOSTICS.
In some embodiments, magnetic-particle as described herein may include biocompatible coating, the bio-compatible
Property coating can with amido or carboxyl activation to promote amide to be coupled.In some embodiments, magnetic-particle as described herein can
To be activated with amido or carboxyl to promote amide to be coupled.
In some embodiments, reporter particles as described herein may include biocompatible coating, the bio-compatible
Property coating can with amido or carboxyl activation to promote amide to be coupled.In some embodiments, reporter particles as described herein can
To be activated with amido or carboxyl to promote amide to be coupled.
In some embodiments, particle as described herein can be particle (such as microballon), and a diameter of about 0.5 micron
To about 500 microns of (for example, about 0.5 micron or about 1 micron or about 10 microns or about 50 microns or about 100 microns or about 250
Micron or about 500 microns).
In some embodiments, particle as described herein can be nano particle (such as nanometer bead), and diameter is small
In 1 micron (for example, about 5 to about 500 nanometers, for example, about 5 nanometers or about 10 nanometers or about 50 nanometers or about 100 nanometers or about
250 nanometers or about 500 nanometers).In some embodiments, the average grain diameter of nano particle (such as nanometer bead) is 25-
500nm+/-5nm、25-500nm+/-10nm、25-500nm+/-15nm、25-500nm+/-20nm、25-500nm+/-25nm、
25-500nm+/- 30nm, 25-500nm+/- 35nm, 25-500nm+/- 40nm, 25-500nm+/- 45nm or 25-500nm+/-
50nm.In some embodiments, the average grain diameter of nano particle (such as nanometer bead) is about 20 to about 200nm.
In some embodiments, the diameter of particle (such as microballon) is about 0.5 micron to about 500 micron (for example, about 0.5
Micron or about 1 micron or about 10 microns or about 50 microns or about 100 microns or about 250 microns or about 500 microns).
In some embodiments, the diameter of nano particle (such as nanometer bead) is less than 1 micron (for example, about 5 to about 500
Nanometer, for example, about 5 nanometers or about 10 nanometers or about 50 nanometers or about 100 nanometers or about 250 nanometers or about 500 nanometers).
In some embodiments, the average grain diameter of nano particle (such as nanometer bead) be 25-500nm+/- 5nm, 25-500nm+/-
10nm、25-500nm+/-15nm、25-500nm+/-20nm、25-500nm+/-25nm、25-500nm+/-30nm、25-500nm
+/- 35nm, 25-500nm+/- 40nm, 25-500nm+/- 45nm or 25-500nm+/- 50nm.In some embodiments, it receives
The average grain diameter of rice grain (such as nanometer bead) is about 20 to about 200nm.
In some embodiments, magnetic-particle can be magnetic nanoparticle (such as nanometer bead), by oxide
(such as ferrite, maghemite, magnetic iron ore or iron oxide) composition, optionally by surfactant, silica, siloxanes or
Phosphoric acid derivatives are modified.In some embodiments, nano particle (such as nanometer bead) is by with shell (such as silica
Shell is optionally modified) ferrite composition.In some embodiments, magnetic nanoparticle (such as nanometer bead) is gold
(such as iron, the cobalt etc.) belonged to.In some embodiments, magnetic nanoparticle (such as nanometer bead) be with shell (such as temperature
With oxidation, surfactant, polymer and noble metal (such as gold, graphene etc.)) metal.
In some embodiments, particle as described herein can be the nano particle comprising one or more quantum dots
(such as nanometer bead).In some embodiments, nano particle includes metal core and one or more quantum dots.In some realities
It applies in scheme, nano particle includes the metal core that can be inlaid with one or more quantum dots.In some embodiments, nanometer
Particle includes the metal core that can be inlaid with multiple quantum dots.Quantum dot is discrete nano particle, their property and body half
Conductor is similar, so that they then issue energy when being exposed to electromagnetic energy.It can be set by changing size and composition, quantum dot
It is calculated as to the energy-sensitive in infrared region, visible spectrum even ultraviolet ray range.In addition, they are designed to luminescence generated by light
Or photovoltaic, generate light or energy respectively.
In some embodiments, reporter can be nano particle (such as nanometer bead), may include one or
Multiple quantum dots.In some embodiments, reporter includes metal core and one or more quantum dots.In some embodiments
In, reporter includes the metal core that can be inlaid with one or more quantum dots.In some embodiments, reporter include can
To be inlaid with the metal core of multiple quantum dots.
In some embodiments, reporter may include one or more quantum dots.In some embodiments, report is sewed
Closing object may include one or more quantum dots.In some embodiments, reporter and/or report conjugate may include multiple amounts
Sub- point.
Such as the example of the quantum dot generated by colloid method includes but is not limited to cadmium selenide (CdSe), cadmium sulfide
(CdS), indium arsenide (InAs) and indium phosphide (InP) cadmium sulfide tellurium (CdTeS).The number range for constituting the atom of quantum dot can be with
Be 100 to 100,000, general diameter range be 2 to 20nm (such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,
16、17、18、19、20、2.5、3.5、4.5、5.5、6.5、7.5、8.5、9.5、10.5、1 1.5、12.5、13.5、14.5、
15.5、16.5、17.5、18.5、19.5、20.5nm)。
In some embodiments, the granular materials including quanta point material includes but is not limited to carbon, colloidal gold, germanium, arsenic
Change indium, indium antimonide, GaAs, gallium nitride, cadmium/selenium/tellurides, lead, lead oxide, vulcanized lead, lead selenide, InGaP, silicon, glue
Body silver, cadmium mercury telluride, iron, iron oxide, cobalt, graphene, lanthanum, cerium, strontium carbonate, manganese, manganese oxide, nickel oxide, platinum, lithium, lithium titanate,
Tantalum, copper, palladium, molybdenum, boron carbide, silicon carbide, titanium carbide, tungsten oxide, aluminium, niobium, thulium, aluminium nitride, tin, aluminium oxide, tin oxide, antimony,
Dysprosium, praseodymium (paseodynium), antimony oxide, erbium, rhenium, barium, ruthenium, beryllium, samarium, bismuth oxide, boron, gadolinium, boron nitride, vanadium oxide, strontium, ytterbium,
Zirconium, diamond (C), silicon (Si), germanium (Ge), silicon carbide (SiC), silicon-germanium (SiGe), aluminium antimonide (AlSb), aluminium arsenide (AlAs),
Aluminium nitride (AlN), aluminum phosphate (AlP), boron nitride (BN), boron phosphide (BP), arsenic boron (BAs), gallium antimonide (GaSb), GaAs
(GaAs), gallium nitride (GaN), gallium phosphide (GaP), indium antimonide (InSb), indium arsenide (InAs), indium nitride (InN), indium phosphide
(InP), aluminum gallium arsenide (AlGaAs), InGaAsP (InGaAs, InxGai_xAs), InGaP (InGaP), aluminum indium arsenide
(AlInAs), indium aluminium antimonide (AllnSb), nitridation GaAs (GaAsN), arsenide phosphide gallium (GaAsP), aluminium gallium nitride alloy
(AlGaN), phosphatization gallium aluminium (AlGaP), InGaN (InGaN), antimony indium arsenide (InAsSb), indium antimonide gallium (InGaSb),
AlGaInP (AlGaInP, also referred to as InAlGaP, InGaAlP, AlInGaP), arsenide phosphide gallium aluminium (AlGaAsP), phosphatization
InGaAsP (InGaAsP), arsenide phosphide aluminium indium (AlInAsP), nitridation aluminum gallium arsenide (AlGaAsN), nitridation InGaAsP
(InGaAsN), indium arsenide aluminium (InAlAsN), nitridation antimony GaAs (GaAsSbN), antimony arsenide nitride gallium indium are nitrogenized
(GalnNAsSb), phosphatization antimony Gallium indium arsenide (GalnAsSbP), cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride
(CdTe), zinc oxide (ZnO), zinc selenide (ZnSe), zinc sulphide (ZnS), zinc telluridse (ZnTe), cadmium zinc telluride (CdZnTe,
" CZT "), cadmium mercury telluride (HgCdTe), telluride mercury zinc (HgZnTe), mercury selenide zinc (HgZnSe), stannous chloride (CuCl), selenizing
Lead (PbSe), vulcanized lead (PbS), lead telluride (PbTe), artificial gold (SnS), telluride tin (SnTe), telluride slicker solder (PbSnTe),
Telluride thallium tin (Ti2SnTe5), telluride thallium germanium (Tl2GeTe5), bismuth telluride (Bi2Te3), cadmium phosphide (Cd3P2), Cadmium arsenide
(Cd3As2), cadmium antimonide (Cd3Sb2), zinc phosphide (Zn3P2), zinc arsenide (Zn3As2), zinc antimonide (Zn3Sb2), lead iodide (II)
(Pbl2), molybdenum disulfide (MoS2), gallium selenide (GaSe), artificial gold (SnS), bismuth sulfide (Bi2S3), copper indium gallium selenide (CIGS),
Platinum silicide (PtSi), bismuth iodide (III) (BiI3), mercuric iodixde (II) (HgI2), thallium bromide (I) (TlBr), titanium dioxide (rutile titania
Mine) (TiO2), copper oxide (I) (Cu2O), copper oxide (II) (CuO), uranium dioxide (UO2), orange oxide (UO3) etc..
In various embodiments, apply magnetic field using external magnets.In various embodiments, magnet is permanent magnet
(such as neodymium iron boron (NdFeB), SmCo (SmCo), alnico alloy, and ceramics or ferrimagnet).In various embodiments
In, magnet is temporal magnet.In various embodiments, magnet is electromagnet.
In various embodiments, the detection of label carries out near magnetic field.In various embodiments, the detection of label
It carries out far from magnetic field, such as is carried out in the chamber separated with the chamber for executing the downward distraction step of magnetic.
The immunoassay kit that can be used according to the method for the present invention
In some embodiments, the present invention includes kit, and the kit includes as described herein for practicing
The immunoassays of one or more methods as described herein.
In some embodiments, the present invention provides pregnant detection kits, and the gestation detection kit includes this
Immunoassays described in text, and it is related to the detection of hCG (such as β-hCG).
In some embodiments, the present invention provides ovulation tests kit, the ovulation tests kit includes this
Immunoassays described in text, and it is related to the detection of LH.
In some embodiments, the present invention provides pregnant detection kits, and the gestation detection kit includes this
Immunoassays described in text, and it is related to the detection of E3G.
In some embodiments, the present invention provides the kit for detecting PSA, the kit includes this paper institute
The immunoassays stated.
In some embodiments, the present invention provides for detecting for the anti-of HSV (such as HSV-1 and/or HSV-2)
The kit of body, the kit include immunoassays as described herein.In one embodiment, HSV-1 antibody is compared, this
Kit described in text has specificity to HSV-2 antibody.
In some embodiments, the present invention provides the kit for detecting A group of streptococcus, the kit includes
Immunoassays as described herein.
In other embodiments, the present invention provides the reagents for detecting infection (such as stranguria syndrome and choamydiae infection)
Box, the kit include immunoassays as described herein.
In other embodiments, the present invention provides for detecting disease or illness (such as, but not limited to inflammation and sugar
Urine disease) kit, the kit includes immunoassays as described herein.
Unless otherwise defined, otherwise all technical and scientific terms used herein all have and neck belonging to the present invention
The identical meaning of the normally understood meaning of the technical staff in domain.The all patents and publications being mentioned above is whole by reference
Body is incorporated to.
Definition
As used herein, "/kind " or " should/described " mean/kind or more than one/kind.
In addition, term " about " means to quote digital indication plus or minus be somebody's turn to do when being used in combination with reference digital indication
Quote most the 10% of digital indication.For example, language " about 50% " covers 45% to 55% range.
As used herein, there are agent or stimulant, if relative to living the case where this adjusting is not present
The reading of property and/or effect reduces significant quantity, such as reduce at least about 10%, at least about 20%, at least about 30%, at least
About 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least
About 97%, at least about 98% or more (up to and including at least about 100%), then something " reduction ".Such as the common skill in this field
Art personnel will be understood that in some embodiments, activity reduces and some downstreams reading will reduce but other readings can
To increase.
On the contrary, there are agent or stimulant, if relative to the case where there is no this dose or stimulants
Activity and/or the reading of effect increase significant quantity, for example, increase at least about 10%, at least about 20%, at least about 30%, extremely
Few about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, extremely
Few about 97%, at least about 98% or more (up to and including at least about 100%), at least about 2 times, at least about 3 times, at least about
4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 50 times,
At least about 100 times, then active " increase ".
As mentioned in this article, unless otherwise specified, all percentage compositions are based on the weight of composition totality.Such as
Used herein, word " comprising " and its variant are intended to be non-limiting, so that being not excluded for the narration of the project in list can
It can also be used for the other similar project in the composition and method of this technology.Similarly, term "available" and " can with " and its become
Body is intended to be non-limiting, so that embodiment may or can be not excluded for not including that including the narration of certain elements or feature
Other embodiments of this technology of a little elements or feature.
As used herein, term " sample ", which can refer to, may include or the solution not comprising analytes of interest analytes, suspension, mixed
Close the body fluid of object or undiluted amount.As used herein, sample may include water and/or buffer.In some embodiments, can add
Add buffer as described herein to reduce or eliminate hook effect (hook effect), hook effect is in the most of of this field
Exist in immunoassays platform.In some embodiments, the sample of " large volume " can be 20 μ L or higher volume of sample
Or 20 μ L to 500 μ L sample.In some embodiments, the sample of " small size " can be sample or 1 μ L less than 20 μ L
To the sample of 15 μ L.
As used herein, term " body fluid " can refer to any fluid that can be separated in vivo from individual, and include but is not limited to
Whole blood, blood plasma, serum, bile, saliva, urine, tear, sweat, cerebrospinal fluid (CSF), sperm, swab samples (such as cheek swab,
Brush,throat etc.), mucus, phlegm, menses, menstrual fluid, vaginal mucus, amniotic fluid, synovia, breast milk, earwax, pre-firing sperm, lochia, hair
Rheuminess object (Rheum), lymph, purulence etc..In some embodiments, body fluid can more specifically refer to whole blood, serum, urine
Liquid, saliva, swab samples, mucus or sperm.In certain embodiments, body fluid can more specifically refer to whole blood, serum, urine
Or saliva.In some embodiments, body fluid may include analytes of interest analytes (such as biomarker).
As used herein, term " analytes of interest analytes " or " target analyte " or " analyte " may be used interchangeably, and
Refer to the antigen and/or biomarker of biological event, including any biomarker as described herein.In some embodiments
In, biological event may include illness events (such as disease biomarker), inflammatory episode (such as inflammation biomarker), life
Grow event (such as reproduction biomarker) and/or aging event (such as aging biomarker).Disease biomarker can
Including to seizure of disease in patient, disease regression and/or morbid state there are related or associated one or more diseases
Biomarker.Disease biomarker may include viral organism marker, bacterial biomarkers object, cancer biomarkers object or
One or more of symptom biomarker.Viral organism marker may include but be not limited to common cold (such as rhinopathy
Poison), influenza, bleb, zika virus (Zika) and/or HIV biomarker.In some embodiments, viral organism mark
Remember that object may include herpes simplex virus (HSV), one or more rhinopathy toxalbumin, one or more first/second/influenza C eggs
White, one or more HSF-1/2 albumen and/or one or more inhibition of HIV albumen.Bacterial biomarkers object may include but unlimited
In the biomarker (i.e. A group of streptococcus (Streptococcus-A/Strep-A)) of Strep throat, the biology of Chlamydia
The biomarker of marker and/or stranguria syndrome.In some embodiments, bacterial biomarkers object may include but be not limited to one kind
Or a variety of streptococcal proteins, one or more Chlamydia proteins and/or one or more Neisseria mycoprotein.Symptom
Biomarker may include but be not limited to cough, wheezes, has a running nose, nausea, spasm, uncomfortable in chest, dizzy, sore throat and/or pectoralgia
Biomarker.Disease biomarker can also include but is not limited to the biomarker of heart disease and/or diabetes.In
In some embodiments, disease biomarker may include troponin, CRP and/or ha1c.Cancer biomarkers object may include
The life of prostate cancer, breast cancer, colorectal cancer, gastric cancer, GIST, leukaemia/lymthoma, lung cancer, melanoma and/or cancer of pancreas
Substance markers object.In some embodiments, prostate cancer biomarker may include PSA.In some embodiments, breast cancer
Biomarker may include one or more of ER/PR and HER-2/neu.In some embodiments, colorectal cancer is raw
Substance markers object may include one or more of EGFR, KRAS and UGT1A1.In some embodiments, gastric cancer biomarker
It may include HER-2/neu.In some embodiments, GIST biomarker may include c-KIT.In some embodiments,
Leukaemia/lymthoma biomarker may include CD20 antigen, CD30, FIP1L1-PDGRF α, PDGFR, PML/RAR α, TPMT
One or more of with UGT1A1.In some embodiments, lung cancer biomarkers object may include in ALK, EGFR and KRAS
One of or more persons.In some embodiments, melanoma biomarker may include BRAF.It may include anti-inflammatory biomarker
Inflammatory biomarker may include one or more inflammatories described in U.S. Patent Application Publication No.2010/0275282
The full content of biomarker, the patent application is herein incorporated by reference.Reproduction biomarker may include ovulation,
The biomarker of fertilization, implantation and/or embryonic development.In some embodiments, reproduction biomarker may include β-people
Human chorionic gonadtropin (β-hCG or hCG), H-hCG, metakentrin (LH), oestrone -3- glucosiduronic acid (E3G),
Early pregnancy factor (EPF) and/or it is implanted into the factor in advance.Aging biomarker or age-dependent biomarker include United States Patent (USP)
Apply for one or more biomarkers described in open No.2008/0124752, the full content of the patent application with
Way of reference is incorporated herein.In addition antigen/biomarker interested include but is not limited to it is following relevant any known
Antigen/biomarker: SARS, hand-foot-and-mouth disease, heart disease biomarker, thyroid hormone, obesity biomarker, with
The relevant biomarker of hemorrhagic conditions such as (vWF, the factor 8, the factor 10), the 5th sick (fifths disease), flu, stream
Sense, Ebola virus, Escherichia coli, Listeria and salmonella.
As used herein, term " magnetic-particle " refer to at least some magnetic characteristics (such as ferromagnetism, paramagnetism and
Superparamagnetism) any particle.Term " bead " and " particle " are used interchangeably.In some embodiments, magnetic-particle can
Including magnetic material such as iron, nickel and cobalt and metal oxide such as Fe3O4、BaFe12O19、Mn2O3、Cr2O3、CoO、NiO
And CoMnP.In some embodiments, magnetic-particle is comprising polymer magnetic material or completely by polymer magnetic material group
At.Polymer magnetic material include the material that for example wherein magnetic material is mixed with polymer material and be coated with polymeric material
The magnetic material of material.Preferably, magnetic material is only a kind of component of particle, and the rest part of particle is by polymer material group
At magnetic response material is fixed on the polymer material (referring to following encoded particles).It is used to prepare or forms magnetism
The illustrative methods of particle are described in such as United States Patent (USP) No.6, in 773,812 and 6,280,618, in the whole of the patent
Appearance is herein incorporated by reference.In some embodiments, magnetic-particle can be magnetic nanoparticle as described herein
Or magnetic particle.
As used herein, term " capture portion " can refer to and can be with selection for the magnetism in conjunction with target analyte
Particle conjugation or combine antibody, single-chain antibody, antigen binding antibody fragment, antigen, receptor, ligand, aptamer, aptamer receptor,
Nucleic acid or small molecule.In certain embodiments, capture portion is capture antibody.
As used herein, term " capture antibody " can refer to and can be with selection for the magnetism in conjunction with target analyte
Antibody particle conjugation or combined.
As used herein, term " report bound fraction " can refer to and can be with selection in conjunction with target analyte
Reporter conjugation or combine antibody, single-chain antibody, antigen binding antibody fragment, antigen, receptor, ligand, aptamer, aptamer by
Body, nucleic acid or small molecule.In certain embodiments, report bound fraction is reporter antibody.
As used herein, term " reporter antibody " can refer to and can be with selection for the report in conjunction with target analyte
Antibody object conjugation or combined.In some embodiments, " capture antibody " and " reporter antibody " can be in conjunction with target analytes
Different piece.
As used herein, term " reporter " and " label " are used interchangeably, and can usually refer to that signal generates chemical combination
Object and/or detectable label or core (such as metal core), one or more of them signal generate compound and/or detectable label
It is connected to the core.In some embodiments, reporter can be fluorescence report object, phosphorescence reporter or colorimetric reporter
(such as coloured particle), the absorption and/or scattering that the reporter is used to measure light by colorimetric analysis (alternatively, for example measured
Certain color in the presence/absence of).In some embodiments, it can be used known in the art any suitable detectable
Label.For example, detectable label can be radioactive label (such as3H、125I、35S、14C、32P and33P), enzyme label is (such as peppery
Root peroxidase, alkaline phosphatase, zwischen-ferment etc.), chemiluminescent labeling (such as acridinium ester, thioesters or sulphur
Amine;Luminol, different luminol, phenanthridines ester etc.), fluorescent marker (such as fluorescein (such as 5- fluorescein, 6- Fluoresceincarboxylic acid,
3 ' 6- Fluoresceincarboxylic acids, 5 (6)-Fluoresceincarboxylic acid, 6- chlordene fluorescein, 6- tetrachlorofluorescein, fluorescein isothiocynate etc.)),
Rhodamine, phycobniliprotein, R-PE, (Mc) point (such as the capped cadmium selenide of zinc sulphide), thermometric containing quantum or metal
Label or Immuno polymerase chain reaction label.In various embodiments, label includes but is not limited to fluorogen, chromophore, puts
Injectivity isotope, magnetic-particle, gold particle, zymolyte etc..In some embodiments, label is chemiluminescence or fluorescence egg
It is white, such as green fluorescent protein (GFP), enhanced green fluorescence protein (EGFP), sea pansy (Renilla
Reniformis) green fluorescent protein, GFPmut2, GFPuv4, yellow fluorescence protein (YFP), enhanced yellow fluorescence protein
(EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), come
From the lemon yellow and red fluorescent protein (dsRED) of mushroom coral (discosoma), luciferase, umbelliferone, rhodamine, glimmering
Light element, dichlorotriazine base amine fluorescein, dansyl Cl, phycoerythrin etc..In some embodiments, label is following any family
The nonprotein organic fluorescence group of race: xanthene derivative, such as fluorescein, rhodamine, Oregon green (Oregon green), she
Red and texas Red (Texas red);Flower cyanines derivative, such as spend cyanines, indoles cyanines, oxygen carbon cyanines (oxacarbocyanine),
Sulphur carbon cyanines (thiacarbocyanine) and portion spend cyanines;Squaric acid derivertives and cyclosubstituted side acid, including Seta, SeTau and
Square dyestuff;Naphthalene derivatives (red sulphonyl and prodan (prodan) derivative);Coumarin derivative;Oxadiazoles is derivative
Object, such as pyridyl group oxazole, nitrobenzoxadiazole and benzoxadiazole;Anthracene derivant, such as anthraquinone, including DRAQ5,
DRAQ7 and CyTRAK orange;Pyrene derivatives, cascade blue (cascade blue) etc.;Oxazines derivative, such as Nile red
(Nile red), Nile blue (Nile blue), cresol-purple, oxazines 170 etc.;Acridine derivatives, such as proflavin, acridine orange, a word used for translation
Pyridine Huang etc.;Arylmethine derivative, such as auramine, crystal violet, malachite green;And tetrapyrrole derivative, such as porphines, phthalein
Cyanines, bilirubin.In various embodiments, label includes but is not limited to enzyme label, such as such as horseradish peroxidase, alkalinity
The enzyme of phosphatase, beta galactosidase, zwischen-ferment etc..In some embodiments, reporter can be such as this
Quantum dot described in text.In some embodiments, reporter may include quantum dot as described herein.In some embodiments
In, reporter may include the metal core (i.e. golden core) with silica shell, wherein silica shell be impregnated with it is multiple (such as
100-600) quantum dot.
Although as include, contain or with etc. the open-ended term "comprising"s of synonym of terms use herein
In being described and claimed the present invention, but the invention or an embodiment thereof be alternatively used substituting term such as " by ...
Composition " or " generally by ... form " describe.
As used herein, word " preferred " and " preferably " refer to the technology that certain benefits are provided in some cases
Embodiment.However, in the case that it is identical or other, other embodiments are also possible to preferably.In addition, to one or more
The narration of preferred embodiment is not meant to that other embodiments are useless, and is not intended to and excludes other embodiments
Except the range of the technology.
To avoid doubt, it is contemplated that be in conjunction with certain aspects of the present disclosure, embodiment or embodiment description spy
Determine feature (such as integer, characteristic, value, purposes, disease, formula, compound or group) to be interpreted as being suitable for as described herein
What his aspect, embodiment or embodiment, unless incompatible.Therefore, these features can be combined in due course and be defined herein
Any definitions, claims or embodiments use.All features disclosed in this specification (including any appended right is wanted
Ask, make a summary and attached drawing) and/or all steps of disclosed any method or process can be combined with any combination,
In except at least some features and/or the mutually exclusive combination of step.The present invention is not limited to appointing for any disclosed embodiment
What details.The present invention extend to this specification (include any accompanying claims, abstract and drawings) disclosed in feature appoint
What novel feature or novel combination, or any novel step or any for the step of extending to disclosed any method or process
Novel combination.
Although the preferred embodiments of the invention have been illustrated and described herein, these embodiments are only with the side of citing
Formula provides, it is no intended to otherwise limit the scope of the invention.Reality of the present invention can be used when practicing the present invention
Apply the various alternative solutions of scheme.
Embodiment
The embodiment covered herein is described referring now to following embodiment.These embodiments are provided only for explanation
Mesh, and never the disclosure covered herein should be considered limited to these embodiments, on the contrary, it is thus understood that cover due to this
Text provide introduction and will become apparent from any and all modification.
Reporter/antibody coupling program
Covalent coupling program is developed with by the reporter particles of antibody coupling to fluorescent marker.Unless otherwise specified,
The reporter particles are that metal core quantum spot inlays particle (Nanocompsix)), with carboxyl surface covering.
Experimental arrangement
The vial containing reserve report particle being ultrasonically treated by being stood in ultrasonoscope bottle, being held
It is 10 seconds continuous.Using the P1000 mixing reporter particles for being set as 600, until without apparent sediment or sediment.
Using the P1000 for being set as 600,600 μ L reserve report objects are transferred in 1.5mL VWR centrifuge tube.
Using the P10 for being set as 10, to 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide (EDC) of 3 1mg
10 μ L are respectively added in pipe.EDC pipe is put into the mini centrifuge of Ezee (Ezee Mini-Centrifuge).Rotation 5 seconds.From two
10uL is taken out in a pipe and is added in third pipe.It is sufficiently mixed with pipette.
Using the P100 for being set as 28.8,28.8 μ L EDC solutions are added in 600 μ L reporter particles.
Using the P20 for being set as 20, respectively added into n-Hydroxysulfosuccinimide (Sulfo-NHS) pipe of 3 2mg
20μL.It is sufficiently mixed with pipette, until can't see solid.20 μ L are taken out from two pipes and are added in third pipe.With
Pipette is sufficiently mixed.
Using the P100 for being set as 57.6,57.6 μ L EDC solutions are added into 600uL reporter.
Using the P200 pipette for being set as 200 μ L, reporter particles-EDC-NHS mixture is mixed.
Reporter particles-EDC-NHS mixture is put into bath ultrasonoscope and is opened 1 minute.
Reporter particles-EDC-NHS is placed on the Rugged rotator (Rugged Rotator) for being set as 40% speed
And rotation 15 minutes of turning upside down.
In the low protein adsorption pipe of 1.5mL Eppendorf (Eppendorf Protein Lo-bind tube), addition
360 μ L 1mg/mL antibody.If antibody is higher than 1mg/mL, use magnetic bead coupling buffer (MB Coupling Buffer)
(i.e. PBS, 0.01%Tween-20, pH 7.4) is diluted to 1mg/mL.
By reporter particles-EDC-NHS solution with 3600rcf rotation 5 minutes in centrifuge.
Using P200, supernatant is carefully removed from pipe.Ensure not interfere the reporter particles sediment of bottom of the tube.
Using P1000,600uL MD reaction buffer is added into reporter particles sediment.
Reporter particles pipe is put into for 10 seconds in ultrasonoscope.
Using the P1000 for being set as 600 μ L, it is sufficiently mixed and 600 μ L reporter particles is transferred in pipe antibody-containing.
Reporter particles-antibody-solutions are placed on the Rugged rotator for being set as 40% speed and are rotated 1 hour.
The deposit hydroxyl of 7 μ L is added by using the P10 for being set as 7 to 63 μ L nuclease-free waters (as measured by P100)
Amine aqueous solution, preparation 1:10 dilute hydroxylamine solution.
Using P100,60 μ L 1:10 dilution hydroxylamine solution is added into reporter particles-antibody-solutions.
Reporter particles-antibody-solutions are placed on the Rugged rotator for being set as 40% speed and are rotated 10 minutes.
By reporter particles-antibody-solutions with 3600rcf rotation 5 minutes in centrifuge.
Using P200, supernatant is carefully removed from pipe.Ensure not interfere the reporter particles sediment of bottom of the tube.
Using P1000,600 μ L magnetic bead store buffer liquid (MB Storage are added into reporter particles sediment
Buffer).It is sufficiently mixed and is ultrasonically treated 10 seconds.
By reporter particles-antibody-solutions with 3600rcf rotation 5 minutes in centrifuge.
Using P200, supernatant is carefully removed from pipe.Ensure not interfere the reporter particles sediment of bottom of the tube.
Using P1000,600 μ L magnetic bead store buffer liquid are added into reporter particles sediment.It is sufficiently mixed and at ultrasound
Reason 10 seconds.By reporter particles-antibody-solutions with 3600rcf rotation 5 minutes in centrifuge.
Using P200, supernatant is carefully removed from pipe.Ensure not interfere the reporter particles sediment of bottom of the tube.
Using P1000,300 μ L magnetic bead store buffer liquid are added into reporter particles sediment.It is sufficiently mixed and at ultrasound
Reason 10 seconds.
Pipette by suitably setting size adds the magnetic bead store buffer of certain volume into the reporter particles of coupling
Liquid (i.e. PBS, 0.01%Tween-20,0.05% sodium azide, pH 7.4), and the reporter particles for ensuring to be coupled are 2.2 moles
Particle/L.
The reporter particles of antibody coupling are stored at 4 DEG C up to one month.
Magnetic bead/antibody coupling program
The program description is for by the exemplary arrangement of antibody and magnetic catch particle (i.e. magnetic bead) covalent coupling.
Magnetic bead used in the program is with superparamagnetism Fe2O3The nano particle of core and biocompatibility external skin.
It is activated with carboxyl on surface.
Experimental arrangement
Protein preparation
Using 7K Zeba desalting column, by the buffer of antibody interested be changed to magnetic bead coupling buffer (i.e. PBS,
0.01%Tween-20, pH 7.4);If antibody concentration is greater than 1mg/mL, it is coupled before using Zeba column using magnetic bead
Antibody-solutions are diluted to 1mg/mL by buffer.Measure IgG concentration.
Magnetic bead (Magbead) cleaning and resuspension
Using the P200 for being set as 175,175 μ L Ocean are added into the low protein adsorption pipe of 0.5mL Eppendorf
The super carboxyl magnetic bead of Nanotech (Ocean Nanotech Super Carboxyl Magbead), addition twice, obtain 350 μ L.
Pipe is placed on Promega magnetic frame (Promega Magstand) and continues 1 minute.
By using 200 P200 is set as twice, remove supernatant (supernatant can be brown).
Using the P200 for being set as 100, adds 100 μ L magnetic beads and activate buffer (Magbead Activation
Buffer it), and by gently liquid relief is resuspended completely and (dark area should be can't see).
Pipe is put back on Promega magnetic frame and continues 1 minute.
By using 200 P200 is set as twice, remove supernatant (supernatant can be brown).
Using the P200 for being set as 100, adds 100 μ L magnetic beads and activate buffer (i.e. MES, 0.01%Tween-20, pH
6.0) it, and by gently liquid relief is resuspended completely and (dark area should be can't see).
Pipe is placed on pipe float and is ultrasonically treated 1 minute.
Magbead activation
Using the P100 for being set as 100, the water of 100 μ L nuclease frees is added in Pierce zero gravity 1mg EDC pipe
(10mg/mL)。
It is vortexed 30 seconds, and is rotated 30 seconds on pipe rotator.
Using the P100 for being set as 25, the 25 μ L EDC to suspend is added in magnetic bead solution.
The water of 200 μ L nuclease frees is added in ThermoFisher zero gravity 2mg Sulfo-NHS pipe, and sufficiently mixed
It closes with dissolved solid (10mg/mL).
Using the P100 for being set as 25, the 25 μ L Sulfo-NHS to suspend is added in magnetic bead solution.
Pipe is placed on rotator and is reacted 15 minutes at room temperature.
Antibody conjugate
Using the P200 for being set as 150, the 150 μ L Magbead activated is transferred to new Eppendorf low adsorption pipe
In.
Pipe is placed on Promega magnetic frame and continues 30 seconds.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and adds a certain amount of activation buffer using P200.The amount be based on antibody concentration and
Variation, to ensure that the final incubation antibody concentration between batch is identical.
Pipe midway is immersed in for 10 seconds to be resuspended in ultrasonoscope.
Using P100, the hammer bacteria antibody of a certain amount of buffering fluid exchange is added, and will be managed using the P200 for being set as 200
It is sufficiently mixed.The amount is based on antibody concentration and changes, to ensure that the final incubation antibody concentration between batch is identical.
It is reacted 2.5 hours on rotator at room temperature.It is mixed at 1:15 time point with P200 pipette.
Using the P100 for being set as 50,50 μ L magnetic beads quenching buffer (Magbead Quenching Buffer) is added.
It is reacted 30 minutes on rotator at room temperature.
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and adds 200 μ L magnetic bead store buffer liquid (Magbead using the P200 for being set as 200
Storage Buffer) and gently liquid relief to be resuspended.
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and is added 200 μ L magnetic bead store buffer liquid using the P200 for being set as 200 and gently moved
Liquid is to be resuspended.
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and using the P200 for being set as 150, adds the storage of 300 μ L magnetic beads twice by liquid relief
Buffer, gently liquid relief is to be resuspended.
Pipe midway is immersed in for 10 seconds to be resuspended completely in ultrasonoscope.
The magnetic bead of coupling is stored at 4 DEG C up to one month.
Magnetic bead/biomarker coupling procedure (oestrone -3- glucosiduronic acid-magnetic bead)
The program description is for by biomarker, (i.e. oestrone -3- glucosiduronic acid (E3G) and magnetic catch particle to be (i.e.
Magnetic bead) covalent coupling exemplary arrangement.
The magnetic bead used is with superparamagnetism Fe2O3The nano particle of core and biocompatibility external skin.Surface amine
Base activation.
Magnetic bead cleaning and resuspension
Using the P1000 for being set as 300 μ L, by the super amine magnetic bead of Ocean Nanotech (Ocean Nanotech Super
Amine Magnetic Bead) liquid relief is into the low protein adsorption pipe of 0.5mL Eppendorf.
Pipe is placed on Promega magnetic frame (Promega Magstand) and continues 1 minute.
By using 200 P200 is set as twice, remove supernatant (supernatant can be brown).
Using the P100 for being set as 90, adds 90 μ L magnetic beads and activate buffer (i.e. MES, 0.01%Tween-20, pH
6.0) it, and by gently liquid relief is resuspended completely and (dark area should be can't see).
Pipe is put back on Promega magnetic frame and continues 1 minute.
It is primary by using the P100 for being set as 100, it removes supernatant (supernatant can be brown).
It using the P100 for being set as 90, adds 90 μ L magnetic beads and activates buffer, and (answered by gently liquid relief to be resuspended completely
It can't see dark area).
Pipe is placed on pipe float and is ultrasonically treated 1 minute.
Reagent mixing
Using the P100 for being set as 100, the water of 100 μ L nuclease frees is added in Pierce zero gravity 1mg EDC pipe.
(10mg/mL)
It is vortexed 30 seconds, and is rotated 30 seconds on pipe rotator.
Using the P100 for being set as 30, the 30 μ L EDC to suspend is added in magnetic bead solution.
The water of 200 μ L nuclease frees is added in ThermoFisher zero gravity 2mg Sulfo-NHS pipe, and sufficiently mixed
It closes with dissolved solid (10mg/mL).
Using the P100 for being set as 30, the 30 μ L Sulfo-NHS to suspend is added in magnetic bead solution.
Using the P100 for being set as 30, the nuclease free aqueous solution of the 1mg/mL E3G of 30 μ L is added.
Activation is incubated for
Pipe is placed on rotator and is reacted 15 hours at room temperature.
Magnetic bead cleaning and final suspension
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using 200 P200 is set as twice, supernatant is removed.
Remove pipe from magnetic frame, and using the P200 for being set as 200 add 200 μ L magnetic bead store buffer liquid (i.e. PBS,
0.01%Tween-20,0.05% sodium azide, pH 7.4) and gently liquid relief to be resuspended.
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and is added 200 μ L magnetic bead store buffer liquid using the P200 for being set as 200 and gently moved
Liquid is to be resuspended.
Pipe is placed on Promega magnetic frame and continues 1 minute.
Using the P200 for being set as 200, supernatant is removed.
Pipe is removed from magnetic frame, and adds 300 μ L magnetic bead store buffer liquid using the P1000 for being set as 300, is gently moved
Liquid is to be resuspended.
Pipe midway is immersed in for 10 seconds to be resuspended completely in ultrasonoscope.
Storage is up to one month at 4 DEG C.
The operation of embodiment 1- immunoassay sandwich mode
Prepare immunoassays as described herein for sandwich mode, this is set for handling the ideal of small fluid volume
It sets.
As shown in Figure 1A to Fig. 1 D, the sandwich mode of immunoassays as described herein can be used.As shown in the figure, may be used
It include reporter (the golden slug particle with the silica shell for being impregnated with 100-600 quantum dot to use
(nanoComposix)) it is detected with the report conjugate of antibody #1 and magnetic conjugate comprising antibody #2 and magnetic-particle
Analyze the hCG analyte (Figure 1A) interested in chamber.
In the first step, can by magnetic conjugate and report conjugate be added to measurement chamber in and by they with contain
The sample of analytes of interest analytes (hCG) mixes (Figure 1B).Magnetic field (" downwards traction ") can be applied by magnet with will be interested
Analyte separates (Fig. 1 C) from sample.Then, light transmission can be made by a part of analysis chamber, so that reporter fluoresces
(Fig. 1 D).This fluorescence can be detected by detector.
In the absence of the analyte, reporter will not be drawn downwards together with analyte, therefore will not be gone out
Existing fluorescence.
Operation is individually added in embodiment 2- immunoassays
Prepare immunoassays as described herein for independent addition mode, this is for handling bulk sample and allowing
Concentrating analysis is preferably provided with.This greatly improves sensitivity.
As shown in Fig. 2A to Fig. 2 F, the independent addition mode of immunoassays as described herein can be used.Such as institute in figure
Show, can be used comprising reporter (the golden slug particle with the silica shell for being impregnated with 100-600 quantum dot
(nanoComposix)) it is detected with the report conjugate of antibody #1 and magnetic conjugate comprising antibody #2 and magnetic-particle
Analyze the hCG analyte (Fig. 2A) interested in chamber.
In the first step, magnetic conjugate can be added to measurement chamber in and by its with contain analytes of interest analytes
(hCG) sample mixes (Fig. 2 B).Downward traction can be executed, and optionally can remove the sample of certain volume, then
The sample (Fig. 2 C) of other volume can be added.After adding other sample, magnetic field can be made to deactivate and can be with
Magnetic conjugate is mixed with sample again.The process can be repeated so that analytes of interest analytes is concentrated.Analysis interested is being concentrated
After object, report conjugate can be added and mix it to combine analytes of interest analytes (Fig. 2 D) with sample.It can execute another
Analytes of interest analytes to be separated (Fig. 2 E) by outer downward traction from sample.Then, light transmission can be made to pass through analysis chamber
A part, so that reporter fluoresces (Fig. 2 F).This fluorescence can be detected by detector.
In the absence of the analyte, reporter will not be drawn downwards together with analyte, therefore will not be gone out
Existing fluorescence.
Embodiment 3- competitive immunoassay operation-first method
Prepare immunoassays as described herein for competing sexual norm, this is when analytes of interest analytes is too small and cannot be by
Two kinds of antibody are preferable to provide when combining.When there is only a kind of antibody to combine analytes of interest analytes, this is also preferred.
As shown in Fig. 3 A to Fig. 3 E, the competition sexual norm of immunoassays as described herein can be used.As shown in the figure,
The analyte of report substance markers can be used and test and analyze the sense in chamber comprising the magnetic conjugate of antibody and magnetic-particle
Interest analysis object (Fig. 3 A).
In the first step, magnetic conjugate can be added to measurement chamber in and by its with containing analytes of interest analytes
Sample mixes (Fig. 3 B).Then the analyte of report substance markers can be added and it is mixed to (Fig. 3 C) with sample.Feel when existing
When interest analysis object, report the analyte of substance markers not in conjunction with magnetic conjugate.In fact, the knot on magnetic conjugate antibody
Coincidence point is occupied by the analytes of interest analytes from sample.However, magnetism is sewed if analytes of interest analytes is not present in sample
Closing object will be in conjunction with the analyte of report substance markers (Fig. 3 C).Traction downwards is executed to separate analytes of interest analytes from sample
(Fig. 3 D).Then, light transmission can be made by a part of analysis chamber, so that reporter fluoresces (Fig. 3 E).Inspection can be passed through
It surveys device and detects this fluorescence.
In the absence of the analyte, report that the analyte of substance markers will be drawn downwards together with magnetic conjugate
And it will test fluorescence.
Embodiment 4- competitive immunoassay operation-second method
Prepare immunoassays as described herein for alternative competition sexual norm
Magnetic-particle can be used together with analytes of interest analytes in connection.In this respect, the magnetic when measuring beginning
Property particle have analytes of interest analytes, rather than as in Example 3 reporter have analytes of interest analytes.Report conjugate packet
Containing antibody and reporter, the reporter can be used for the analytes of interest analytes in analysis chamber in test sample.
In the first step, report conjugate is added in analysis chamber and by itself and the sample containing analytes of interest analytes
Mixing.Magnetic-particle and the analyte in conjunction with the magnetic-particle are added in analysis chamber and mixed with sample.Then
Traction downwards is executed to separate magnetic-particle and the analyte in conjunction with the magnetic-particle from sample.Then take out sample
Product.Then light transmission can be made by sample, so that reporter fluoresces.This fluorescence can be detected by detector.
In the absence of the analyte, report conjugate will be drawn downwards and will not be existed in the sample, be led
Cause no fluorescence.
Embodiment 5- three-level immunoassay procedures
Prepare immunoassays as described herein for three-level schema, enhances the power of system using three binding events
It learns.Three-level binding motif can be applied to sandwich mode (embodiment 1), individually add mode (embodiment 2) and competitive survey
Mould-fixed (embodiment 3 and 4).
As shown in Fig. 4 A to Fig. 4 G, the three-level schema of immunoassays as described herein can be used.As shown in the figure, may be used
It include reporter (with the fluorescence quantum of streptavidin functionalization) and antibody #1 to use (with biotin labeling)
Report conjugate and magnetic conjugate comprising antibody #2 and magnetic-particle test and analyze the hCG interested in chamber points
It analyses object (Fig. 4 A).
In the first step, magnetic conjugate can be added to measurement chamber in and by its with containing analytes of interest analytes
Sample mixes (Fig. 4 B).Magnetic field (" traction downwards ") can be applied by magnet to separate analytes of interest analytes from sample
(Fig. 4 C).It can remove the sample of certain volume and analyte concentration step can be executed as described in example 2 above.It can make
Magnetic field deactivation, and antibody #1 can be added in analysis chamber, the antibody #1 then can be with analysis interested
Object combines, the analytes of interest analytes (Fig. 4 D) also in conjunction with magnetic conjugate.Then reporter can be added to analysis cavity
In room, the reporter may then pass through strepavidin-biotin binding interactions (figure in conjunction with antibody #1
4E).Then traction downwards can be executed so that analyte to be separated to (Fig. 4 F) from sample again.
Then, light transmission can be made by a part of analysis chamber, so that reporter fluoresces (Fig. 4 G).Inspection can be passed through
It surveys device and detects this fluorescence.
In the absence of the analyte, reporter will not be drawn downwards together with analyte, therefore will not be gone out
Existing fluorescence.
Embodiment 6- is used to detect the demonstration of the measurement of human chorionic gonadotrophin
Immunoassays are provided for using the scheme of embodiment 15 to detect human chorionic gonadotropin's gland in urine sample
Hormone (hCG).As shown in Figure 5, which shows femtomole grade sensitivity.
Embodiment 7- is used to detect the demonstration of the measurement of metakentrin
Immunoassays are provided to detect the metakentrin in urine sample (LH).As shown in Figure 6, which shows
Femtomole grade sensitivity out.
Scheme for detecting the LH in urine is as follows.
Magnetic bead in the measurement is the magnetic nanoparticle coated with the antibody (Medix 5304) in conjunction with LH.
Reporter in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated in conjunction with the anti-of LH
Body (Medix 5304).
Device therefor
Use following equipment.
Experimental arrangement
It is dilute in Chon Block according to the following formula (for example, about at 5 points in the afternoon before collecting day) before collecting day
Magnetic bead and reporter are released, the definite volume used is recorded:
·
ChonBlock volume (uL)=(magnetic bead volume) × (the 51X coefficient of dilution)-(magnetic bead volume)
·
ChonBlock volume (uL)=(magnetic bead volume) × (the 6X coefficient of dilution)-(magnetic bead volume)
Reporter is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes reporter.
The Chon Block for calculating volume is added to 0.5mL Eppendorf egg using the pipette for suitably setting size
In white low adsorption pipe.
The reporter for calculating volume is added in Chon Block.Upper and lower liquid relief is to ensure to have removed report in pipette
Object.
It diluted reporter will be mixed now with the pipette for suitably setting size.
Magnetic bead is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using the P100 for being set as 100,
Mix magnetic bead.
The Chon Block for calculating volume is added to 0.5mL Eppendorf egg using the pipette for suitably setting size
In white low adsorption pipe.
The magnetic bead for calculating volume is added in ChonBlock.Upper and lower liquid relief is to ensure to have removed magnetic bead in pipette.
It diluted magnetic bead will be mixed now with the pipette for suitably setting size.
Diluted reagent is stored in metallic support at 4 DEG C.Reagent can save 20 hours.
While collecting urine sample, 3 μ L magnetic beads are assigned to three 0.5mL using the P10 pipette for being set as 3 μ L
In Eppendorf albumen low adsorption pipe, label has number, cycle date and repetition number (1,2,3) to these pipes in advance.
When sample reaches, 3 μ L urines are added in the first repeating pipe.
And then, start timer, count a few minutes.
After 30 seconds, 3 μ L reporters are added in the first repeating pipe using P10 pipette.Use the P10 for being set as 3 μ L
Mixing.
When timer reaches 1 minute, 3 μ L urines are added in the second repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
When timer passes through 1:30 seconds, 3 μ L reporters are added in the second repeating pipe using P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
When timer reaches 2 minutes, 3 μ L urines are added in the third repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
When timer passes through 2:30 seconds, 3 μ L reporters are added in the third repeating pipe using P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
By after five minutes, the first repeating pipe being placed on Promega magnetic frame, and check timer on timer, count
1 minute.
6 minutes time points, the second repeating pipe is placed on the 2nd Promega magnetic frame.
Supernatant is removed from the first repeating pipe using the P20 pipette for being set as 20 μ L in 6:05, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the first repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the first weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
7 minutes time points, the third repeating pipe is placed on the first Promega magnetic frame.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 7:05, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 8:05, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Measurement pipe is stored in the metallic support of cooler, in case subsequent analysis.
Spectra Max analysis
Using P20 pipette, each 20uL of replicate sample is loaded on 384 orifice plates.
Using P10 pipette, 20uL TBS is loaded in 2 holes on same 384 orifice plate.
384 orifice plates are loaded in SpectraMax.
Embodiment 8- is used to detect the demonstration of the measurement of prostate-specific antigen
Immunoassays are provided to detect the prostate-specific antigen (PSA) in serum/whole blood sample.Such as institute in Fig. 7
Show, which shows femtomole grade sensitivity.
Exemplary arrangement for detecting the PSA in whole blood and serum is as follows.
Magnetic bead in the measurement is the magnetic Nano coated with the antibody (Medix Anti-h PSA 8311) in conjunction with PSA
Particle.Magnetic bead solution is prepared, magnetic bead is mixed and sonicated 10 seconds.200pM magnetic bead solution includes 2.5 μ L magnetic beads and 22.5 μ L
Chon Block。
Reporter in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated in conjunction with PSA's
Antibody (Medix Anti-h PSA 8301)
PSA removes antibody-solutions
8301 antibody of PSA: deposit 5.3mg/mL (35.35 μM).For 6.66 μM, the deposit 8301 of 5.65 μ L of mixing is anti-
The Chon Block of body and 24.35 μ L.For 660nM, 6.66 μM of solution of 10 μ L and the Chon Block of 90 μ L are added.
For serum stripping solution, the 660nM solution of 2 μ L and the Chon Block of 98 μ L are added.
For whole blood stripping solution, the 660nM solution of 10 μ L and the Chon Block of 90 μ L are added.
Serum preparation
Standard serum preparation includes adding 20 μ L serum into 80 μ L Chon Block.
The serum of PSA removing is prepared by the way that 20 μ L serum are added in 80 μ L serum stripping solutions.
Whole blood prepares
Standard whole blood preparation includes 76 μ L whole bloods of mixing, 19 μ L Chon Block and 4.75 μ L10%Triton X-100.
PSA stripping is prepared by 76 μ L whole bloods of mixing, 19 μ L whole blood stripping solutions and 4.75 μ L 10%Triton X-100
From whole blood.
It is incubated for
By adding 25 μ L magnetic beads and 9 μ L reporters into the serum of 100 μ L preparation first, then sample incubation 30 is divided
Clock is incubated for.
Same pass through adds 25 μ L magnetic beads and 9 μ L reporters into 100 μ L whole bloods first, then by sample incubation 30 minutes
To be incubated for.
To lower traction after incubation
Downward traction is executed by the way that the solution of incubation is exposed to magnet 5 minutes.
100 μ L PBS of the result drawn downwards are rinsed twice, while being stilled remain on magnet and with 27 μ L PBS
It is resuspended.
Magnetic bead/reporter of fluorescent spectrometry analysis resuspension be may then pass through to detect the PSA in whole blood and serum.Mark
Signal difference between quasi- serum (or whole blood) and the serum (or whole blood) of removing will provide the measurement of PSA level, which disappears
Except the matrix effect of individual serum.
The measurement operation of embodiment 9- bacterial immune
Prepare immunoassays as described herein for the bacterium in test sample.
As shown in Fig. 8 A to Fig. 8 C, it can be used thin in the sandwich mode test sample of immunoassays as described herein
Bacterium (such as A group of streptococcus).As shown in the figure, can be used (has comprising reporter and is impregnated with the two of 100-600 quantum dot
The golden slug particle (nanoComposix) of silica shell) and antibody #1 report conjugate and include antibody #2 and magnetic-particle
Magnetic conjugate test and analyze the bacterial analysis object interested (Fig. 8 A) in chamber.
In the first step, can by magnetic conjugate and report conjugate be added to measurement chamber in and by they with contain
The sample of analytes of interest analytes (bacterium) mixes (Fig. 8 B).Magnetic field (" downwards traction ") can be applied by magnet with will be interested
Analyte separates (Fig. 8 C) from sample.Then, light transmission can be made by a part of analysis chamber, so that reporter fluoresces
(Fig. 8 D).This fluorescence can be detected by detector.
In the absence of the analyte, reporter will not be drawn downwards together with analyte, therefore will not be gone out
Existing fluorescence.
Embodiment 10- detects the demonstration of the bacterial immune measurement of A streptococci bacteria
If embodiment 9 provides bacterial immune measurement, bacterial immune measurement is deposited for A group of streptococcus in test sample
It include that subject and 15 suspection from 26 with Strep throat is not infected by A group of streptococcus in, the sample
Subject throat, cheek and saliva sample.As shown in Figure 9, which shows the grade sensitivity of 200 organisms.
In addition, about sensitivity, compared with Quidel clinical-grade tests 92% sensitivity and 98% specificity, this
Measurement shows the sensitivity of 93%ND and 100% specificity.
It is as follows for detecting streptococcic measurement.
Magnetic bead in the measurement is the magnetic coated with the antibody (Biospacific G47091041) in conjunction with A group of streptococcus
Property nano particle.300pM magnetic bead solution is prepared by adding 4 μ L magnetic beads (2.89nM) into flask, it is with magnet that it is downward
Traction, is then resuspended in 38.5 μ L Chon Block
The reporter used in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated with combination
The antibody (Biospacific G47091041) of A group of streptococcus.Pass through 4.5 μ L reporters (2.2nM) of mixing and 35.5 μ L
Chon Block prepares 250pM reporter solution.
Deposit closing mixture preparation
Biospacific G47091041 (1m/mL) is prepared as 6.67 μM of stock solution, and prepares 1 in PBS
μM, the solution of 500nM and 100nM.
Swab processing
In the first step, the tonsillotome of subject is wiped.Then 150 μ L PBS are added in extrudable pipe.Then will
Swab is immersed in PBS solution.After 1 minute, swab is pulled out among pipe and is clamped with finger with the extracting liq from swab.
Streptomycete solution is prepared by the mixture and 2 μ L Chon Block of mixing 16 μ L swabs processing.
It is closed to prepare that mixture is closed by the mixture of mixing 16 μ L swabs processing and the 500nM streptococcus of 2 μ L
Streptomycete solution.
Sample incubation
Streptococcus is incubated for: 16 μ L streptococcus solution are mixed with 3 μ L magnetic beads and 3 μ L reporters.It is incubated for and carries out 10 minutes.
Closing is incubated for: the closed streptococcus solution of 16 μ L is mixed with 3 μ L magnetic beads and 3 μ L reporters.It is incubated for and carries out 10 points
Clock.
To lower traction after incubation
Then traction downwards is executed to the solution of incubation on magnet, continues 5 minutes.Gained is drawn downwards to object with 100 μ
L PBS is rinsed twice, while being stilled remain on magnet, is then suspended with 27 μ L PBS.
Magnetic bead/reporter of fluorescent spectrometry analysis resuspension be may then pass through with the A group of streptococcus in test sample.Chain
Signal difference between the signal of coccus incubation tube and closed incubation tube will provide the measurement of A group of streptococcus, which eliminates
Matrix effect in Different Individual swab.
The method of embodiment 11- competitiveness E3G detection assay
E3G can be detected in the sample analyzed by competitive immunoassay as described herein.
E3G coupled bead and anti-E3G coupling quantum dot, which can be used, (has the dioxy for being impregnated with 100-600 quantum dot
The golden slug particle (nanoComposix) of SiClx shell).Buffer can be used in analysis chamber, the buffer may include
Chonblock and PBS.Quantum dot working solution may include that 1 μ L lays in anti-E3G quantum dot and 6 μ L Chonblock.
The program can be as follows:
A. 6.25 μ L urines are mixed with 1.25 μ L quantum dot working solutions;
B. it is incubated for 5 minutes;
C. 5 μ L E3G coupled beads are added;
D. it is incubated for 30 minutes;
E. it is placed on magnetic frame 1 minute;
F. supernatant is removed;
G. it is rinsed twice with 50 μ L PBS;
H. it is resuspended in 27 μ L PBS;And
I. 20 μ L are pipetted into orifice plate.
Then the material pipetted can be imaged, as shown in Figure 10, compared with blank, is detected in two samples
To 44 μM and 200nM E3G.
The measured value and the phase between the measured value realized in clinical labororatory that embodiment 12- measures the immunoassays
The clinical research of closing property
Carry out clinical research, measure embodiment 8 described in this PSA detection immunoassays platform on measured value with facing
The correlation between measured value realized in bed laboratory, shows very high correlation as shown in Figure 11.
The clinical verification for the β-hCG detection that embodiment 13- is carried out using immunoassay
In this embodiment, the method used in the clinical verification of β-hCG detection platform and standard operation journey are described
Sequence.
IRB approval starts a clinical research, which receives nearly 500 investigation, share 76 subjects
Enter group, wherein 15 people are pregnant.13 in 15 pregnancies collect urines using following frozen samples schemes, and 15 by
2 in examination person are collected sample using fresh scheme.
Frozen samples scheme.Urine cup, label, Ziploc bags and sample record are provided to the subject for collecting frozen samples,
So that they can collect daily sample.Urine collecting is recorded urination time in urine cup by subject every morning, gives cup
Labelled, pack is put into refrigerator immediately after.It (is obtained by using one of provided test after the completion of subjectxperiod
Positive pregnancy test result was obtained, or has known their period), researcher will collect sample from subject family, he
Sample and is transported in the cooler for filling ice bag in laboratory sample by retrieval.It is cold that sample is stored in laboratory immediately
(material and method are detailed in) in jelly machine.
Fresh sample scheme.Single urine cup and small-sized vacuum flask are provided to the subject for collecting fresh sample.Subject is every
Its morning collects urine and urine cup is put into vacuum flask.Then, they can send short message to clinical research expeditor, and clinic is ground
Study carefully expeditor and can take back sample in 2 hours after urine collecting and analyzes.
Sampling test example.Sample is tested to determine that First Response generates first day of positive findings
(i.e. First Response urine reading on the day before this day is feminine gender, but urine reading is the positive on the day of this day).This
It is referred to as First Response days.Then immunoassays platform is carried out before First is Response days and at least three days
Test.In addition, testing two baseline days (date before participant's ovulation, it is ensured that she cannot be pregnant).
Typical test seems that wherein the first row and the second row are repeat samples as shown in Figure 12.Darker circle
Indicate that the hCG concentration in sample is higher.As expected, low-down signal is shown baseline day, and First
Response determines that the intensity on the date before day increases.The image of Figure 12 is quantified and is drawn in Figure 13, wherein error
Item represents 1 standard deviation.
Performance compares.Figure 14 shows staircase curve, and this graph illustrate the letters as ovulation (LH peak value day) number of days afterwards
Several fraction positives, wherein Confer Magneto is immunoassays as described herein.
Baseline compares.The daily of analysis and the signal on the date (also referred to as baseline day) for collecting urine before preovulatory are compared
Compared with.This is because everyone has characteristic baseline hCG amount.Signal by calculating acquisition, which is greater than, has been directed to the subject
The probability of the maximum background signal of measurement, makes a determination Yes/No gestation.
Assuming that daily signal is described by gaussian probability, wherein average value is equal to the measuring signal and standard of each corresponding day
Estimation of deviation value calculates probability with this.Baseline day signal probability PBL(S), it, is given by the following formula:
Data used are signal (S), average value (μBL) and standard deviation (σBL).The probability P for the same day signal analyzedCD
(S) it is:
Data used are signal (S), average value (μCD) and standard deviation (σCD)。
The probability that same day signal is greater than BL signal is 1 probability for subtracting same day signal less than or equal to baseline day signal
PCD≤BL, it is given by:
These integrals are with Numerical value.If the difference between any two days is greater than 4 (σBL+σCD), then we set PCD
≤BL=0.
PCD≥BL=1-PCD≤BL, so if the difference between any two days is greater than 4 (σBL+σCD), then we set PCD≤BL=
1 (or 100%).For convenience, these numerical value are for determining the % certainty in the number reappeared herein.
The summary of gestation data is shown in Figure 15, is shown and is given number of days reading before First Response as sun
The percentage of the sample of property.
Material and method.There is provided (Fig. 2A to Fig. 2 F) as described in example 2 above.
HCG:2 stage -7 minutes schemes of association schemes.
Buffer:
A.CD=Conjugate Diluent (0.1x PBS, 0.5%BSA, 0.05% sodium azide)
B.Chon Block is commercially available lock solution
Antibody and quantum dot are conjugated:
Reaction buffer: 5mM potassium phosphate, pH 7.4,0.5%20K MW PEG.
Program:
A. 100mg/mL EDC and Sulfo-NHS solution is prepared
i.2mg sulfo-NHS+20uL H2O。
ii.4.8mg EDC+48μL H2O。
B. 0.8 μ L EDC and 1.6 μ L sulfo-NHS are added to 16.7 μ L quantum dots (has and is impregnated with 100-600
The golden slug particle (nanoComposix) of the silica shell of quantum dot).
C. it rotates 30 minutes.
D. with 3600RCF centrifugation 5 minutes.
E. it removes supernatant and is resuspended with 16.7 μ L reaction buffers.
F. it is ultrasonically treated 10 seconds and is vortexed to complete and be resuspended.
G. it is resuspended in the 1.7mg/mL 4F9 antibody of 17 μ L.
H. it is incubated for 1 hour.
I. 3.75 μ L azanols are added, are rotated 10 minutes.
J. it with 3600RCF centrifugation 5 minutes, removes supernatant and is resuspended with 100 μ L reaction buffers.
K. it with 3600RCF centrifugation 5 minutes, removes supernatant and is resuspended with the Conjugate Diluent of proper volume.
Preparation work solution:
Following substance is mixed in the low protein adsorption pipe of 0.5mL Eppendorf (can be saved by storing at 4 DEG C
Solution in case use in the future):
A.1.1 μM 5011_ biotin antibody (being dissolved in Conjugate Diluent)
I.10 the Conjugate Diluent of μ l
Deposit (6.6 μM) 5011_ biotin antibody of ii.2 μ l
B.2.5x diluted 4F9_ quantum dot
I.3.75 the Chon Block of μ L
The deposit 4F9_ quantum dot of ii.2.5 μ L
Magnetic bead preparation:
A. following substance is mixed in the low protein adsorption pipe of 0.5mL Eppendorf:
I.4.4 μ L 100nm lays in Streptavidin magnetic beads.
1.1 μM of 5011_ biotin antibodies (being dissolved in Conjugate Diluent) of ii.2.2 μ L.
B. it is incubated for 5 minutes on rotator at room temperature.
C. 4.14 μ L saturation biotin solution is added.
D. it is sufficiently mixed with pipette.
E. it is incubated for again 1 minute.
F. pipe is placed on magnetic frame and is allowed to rest for (noting: if to be a large amount of sample preparation magnetic beads, working as totality for 1 minute
When product is more than 100 μ L, this way can be amplified, need to increase the time on magnetic frame, such as 300 μ L volumes will be needed in magnetic
Kept on power frame 4 minutes to restore magnetic bead completely).
G. supernatant is removed using P20 pipette (or appropriate pipette that volume will be removed in amplification).
H. 20uL Chon Block is added into sediment and is mixed with pipette to be resuspended
I. pipe is placed on and is allowed to rest for (please referring to saying for the time of the volume above for > 100 μ l in 1 minute on magnetic frame
It is bright).
J. supernatant is removed using P20 pipette (or appropriate pipette that volume will be removed in amplification).
K. 4.4 μ L Chon Block are added into sediment and are mixed with pipette to be resuspended.
This method provides the 4.4 prefabricated magnetic beads of μ L.
Stage 1 combines: magnetic beads analysis object combines
A. following substance is mixed in the low protein adsorption pipe of 0.5mL Eppendorf:
I.4 the prefabricated magnetic bead of μ L
Ii.25 μ L urine
B. it is incubated for 1 minute on the table at room temperature.
C. pipe is placed on magnetic frame and is allowed to rest for 45 seconds.
D. supernatant is removed using P200 pipette (or appropriate pipette that volume will be removed in amplification).
E. 4 μ L Chon Block are added into sediment and are mixed with pipette to be resuspended.
This method provides 4 μ L stage, 1 binding mixtures.
Stage 2 combines: quantum dot combines
A. following substance is mixed in the low protein adsorption pipe of 0.5mL Eppendorf:
I.4 1 binding mixture of μ L stage
The diluted 4F9_ quantum dot of the 2.5x of ii.1 μ L
B. it is incubated at room temperature 5 minutes.
This method provides 5 μ L stage, 2 binding mixtures.
Traction downwards and flushing
A. the pipe containing 5 μ L stage, 2 binding mixture is placed on magnetic frame and is allowed to rest for 20 seconds
B. pipe is maintained on magnetic frame in remaining 5 step
I. supernatant is removed using P20 pipette (or appropriate pipette that volume will be removed in amplification).
Ii. the 1xTBS buffer of 100 μ L is added with P200 pipette.
Iii. the TBS buffer of 100 μ L is removed with P200 pipette.
Iv. the TBS buffer of 100 μ L is added with P200 pipette.
V. the TBS buffer of 100 μ L is removed with P200 pipette.
C. pipe is removed from magnetic frame.
D. 10 μ L Conjugate Diluents are added into sediment and are mixed with P20 pipette to be resuspended.
This method provides the analytes that 10 μ L and both magnetic-particle and fluorescence quantum combine, and prepare imaging.
Embodiment 14- is for analyzing urine sample to detect the scheme of oestrone -3- glucosiduronic acid
It is presented below to analyze urine sample according to methods described herein to detect the example of oestrone -3- glucosiduronic acid (E3G)
Property scheme.
According to aforementioned schemes, the magnetic bead in the measurement is the magnetic nanoparticle coated with E3G.
Reporter in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated in conjunction with E3G's
Antibody (Absolute Antibody 4115).
Device therefor
Use following equipment.
The desk-top cleaner of CPX2800 ultrasonic digital |
Rugged rotator: #099A-RD4612 |
SpectraMax i3x |
Pipetman P200 pipette |
Pipetman P100 pipette |
Pipetman P20 pipette |
Pipetman P10 pipette |
Promega magnetic separation frame (0.5mL) |
Promega magnetic separation frame (0.5mL) |
Timer |
Experimental arrangement
It is dilute in Chon Block according to the following formula (for example, about at 5 points in the afternoon before collecting day) before collecting day
Release magnetic bead and reporter:
ChonBlock volume (μ L)=(magnetic bead volume) × (the 50X coefficient of dilution)-(magnetic bead volume)
ChonBlock volume (uL)=(magnetic bead volume) × (the 21.74X coefficient of dilution)-(magnetic bead volume).
A. reporter is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes reporter.
B. using the pipette for suitably setting size, the Chon Block of certain volume is added to 0.5mL
In Eppendorf albumen low adsorption pipe.
C. P10 is used, the E3G coupled bead of certain volume is added into Chon Block.Upper and lower liquid relief is to ensure liquid relief
Guan Zhongyi removes deposit magnetic bead.
D. it diluted E3G magnetic bead will be mixed now with the pipette for suitably setting size.
E. E3G coupled bead is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as
100 P100 mixes magnetic bead.
F. using the pipette for suitably setting size, the Chon Block of certain volume is added to 0.5mL
In Eppendorf albumen low adsorption pipe.
G. using the pipette for suitably setting size, the E3G coupled bead of certain volume is added into Chon Block.On
Lower liquid relief is to ensure to have removed magnetic bead in pipette.
H. it diluted E3G magnetic bead will be mixed now with the pipette for suitably setting size.
I. diluted reagent is stored in metallic support at 4 DEG C.Reagent will use in 20 hours.
While collecting urine sample, 3 μ L magnetic beads are assigned to three 0.5mL using the P10 pipette for being set as 3 μ L
In Eppendorf albumen low adsorption pipe, label has number, cycle date and repetition number (1,2,3) to these pipes in advance.
When sample reaches, 3 μ L urines are added in the first repeating pipe.
And then, start timer, count a few minutes.
After 30 seconds, 3 μ L reporters are added in the first repeating pipe using P10 pipette.Use the P10 for being set as 3 μ L
Mixing.
When timer reaches 1 minute, 3 μ L urines are added in the second repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
On timer after 1:30 seconds, 3 μ L reporters are added in the second repeating pipe with P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
When timer reaches 2 minutes, 3 μ L urines are added in the third repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
On timer after 2:30 seconds, 3 μ L reporters are added in the third repeating pipe with P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
By after five minutes, the first repeating pipe being placed on Promega magnetic frame, and check timer on timer, count
1 minute.
6 minutes time points, the second repeating pipe is placed on the 2nd Promega magnetic frame.
Supernatant is removed from the first repeating pipe using the P20 pipette for being set as 20 μ L in 6:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, the Tris buffered saline (TBS) of 100 μ L is added with P200 pipette
Into the first repeating pipe, the TBS of 100 μ L is then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the first weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
7 minutes time points, the third repeating pipe is placed on the first Promega magnetic frame.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 7:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 8:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Storage measurement pipe is in case subsequent analysis.
Spectra Max analysis
Using P20 pipette, each 20 μ L of sample is loaded on 384 orifice plates.
Using P10 pipette, 20 μ L TBS are loaded in 2 holes on same 384 orifice plate.
384 orifice plates are loaded in SpectraMax and are analyzed.
Embodiment 15- is for analyzing urine sample to detect the scheme of human chorionic gonadotrophinIt is presented below according to this
Literary the method analyzes urine sample to detect the exemplary arrangement of human chorionic gonadotrophin (HCG).
Magnetic bead in the measurement is the magnetic nanoparticle coated with the antibody (Scripps GC099) in conjunction with HCG.
Reporter in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated in conjunction with HCG's
Antibody (Scripps GC099).
Device therefor
Use following equipment.
Experimental arrangement
Before measurement, magnetic bead and reporter are diluted in Chon Block according to following way:
A. reporter is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes reporter.
B. using the P200 for being set as 50 μ L, 50 μ L Chon Block are added to the low suction of 0.5mL Eppendorf albumen
In attached pipe.
C. 5 μ L magnetic beads are added in 50 μ L Chon Block.Upper and lower liquid relief is to ensure to have removed magnetic bead in pipette.
D. it diluted magnetic bead will be mixed now with the P200 for being set as 150.
E. magnetic bead is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes magnetic bead.
F. P200 is used, 59.4 μ L Chon Block are added in 0.5mL Eppendorf albumen low adsorption pipe.
G. 6 μ L magnetic beads are added in 59.4 μ L Chon Block.Upper and lower liquid relief is to ensure to have removed magnetic in pipette
Pearl.
H. it diluted magnetic bead will be mixed now with the P200 for being set as 125.
I. diluted reagent is stored in metallic support at 4 DEG C.
16 μ L urines are added to the low suction of three 0.5mL Eppendorf albumen using the P20 pipette for being set as 16 μ L
In attached pipe, label has number, cycle date and repetition number (1,2,3) to these pipes in advance.
When sample reaches, 2 μ L reporters are added in the first repeating pipe.
And then, start timer, count a few minutes.
After 30 seconds, 2 μ L magnetic beads are added in the first repeating pipe using P10 pipette.It is mixed using the P20 for being set as 16 μ L
It closes.
When timer reaches 1 minute, 2 μ L reporters are added in the second repeating pipe using P10 pipette.Using setting
It is set to the P10 mixing of 2 μ L.
When timer passes through 1:30 seconds, 2 μ L magnetic beads are added in the second repeating pipe using P10 pipette.Using setting
It is set to the P10 mixing of 2 μ L.
When timer reaches 2 minutes, 2 μ L reporters are added in the third repeating pipe using P10 pipette.Using setting
It is set to the P10 mixing of 2 μ L.
When timer passes through 2:30 seconds, 2 μ L magnetic beads are added in the third repeating pipe using P10 pipette.Using setting
It is set to the P10 mixing of 2 μ L.
By after twenty minutes, the first repeating pipe being placed on Promega magnetic frame, and check timer on timer,
Meter 1 minute.
21 minutes time points, the second repeating pipe is placed on the 2nd Promega magnetic frame.
Supernatant is removed from the first repeating pipe using the P20 pipette for being set as 20 μ L in 21:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the first repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the first weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
22 minutes time points, the third repeating pipe is placed on the first Promega magnetic frame.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 22:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 23:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Storage measurement pipe is in case subsequent analysis.
Spectramax analysis
Using P20 pipette, each 20 μ L of sample is loaded on 384 orifice plates.
Using P10 pipette, 20 μ L TBS are loaded in 2 holes on same 384 orifice plate.
384 orifice plates are loaded in SpectraMax and are analyzed.
Embodiment 16- is for analyzing urine sample to detect the scheme of metakentrin
It is presented below to analyze urine sample according to methods described herein to detect the exemplary arrangement of metakentrin (LH).
Magnetic bead in the measurement is the magnetic nanoparticle coated with the antibody (Medix 5304) in conjunction with LH.
Reporter in the measurement is the nano particle of fluorescent marker, and the nano particle is already coated in conjunction with the anti-of LH
Body (Medix 5304).
Device therefor
Use following equipment.
Experimental arrangement
It is (for example, at 5 points in pact afternoon on the day before collection) before being collected, dilute in Chon Block according to following way
Release magnetic bead and reporter:
A. reporter is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes reporter.
B. using the P200 for being set as 150,150 μ L ChonBlock are added to the low suction of 0.5mL Eppendorf albumen
In attached pipe.
C. 3 μ L magnetic beads are added in 150 μ L Chon Block.Upper and lower liquid relief is to ensure to have removed report in pipette
Object.
D. it diluted magnetic bead will be mixed now with the P200 for being set as 150.
E. magnetic bead is ultrasonically treated by the way that pipe to be kept to 10 seconds in ultrasonoscope.Using being set as 100
P100 mixes magnetic bead.
F. using the P200 for being set as 125,125 μ L Chon Block are added to the low suction of 0.5mL Eppendorf albumen
In attached pipe.
G. 25 μ L magnetic beads are added in 125 μ L Chon Block.Upper and lower liquid relief is to ensure to have removed deposit in pipette
Magnetic bead.
H. it diluted magnetic bead will be mixed now with the P200 for being set as 125.
I. diluted reagent is stored in metallic support at 4 DEG C.
While collecting urine sample, 3 μ L magnetic beads are assigned to three 0.5mL using the P10 pipette for being set as 3 μ L
In Eppendorf albumen low adsorption pipe, label has number, cycle date and repetition number (1,2,3) to these pipes in advance.
When sample reaches, 3 μ L urines are added in the first repeating pipe.
And then, start timer, count a few minutes.
After 30 seconds, 3 μ L reporters are added in the first repeating pipe using P10 pipette.Use the P10 for being set as 3 μ L
Mixing.
When timer reaches 1 minute, 3 μ L urines are added in the second repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
On timer after 1:30 seconds, 3 μ L reporters are added in the second repeating pipe with P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
When timer reaches 2 minutes, 3 μ L urines are added in the third repeating pipe using P10 pipette.Use setting
It is mixed for the P10 of 3 μ L.
On timer after 2:30 seconds, 3 μ L reporters are added in the third repeating pipe with P10 pipette.It uses
It is set as the P10 mixing of 3 μ L.
By after five minutes, the first repeating pipe being placed on Promega magnetic frame, and check timer on timer, count
1 minute.
6 minutes time points, the second repeating pipe is placed on the 2nd Promega magnetic frame.
Supernatant is removed from the first repeating pipe using the P20 pipette for being set as 20 μ L in 6:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the first repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the first weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
7 minutes time points, the third repeating pipe is placed on the first Promega magnetic frame.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 7:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Supernatant is removed from the second repeating pipe using the P20 pipette for being set as 20 μ L in 8:10, while pipe being protected
It holds on magnetic frame.
While pipe is maintained on magnetic frame, 100 μ L TBS are added to the second repeating pipe using P200 pipette
In, 100 μ L TBS are then removed by excessive liquid relief.
While pipe is maintained on magnetic frame, other 100 μ L TBS is added to the second weight using P200 pipette
In multiple pipe, 100 μ L TBS are then removed by excessive liquid relief.
Eppendorf pipe is removed from magnetic frame.
20 μ L TBS are added using P20 pipette, and are sufficiently mixed by upper and lower liquid relief.
Storage measurement pipe is in case subsequent analysis.
Spectra Max analysis
Using P20 pipette, each 20 μ L of sample is loaded on 384 orifice plates.
Using P10 pipette, 20 μ L TBS are loaded in 2 holes on same 384 orifice plate.
384 orifice plates are loaded in SpectraMax and are analyzed.
Embodiment 17- is for analyzing blood serum sample to detect the scheme of c reactive protein
It is presented below to analyze blood serum sample according to methods described herein to detect the exemplary arrangement of c reactive protein (CRP).
Magnetic bead in the measurement is the magnetic nanoparticle coated with the antibody (anti crp-C2) in conjunction with CRP.
Reporter in the measurement is the nano particle of fluorescent marker, and it is (anti-that the nano particle is already coated with antibody
CRP-C6)。
Experimental arrangement
The preincubate of Streptavidin magnetic beads and biotinylated anti crp-C2 antibody:
A. magnetic bead is mixed 5 minutes with the antibody for being diluted to 1 μM in Conjugate Diluent on rotator as described above.
B. addition is saturated biotin solution and is incubated for the combination 1 minute.
C. it is placed on magnet and draws downwards, then washed with Chon Block.
Magnetic bead, is then resuspended in Chon Block by d. traction downwards again.
Reporter preparation
The concentration of reporter solution for the measurement is 440pM, which is the stock solution Chon from 2.2nM
Block dilution.
Serum preparation
Prepare serum in the following manner: then the 20 above-mentioned magnetic beads of μ L of traction downwards are resuspended in 10 μ L serum, then incubate
It educates 10 minutes.
Another traction 30 seconds downwards are carried out, then supernatant is transferred in new pipe.
CRP dilution
Exist from the CRP that the CRP stock solution of 21.28mg/mL preparation concentration is 2.5mg/L, 1.25mg/L and 0.5mg/mL
Dilution in Chon Block.
The preparation of mark-on blood serum sample
According to following addition standard serum samples to prepare mark-on sample:
A.10mg/L:8+2 μ L standard serum (final 2mg/L) of μ L 2.5mg/L CRP dilution;
B.5mg/L:8+2 μ L standard serum (final 1mg/L) of μ L 1.25mg/L CRP dilution;
C.2mg/L:8+2 μ L standard serum (final 0.4mg/L) of μ L 0.5mg/L CRP dilution;And
D.0mg/L:8 μ L Chon Block+2 μ L standard serum (final 0mg/L).
Analytical plan
In one group of four pipe, above-mentioned mark-on standard serum samples are added.Then, 3 μ L magnetic beads are allocated in advance each
Guan Zhong.After magnetic bead, 3 μ L sample to be analysed are added in each pipe.After about 30 seconds, 3 μ L reporters are added, are then incubated for
About 30 minutes.
After being incubated for a period of time, traction downwards is executed to sample cell on magnetic frame, is rinsed twice with 100 μ L, is then weighed
It is suspended from the TBS that final volume is 20 μ L.
The measurement as the result is shown in Figure 17 A and Figure 17 B, the figures illustrate the CRP concentration series in buffer solution
CRP concentration (Figure 17 B) in (Figure 17 A) and mark-on serum.
Claims (34)
1. it is a kind of for the presence of analytes of interest analytes in test sample, be not present or horizontal method, the method includes with
Lower step:
Contact sample with the magnetic conjugate comprising magnetic-particle and capture portion, the capture portion is configured as combining institute
State the analytes of interest analytes in sample;
Contact the sample with the report conjugate comprising reporter and report bound fraction, the report bound fraction is matched
It is set in conjunction with the analytes of interest analytes in the sample;
Make the analytes of interest analytes in conjunction with the capture portion and the report bound fraction;
The analytes of interest analytes is separated from the sample by applying magnetic field to analysis chamber;And
The presence of the analytes of interest analytes is detected by detecting the reporter, is not present or horizontal.
2. the method as described in claim 1, wherein the reporter includes metal core and silica shell.
3. method according to claim 2, wherein the silica shell is impregnated with multiple quantum dots.
4. method as claimed in claim 2 or claim 3, wherein the metal core includes gold.
5. the method as described in claim 1, wherein the reporter includes multiple quantum dots.
6. method as described in any one of the preceding claims, wherein the reporter is fluorescence report object, phosphorescence reporter
Or colorimetric reporter.
7. method as described in any one of the preceding claims, the method also includes following steps: make the sample with
It is described interested in the sample to be concentrated by applying magnetic field to the analysis chamber after the magnetism conjugate contact
Analyte;Then the volume of sample described in the analysis chamber is reduced.
8. the method for claim 7, the method also includes so that the sample is contacted it with the report conjugate
Before make the magnetic field deactivate the step of.
9. method as described in any one of the preceding claims, the method also includes following steps: make the sample with
It is described interested in the sample to be concentrated by applying magnetic field to the analysis chamber after the magnetism conjugate contact
Analyte;The sample of certain volume is removed from the analysis chamber;And by the buffer of certain volume and other body
One or both of long-pending described sample is added in the analysis chamber.
10. method as claimed in claim 9, the method also includes so that the sample is contacted it with the report conjugate
Before make the magnetic field deactivate the step of.
11. the method as described in claim 1, wherein the report conjugate biotin labeling, and the reporter is used
Streptavidin functionalization.
12. method as described in any one of the preceding claims, wherein the analytes of interest analytes is selected from and to be made up of
Group: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA), list
Pure herpesviral (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation biology
Marker, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol are raw
Substance markers object, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biology mark
Remember object, liver function biomarker, estrogen, progesterone, lactic acid and their combination.
13. it is a kind of for the presence of analytes of interest analytes in test sample, be not present or horizontal method, the method includes with
Lower step:
Contact sample with the magnetic conjugate comprising magnetic-particle and capture portion, the capture portion is configured as combining institute
State the analytes of interest analytes in sample;
Make the analytes of interest analytes in conjunction with the capture portion;
By applying magnetic field to analysis chamber with by the magnetic conjugate and associate with the magnetic conjugate interested point
Analysis object is drawn downwards, so that the analytes of interest analytes be separated from the sample;
Contact the sample with the report conjugate comprising reporter and report bound fraction, the report bound fraction is matched
It is set in conjunction with the analytes of interest analytes in the sample;
Make the analytes of interest analytes in conjunction with the report bound fraction;
By applying magnetic field for the analytes of interest analytes and the institute in conjunction with the analytes of interest analytes to the analysis chamber
Report bound fraction is stated to separate from the sample;And
Detect the presence of the analytes of interest analytes by detecting the reporter with light source and photoelectric detector, be not present or
It is horizontal.
14. method as claimed in claim 13, wherein the reporter includes fluorescence report object, phosphorescence reporter or colorimetric report
Accuse object.
15. method as claimed in claim 13, wherein the report conjugate includes multiple quantum dots.
16. the method as described in any one of claim 13 to 15 is made up of wherein the analytes of interest analytes is selected from
Group: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA),
Herpes simplex virus (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation are raw
Substance markers object, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol
Biomarker, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biology
Marker, liver function biomarker, estrogen, progesterone, lactic acid and their combination.
17. it is a kind of for the presence of analytes of interest analytes in test sample, be not present or horizontal method, the method includes with
Lower step:
Contact sample with the magnetic conjugate comprising magnetic-particle and capture portion, the capture portion is configured as combining institute
State the analytes of interest analytes in sample;
Make the analytes of interest analytes in conjunction with the capture portion;
Contact the sample with the analyte for reporting substance markers, the analyte of the report substance markers is configured as in the sample
In product there is no in the case where the analytes of interest analytes in conjunction with the magnetic conjugate;
The analytes of interest analytes is separated from the sample by applying magnetic field to the sample;And
The presence of the analytes of interest analytes is detected by detecting the reporter, is not present or horizontal.
18. method as claimed in claim 17, wherein the reporter includes fluorescence report object, phosphorescence reporter or colorimetric report
Accuse object.
19. the method as described in claim 17 or 18, wherein the analyte of report substance markers includes multiple quantum dots.
20. the method as described in any one of claim 17 to 19 is made up of wherein the analytes of interest analytes is selected from
Group: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA),
Herpes simplex virus (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation are raw
Substance markers object, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol
Biomarker, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biology
Marker, liver function biomarker, estrogen, progesterone, lactic acid and their combination.
21. it is a kind of for the presence of analytes of interest analytes in test sample, be not present or horizontal method, the method includes with
Lower step:
Contact sample with the report conjugate comprising reporter and report bound fraction, the report bound fraction is configured as
In conjunction with the analytes of interest analytes in the sample;
Make the analytes of interest analytes in conjunction with the report bound fraction;
Contact the sample with the analyte of magnetic particle labels, the analyte of the magnetic particle labels is configured as in institute
It states in the case where the analytes of interest analytes is not present in sample in conjunction with the report conjugate;
The analyte of the magnetic particle labels is separated from the sample by applying magnetic field to the sample;And
The presence of the analytes of interest analytes is detected by detecting the reporter, is not present or horizontal.
22. method as claimed in claim 21, wherein the reporter includes fluorescence report object, phosphorescence reporter or colorimetric report
Accuse object.
23. the method as described in claim 21 or 22, wherein the report conjugate includes multiple quantum dots.
24. the method as described in any one of claim 21 to 23 is made up of wherein the analytes of interest analytes is selected from
Group: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA),
Herpes simplex virus (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation are raw
Substance markers object, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol
Biomarker, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biology
Marker, liver function biomarker, estrogen, progesterone, lactic acid and their combination.
25. it is a kind of for the presence of analytes of interest analytes in test sample, be not present or horizontal method, the method includes with
Lower step:
Contact sample with the magnetic conjugate comprising magnetic-particle and capture portion, the capture portion is configured as combining institute
State the analytes of interest analytes in sample;
Make the analytes of interest analytes in conjunction with the capture portion;
Contact the sample with the report bound fraction comprising biotin labeling, the report bound fraction is configured as combining
The analytes of interest analytes in the sample;
Contact the sample with the reporter marked comprising streptavidin, the streptavidin label
It is configured as in conjunction with the biotin labeling;
The analytes of interest analytes is separated from the sample by applying magnetic field to the sample;And
The presence of the analytes of interest analytes is detected by detecting the reporter, is not present or horizontal.
26. method as claimed in claim 25, wherein the reporter includes fluorescence report object, phosphorescence reporter or colorimetric report
Accuse object.
27. the method as described in claim 25 or 26, wherein the reporter includes multiple quantum dots.
28. the method as described in any one of claim 25 to 27 is made up of wherein the analytes of interest analytes is selected from
Group: human chorionic gonadotrophin (hCG), metakentrin (LH)/lutropin, prostate-specific antigen (PSA),
Herpes simplex virus (HSV) antibody, oestrone -3- glucosiduronic acid (E3G), bacterium, HbA1 C, c reactive protein, inflammation are raw
Substance markers object, troponin, Lyme disease antigen, Antibodies Against Lyme Disease, LDL biomarker, HDL biomarker, total cholesterol
Biomarker, thyrotropic hormone, Hepatitis C Virus biomarker, rhinovirus biomarker, influenza virus biology
Marker, liver function biomarker, estrogen, progesterone, lactic acid and their combination.
29. the method as described in any one of claim 1 to 28, the method includes the sample is added to analysis chamber
In step.
30. method as claimed in claim 29, wherein the step packet for contacting the sample with the magnetic conjugate
Including contacts the sample with the magnetic conjugate in the analysis chamber.
31. the method as described in claim 29 or 30, wherein the sample is added to the step in the analysis chamber
Suddenly include the sample is added to it is described analysis chamber in fluid communication sample divider in.
32. method as claimed in claim 31, wherein the step packet for contacting the sample with the magnetic conjugate
Including contacts the sample with the magnetic conjugate in the sample divider.
33. a kind of kit, the kit is used to provide the method as described in any one of claims 1 to 32.
34. a kind of method for assessing biological event, the method includes using as described in any one of claims 1 to 32
Method measure biomarker.
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US62/544,393 | 2017-08-11 | ||
PCT/US2018/015440 WO2018140719A1 (en) | 2017-01-26 | 2018-01-26 | Magnetic particle-based immunoassay and methods of using the same |
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EP (1) | EP3574309A4 (en) |
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WO2018140719A1 (en) | 2018-08-02 |
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CA3051475A1 (en) | 2018-08-02 |
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