CN103575882B - The labelling immunoassay method of whole blood and instant detection system - Google Patents
The labelling immunoassay method of whole blood and instant detection system Download PDFInfo
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- 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
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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Abstract
The present invention is specifically related to the labelling immunoassay method of the whole blood marking object by specific binding reaction Quick Acquisition object and by labelling technique from whole blood and monitor, and a kind of instant detection system.Said method comprising the steps of: 1) magnetic capture; 2) blood wash-out; 3) mark of antibody is detected; 4) wash-out of antibody is detected; With 5) detect.Described system comprises micro-fluidic chip, is arranged on magnetic field generation device and the pick-up unit of next side or two sides on micro-fluidic chip, described micro-fluidic chip comprises microchannel, the magnetic bead be arranged in microchannel, waste liquid outlet and eluent liquid storage tank, and described microchannel is isolated into the first conversion zone, second reaction zone and detection zone by separating mechanism stage.Technical scheme provided by the invention has the features such as highly sensitive, reproducible, system architecture is simple, cost is low, quantitatively can detect object fast from micro blood sample.
Description
Technical field
The invention belongs to Fast Labeling immuno analytical method, be specifically related to the method by specific binding reaction Quick Acquisition object and by labelling technique object being marked from whole blood and monitor, and a kind of instant detection system.
Background technology
Immuno-labelling technique has the advantages such as selectivity is high, highly sensitive, universality is good, mainly comprise radioimmunoassay technique, Immunoenzyme techniques, immunofluorence technic, Chemiluminescence Immunoassay, the probe material labelled antibodies (or antigen) such as fluorescein, isotope or enzyme are adopted to carry out antigen-antibody reaction, by the mensuration to the label in immune complex, reach the object that immune response is monitored.
Instant detection (Point-of-caretesting, POCT) refers to be analyzed at once in sampling location, saves the complex process program of sample when laboratory inspection, obtains class methods of assay fast.In POCT, immune analysis method has been widely used in the quick detection in the fields such as health care, environmental monitoring, anti-terrorism at present, POCT detect conventional to as if whole blood sample, and sample size is few, its method comparatively commonly used realizes being separated of haemocyte and blood plasma by capillary-driven blood through perforated membrane, be separated the blood plasma obtained and under capillarity, enter reaction unit further, complete immune detection.
By convention, blood plasma can be separated from whole blood by centrifuge method, but centrifuge method is very consuming time, needs special equipment, is not suitable in instant test.In order to avoid this problem, general employing has mechanical type filter separated plasma and the red blood cell of micropore filtering medium, a kind of tripping device of similar employing filtering material is disclosed in document CN101918137A, but due to the problem that micropore blocks, this separation method can cause and detect the loss of sample, therefore for only having the POCT of minute quantity sample and inapplicable.
POCT device is owing to being subject to small size, low cost and the restriction required such as easy to use, and when few sample size, accurately cannot control testing process and reject background interference, reliability and the precision of detection reduce greatly.Such as, with absorbing film as the method for sample and reaction reagent carrier in be difficult to accurately control the flow velocity that reaction reagent enters pick-up unit and the time stopped in each specific reaction chamber, and the normal background interference lacking sufficient wash-out and cause to eliminate non-specific adsorption.Based in the detection of micro-fluidic chip, in order to the flow velocity of abundant wash-out, accurately Quality control and reaction reagent and reaction time often need complicated valve control structure, add the volume of device, cost and complexity, reduce the reliability of detection system simultaneously.
Therefore only have the large-scale instrument of centralab just to have the ability of high sensitivity, high duplication and accurate quantitative analysis, can obtain experimental data accurately, but it is compared with method for quick, equipment volume is large, and cost is high, and detection time is long.
Super-paramagnetism nano microballoon (superparamagneticsnanospheres/particles/beads) is called for short magnetic bead, is a kind of novel functional material first developed in 1978 by SenyeiAE.Its inside is a magnetic core, and thus under the effect of external magnetic field, microballoon can displacement; And microballoon outside is one deck clad, surface distributed many reactive groups, can with the biochemical reagents generation couplings such as cell, protein, nucleic acid, enzyme.Magnetic bead for biochemical analysis has following characteristics: 1) superpower paramagnetism, just refer to and to assemble rapidly under the existence in magnetic field, leaving magnetic field can be dispersed, do not occur that gathering manifests phenomenon, in document CN102333595A, namely openly one makes multiple paramagnetic particle reunite in small volumes of liquids also equally distributed method subsequently; 2) suitable particle diameter and particle size distribution range is narrow, makes microballoon have enough strong magnetic responsiveness, because particle diameter is too large, sedimentation can not occur again; 3) there is abundant surface active groups, so as microballoon can with biochemical substances coupling, and to realize and being separated by testing sample under the effect of outside magnetic field.
Summary of the invention
Technical matters to be solved by this invention is that current instant search method sensitivity, stability and repeatability are low, in order to overcome above deficiency, provides a kind of rapid immunoassay method of whole blood and instant detection system.
In order to solve the problems of the technologies described above, technical scheme of the present invention is: the Fast Labeling immune analysis method of described whole blood, comprises the following steps:
1) magnetic capture: the whole blood sample of anti-coagulants process mixes at the first conversion zone with the magnetic bead of load capture antibody, described capture antibody determined antigen in whole blood sample is combined and forms primary antibodie immune complex;
2) blood wash-out: apply magnetic field and the gathering of primary antibodie immune complex, eluent are replaced fluid;
3) mark of antibody is detected: apply magnetic field and magnetic bead is transferred to second reaction zone, adopt immuno-labelling technique, the determined antigen detecting antibody and primary antibodie immune complex reacts the anti-immune complex of formation two;
4) wash-out of antibody is detected: apply magnetic field and assembled by two anti-immune complexs, add eluent by unconjugated detection antibody elution; With
5) detect: apply magnetic field and two anti-immune complexs after step 4) cleaning are moved into detection zones and detect.
The mechanical type filter separation method of blood has been abandoned, as by multi-hole filtering film in analytical approach of the present invention.Although multi-hole filtering film is more suitable for the demand of POCT compared with centrifugal separation technology, but the blood plasma after being separated is generally less than 15% of initial blood volume, large class object is lost in detachment process, thus the sensitivity of detection system is reduced, and the use of multi-hole filtering film adds the complexity of system, reduce the repeatability of detection.And the magnetic bead of load capture antibody directly mixes with anticoagulated blood by the present invention, capture antibody combines with object in blood, then assembled fixing by magnetic field, by eluent, it is separated from blood, decrease the object loss in detachment process, substantially increase detection sensitivity.
Preferably, in step 1) and/or step 3) in, make magnetic bead rapid movement by magnetic force change, promote between capture antibody and determined antigen and/or the combination detected between antibody and determined antigen.By the change of magnetic force, the change of such as magnetic pole, the applying in magnetic field and cancel, thus magnetic bead rapid movement in system.
Preferably, described immuno-labelling technique is selected from enzyme immunolabelling technique (ELISA) or Chemiluminescence Immunoassay (CLIA).Described enzyme immunolabelling technique or Chemiluminescence Immunoassay all can be used for quantitative measurement.
Described enzyme immunolabelling technique refers to and the antigen-antibody reaction that enzymic-labelled antibody or enzymic-labelled antibody carry out then produces color reaction by enzyme-to-substrate, for quantitative measurement.
Described Chemiluminescence Immunoassay utilizes chemistry or bio-luminescence system as the indication mechanism of antigen-antibody reaction, so as to the method for quantitative detectable antigens or antibody, luminescent substance can directly as the label of antigen-antibody, also can in a free form in catalyzer (enzyme) and the antigen of assistant mark or the luminescence-producing reaction of antibody.
Preferably, the volume of described whole blood sample is 2 ~ 20 microlitres.Owing to have employed new separation of whole blood method, the volume of whole blood sample is greatly reduced, the POCT being very beneficial for miniaturization detects.
Present invention also offers a kind of instant detection system, comprise micro-fluidic chip, be arranged on magnetic field generation device and the pick-up unit of next side or two sides on micro-fluidic chip, described micro-fluidic chip comprises microchannel, be arranged on the magnetic bead of the load capture antibody in microchannel, be arranged on the waste liquid outlet of one end, microchannel and be arranged on the eluent liquid storage tank of the microchannel other end, described microchannel is isolated into the first conversion zone by separating mechanism stage, second reaction zone and detection zone, described first conversion zone arranges blood injection port, the liquid storage tank that described second reaction zone is relevant to mark desired substance is connected, described pick-up unit detects material in detection zone.
Detection system provided by the invention no longer relies on the flowing that capillary force drives sample.Rely on capillary drive and need carry out surface treatment to microchannel, and this surface treatment is often difficult to accurate Quality control flow velocity and the residence time at each ad-hoc location, and this surface modification is difficult to permanent maintenance, thus cause the repeatability of detection and stability low.The present invention accurately guides movement and the location of sample by the control in magnetic field, ensures that sample is in the residence time of ad-hoc location, avoids cross jamming by separating mechanism, and system is simple and control easy, system stability and reproducible.
Preferably, described separating mechanism is valve or inserted sheet.Partition method is simple, Be very effective.
Preferably, described waste liquid outlet is arranged on one end residing for the first conversion zone on microchannel, and described eluent liquid storage tank is arranged on one end residing for detection zone on microchannel.
Preferably, be also provided with physics slope between the first conversion zone and second reaction zone in described microchannel, the vertical barrier height on described physics slope is 0.1-2mm.Described physics slope prevents remaining retentate from flowing into second reaction zone from the low portion of the first conversion zone, only has the magnetic bead shifted by magnetic field can enter second reaction zone from the first conversion zone smoothly, reduce further the background noise of system.
Preferably, the volume of the described first conversion zone part of described first conversion zone part is 2-40 microlitre.
Preferably, the magnetic field of described magnetic field generation device is provided by permanent magnet or electromagnet.
Although it is pointed out that whole blood test adopts the technique effect of the technical scheme of invention the most remarkable, those skilled in the art can understand, and the sample in serum or blood plasma source is applicable to technical scheme provided by the invention equally.
Technical scheme provided by the invention has the features such as highly sensitive, reproducible, cost is low, quantitatively can detect object fast from micro blood sample.
Accompanying drawing explanation
Fig. 1 is the structural representation of instant detection system one embodiment of the present invention;
Fig. 2 is the structural representation of microchannel one of the present invention embodiment;
Fig. 3 is the process schematic of step 1) in labelling immunoassay method one embodiment of whole blood of the present invention;
Fig. 4 is step 2 in labelling immunoassay method one embodiment of whole blood of the present invention) process schematic;
Fig. 5 is the process schematic of step 3) in labelling immunoassay method one embodiment of whole blood of the present invention;
Fig. 6 is the process schematic of step 4) in labelling immunoassay method one embodiment of whole blood of the present invention;
Fig. 7 is the process schematic of step 5) in labelling immunoassay method one embodiment of whole blood of the present invention.
Shown in figure: 11-microchannel, 111-separating mechanism, 112-first conversion zone, 113-second reaction zone, 114-detection zone, 115-physics slope, 116-detects antibody liquid storage tank, 117-chromophoric substrate liquid storage tank, 118-detection window, the magnetic bead of 12-load capture antibody, 13-waste liquid outlet, 14-eluent liquid storage tank, 15-blood injection port, the upper electromagnet of 21-, 22-lower electromagnet, 31-whole blood sample, 32-determined antigen, 33-detects antibody, the anti-immune complex of 34-bis-, 35-primary antibodie immune complex.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in detail:
In embodiment, the source of sample, reaction reagent and detecting instrument is as follows:
Whole blood sample: 30 parts of blood with prostate specific antigen (PSA), antigen concentration is respectively 0pg/ml, 5pg/ml, 10pg/ml, 50pg/ml, 150pg/ml, 500pg/ml, 1.5ng/ml, 5ng/ml, 50ng/ml and 150ng/ml, often kind of concentration 3 samples, prostate specific antigen (PSA) standard items (R & DSystems company of the U.S.) that whole blood sample is measured by difference join in the blood of the Healthy People after ethylenediamine tetraacetic acid (EDTA) anti-freezing process and obtain.Available anti-freezing treating agent also comprises citric acid, heparin etc.
The magnetic bead of the load capture antibody adopted in method or system is the NanoLink of solulink company of the U.S.
tMstreptavidinMagneticBeads(diameter about 0.8 μm) and MagnaLink
tMstreptavidinMagneticBeads(diameter about 2.8 μm), the biotinylated capture antibody for PSA (purchased from American R & DSystems company) realizes the load of capture antibody on magnetic bead by directly mixing with magnetic bead, enzyme mark detects the same purchased from American of the antibody for the PSA R & DSystems company that antibody is horseradish peroxidase-labeled, chromophoric substrate (SuperSignalELISAFemtoMaximumSensitivitySubstrate) the purchased from American Pierce company of horseradish peroxidase.
Photomultiplier (PMT) is purchased from Japanese Bin Song company.
As illustrated in fig. 1 and 2, a kind of instant detection system of the present invention comprises micro-fluidic chip, is arranged on the magnetic field generation device of micro-fluidic chip both up and down and pick-up unit (not shown).
Described micro-fluidic chip comprises microchannel 11, be arranged on the magnetic bead 12 of the load capture antibody in microchannel 11, waste liquid outlet 13 and eluent liquid storage tank 14, described microchannel 11 is from left to right isolated into the first conversion zone 112 by separating mechanism 111 stage, second reaction zone 113 and detection zone 114, described waste liquid outlet 13 is arranged on the left end on microchannel 11 residing for first conversion zone 112, described eluent liquid storage tank 14 is arranged on the right-hand member on microchannel 11 residing for detection zone 114, physics slope 115 is provided with between described first conversion zone 112 and second reaction zone 113.
The microchannel inner width 1mm of described first conversion zone 112 part, interior high 0.5mm, length 20mm, the vertical barrier height on physics slope 115 is 0.2mm, and the blood flow volume of carrying is 10 microlitres; The microchannel inner width 1mm of the first conversion zone 112 part in another embodiment, interior high 4mm, length 10mm, the vertical barrier height on physics slope 115 is 2mm, and the blood flow volume of carrying is 40 microlitres; The microchannel inner width 1mm of the first conversion zone 112 part in another embodiment, interior high 0.2mm, length 10mm, the vertical barrier height on physics slope 115 is 0.1mm, and the blood flow volume of carrying is 2 microlitres.
Described first conversion zone 112 arranges blood injection port 15, and described second reaction zone 113 is provided with and detects antibody liquid storage tank 116 and chromophoric substrate liquid storage tank 117, and described detection zone 114 is provided with detection window 118.It is to be noted, detect antibody liquid storage tank 116 and all belong to the liquid storage tank relevant to mark desired substance with chromophoric substrate liquid storage tank 117, this liquid storage tank can according to circumstances increase or reduce, when such as Chemiluminescence Immunoassay or immunofluorence technic, may only need luminescent substance to mark or fluorescently-labeled detection antibody liquid storage tank, in addition, chromophoric substrate liquid storage tank 117 also can be arranged on detection zone 114, add chromophoric substrate again when immune complex moves in detection zone 114, detect after colour developing.
Those skilled in the art are very clear, and waste liquid outlet 13, eluent liquid storage tank 14, blood injection port 15, detection antibody liquid storage tank 116 and chromophoric substrate liquid storage tank 117 are all communicated with microchannel 11 by one-way valve structures, can realize the flowing of unidirectional liquid.
Described separating mechanism 111 is inserted sheet, and inserted sheet inserts microchannel 11 as required and intercepts two adjacent regions, prevents mutual interference, when sample and reaction reagent transfer need adjacent area to communicate, only inserted sheet need be extracted out.Adjacent area arranges valve arrangement also can realize similar effect, and plate-inserting structure is simpler, and cost is lower.
The magnetic field of described magnetic field generation device is provided by electromagnet, it comprises the upper electromagnet 21 be arranged on above microchannel 11 and the lower electromagnet 22 be arranged on below microchannel 11, upper electromagnet 21 pairs of magnetic beads apply the first magnetic field, lower electromagnet 22 pairs of magnetic beads apply the second magnetic field, the pole axis in described first magnetic field is parallel with microchannel or vertical, the pole axis in the second magnetic field and the angle of microchannel can change between 0 ~ 90 °, described upper electromagnet 21 and lower electromagnet 22 can prolong direction, microchannel 11 and move, thus driving and control magnetic bead move on request and locate in microchannel 11.Electromagnet applies fast by the control of electric current and cancels magnetic field, and effect is better, but permanent magnet also can by the mobile relative position with microchannel, magnetic bead is in or the magnetic field that departs from permanent magnet realizes similar technique effect.
Described pick-up unit comprises photomultiplier (PMT), fluorospectrophotometer, charge-coupled device (CCD) imageing sensor and cmos image sensor.
The labelling immunoassay procedure of whole blood of the present invention is described in conjunction with above-mentioned instant detection system:
Step 1), as shown in Figure 3, insert inserted sheet 111, by the first conversion zone 112 and second reaction zone domain separation 113, whole blood sample 31 joins in microchannel 11, and the capture antibody of the determined antigen 32 (i.e. PSA) in whole blood sample 31 on the magnetic bead 12 of load capture antibody is combined and forms primary antibodie immune complex 35; Applying first magnetic field and the second magnetic field is replaced by upper electromagnet 21 and lower electromagnet 22 pairs of magnetic beads 12, magnetic bead moves up and down fast in microchannel 11, accelerate the combination with determined antigen 32, shown in figure, the pole axis in the first magnetic field is parallel with microchannel 11, and the pole axis in the second magnetic field is parallel with microchannel 11.
Step 2), as shown in Figure 4, the capture antibody of determined antigen 32 (i.e. PSA) on the magnetic bead 12 of load capture antibody is combined completely, apply the first magnetic field, primary antibodie immune complex 35, by the upside of adsorpting aggregation in microchannel 11, extracts inserted sheet 111 out, pass into eluent from right to left, described eluent is TBS+0.05%Tween-20+0.05%BSA, and eluent substituted for the fluid in whole blood sample, and determined antigen 32 is retained.It may be noted that, also can apply the second magnetic field primary antibodie immune complex 35 is gathered in downside carry out wash-out.
Step 3), as shown in Figure 5, lower electromagnet 22 pairs of magnetic beads apply the second magnetic field, primary antibodie immune complex 35 adsorpting aggregation is to the downside of microchannel 11, mobile lower electromagnet 22 from left to right moves along microchannel 11, primary antibodie immune complex 35 arrives second reaction zone 113 through one section of physics slope 115 upwards, and the pole axis in the second magnetic field and the angle of microchannel 11 become 30 °; Further, insert inserted sheet 111, by the first conversion zone 112 and second reaction zone 113, and cut off between second reaction zone 113 and detection zone 114, pass into the detection antibody 33 of enzyme labeling, detect antibody 33 and react the anti-immune complex 34 of formation two with the determined antigen of primary antibodie immune complex 35, in movement, magnet 21 and lower electromagnet 22 are to second reaction zone 113, applying first magnetic field and the second magnetic field are replaced to magnetic bead, magnetic bead moves up and down fast in microchannel 11, accelerates the formation of two anti-immune complexs 34.
Step 4), as shown in Figure 6, applies the second magnetic field, two anti-immune complexs 34, by the downside of adsorpting aggregation in microchannel 11, are extracted inserted sheet 111 out, are passed into eluent and removed from system by unreacted detection antibody 33, after wash-out repeatedly, pass into chromophoric substrate, develop the color with enzyme effect.
Step 5), as shown in Figure 7, mobile lower electromagnet 22 from left to right moves along microchannel 11, two anti-immune complexs 34 arrive detection zone 114, the pole axis in the second magnetic field and the angle of microchannel 11 become 30 °, pick-up unit carries out chemiluminescence detection by detection window 118, completes whole analytic process.
Adopt analytical approach of the present invention and instant detection system to detect 35 parts of whole blood samples, the sample size of the whole blood sample of often kind of concentration is 20 microlitres.
In whole blood sample, the result of PSA is as shown in table 1 below.Detection sensitivity can be low to moderate 10pg/ml, and the dynamic range of detection is 10pg/ml to 150ng/ml, detects CV value lower than 15% in the scope of more than 50pg/ml, more than 500pg/ml scope in detect CV value lower than 10%.
Table 1
| Concentration (pg/ml) | Relative light unit (RLU) | Corrected value | The coefficient of variation (CV) |
| 0 | 3.8 | ||
| 5 | 42.4 | 4.4 | 30.2％ |
| 10 | 42.6 | 4.6 | 25.2％ |
| 50 | 68.3 | 30.3 | 14.8％ |
| 100 | 130.3 | 92.3 | 11.1％ |
| 500 | 361.4 | 323.4 | 9.8% |
| 1500 | 1208.8 | 1170.8 | 7.3% |
| 5000 | 3892.2 | 3854.2 | 7.1% |
| 50000 | 11177.1 | 11139.1 | 3.3% |
| 150000 | 15409.4 | 15371.4 | 4.6% |
Claims (10)
1. a Fast Labeling immune analysis method for whole blood, is characterized in that, comprise the following steps:
1) magnetic capture: the whole blood sample of anti-coagulants process mixes at the first conversion zone with the magnetic bead of load capture antibody, described capture antibody determined antigen in whole blood sample is combined and forms primary antibodie immune complex;
2) blood wash-out: apply magnetic field and the gathering of primary antibodie immune complex, eluent are replaced fluid;
3) mark of antibody is detected: apply magnetic field and magnetic bead is transferred to second reaction zone, adopt immuno-labelling technique, the determined antigen detecting antibody and primary antibodie immune complex reacts the anti-immune complex of formation two;
4) wash-out of antibody is detected: apply magnetic field and assembled by two anti-immune complexs, add eluent by unconjugated detection antibody elution; With
5) detecting: apply magnetic field by step 4) two anti-immune complexs after cleaning move into detection zones and detect.
2. the Fast Labeling immune analysis method of a kind of whole blood according to claim 1, it is characterized in that, in step 1) and/or step 3) in, make magnetic bead rapid movement by magnetic force change, promote between capture antibody and determined antigen and/or the combination detected between antibody and determined antigen.
3. a kind of Fast Labeling immune analysis method of whole blood according to claim 1 or 2, it is characterized in that, described immuno-labelling technique is selected from enzyme immunolabelling technique (ELISA) or Chemiluminescence Immunoassay (CLIA).
4. a kind of Fast Labeling immune analysis method of whole blood according to claim 1 or 2, it is characterized in that, the volume of described whole blood sample is 2 ~ 20 microlitres.
5. an instant detection system, it is characterized in that, comprise micro-fluidic chip, be arranged on magnetic field generation device and the pick-up unit of next side or two sides on micro-fluidic chip, described micro-fluidic chip comprises microchannel, be arranged on the magnetic bead of the load capture antibody in microchannel, be arranged on the waste liquid outlet of one end, microchannel and be arranged on the eluent liquid storage tank of the microchannel other end, described microchannel is isolated into the first conversion zone by separating mechanism stage, second reaction zone and detection zone, described first conversion zone arranges blood injection port, the liquid storage tank that described second reaction zone is relevant to mark desired substance is connected, described pick-up unit detects material in detection zone.
6. a kind of instant detection system according to claim 5, it is characterized in that, described separating mechanism is valve or inserted sheet.
7. a kind of instant detection system according to claim 5 or 6, it is characterized in that, be also provided with physics slope in described microchannel between the first conversion zone and second reaction zone, the vertical barrier height on described physics slope is 0.1-2mm.
8. a kind of instant detection system according to claim 7, it is characterized in that, the volume of described first conversion zone part is 2-40 microlitre.
9. a kind of instant detection system according to claim 5,6 or 8, it is characterized in that, described waste liquid outlet is arranged on one end residing for the first conversion zone on microchannel, and described eluent liquid storage tank is arranged on one end residing for detection zone on microchannel.
10. a kind of instant detection system according to claim 9, it is characterized in that, the magnetic field of described magnetic field generation device is provided by permanent magnet or electromagnet.
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