CN106053794A - Reagent card for accurately detecting test object, kit and application - Google Patents
Reagent card for accurately detecting test object, kit and application Download PDFInfo
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- CN106053794A CN106053794A CN201610503539.8A CN201610503539A CN106053794A CN 106053794 A CN106053794 A CN 106053794A CN 201610503539 A CN201610503539 A CN 201610503539A CN 106053794 A CN106053794 A CN 106053794A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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
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Abstract
The invention discloses a reagent card for accurately detecting a test object, a kit and application. A sample pad (1), an antibody carrier film (2) and water absorption paper (3) are successively adhered to a PVC bottom plate (6) from one end to the other end, wherein the sample pad is a sample loading area, and the sample pad is sprayed with a test object antibody-1 coupling marker and a biotin coupling marker; the antibody carrier film is provided with three lines which are successively a line T1 (4), a line T2 and a line C (5) from the sample pad to the water absorption paper, or are the line T1 (4), the line C (5) and the line T2; the line T1 is a detection line, and a test object antibody 2 is smeared on the position of the line T1; an antigen of the test object is smeared on the position of the line T2; streptavidin is smeared on the position of the line C. By adopting the reagent card, the false negative rate caused by the high-dose hook effect can be greatly reduced, the clinical detection accuracy is improved, and the adverse impact of the hook can be effectively solved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of for solving the reagent card of high dose hook effect, examination
Agent box and purposes.
Background technology
Immunochromatography experiment clinically, when high dose occurs, it is easy to high dose hook effect occurs, i.e. HD-
HOOK effect.It refers in two-site sandwich immunity test, the high dose section of its dose-effect curve, linear trend be not in
Prolong after platform-like is unlimited, but curved in being turned under, like a hook or a reaping hook, according to this phenomenon, referred to as " HD-
HOOK " effect.The existence of this effect causes detected sample can not correctly distinguish owing to its concentration is beyond detection kit
The range of linearity or concentration own are exactly that this value causes this phenomenon, to such an extent as to test mistaken diagnosis, especially cause probability of false negative to rise.
The method that there is presently no the impact effectively avoiding HOOK effect to produce, great majority, by increasing coating protein amount, increase mark
Note albumen or metal spraying amount improve this situation, or play assosting effect by the adjustment of technical recipe, but fail to
Well improve the impact that this effect is brought.
Summary of the invention
It is an object of the invention to provide the reagent card of a kind of accurate detection determinand.
For achieving the above object, the present invention provides a kind of reagent card for accurately detecting determinand, it is characterised in that
It is bonding with sample pad (1), antibody carrier film (2) and absorbent paper (3), wherein sample from one end toward the other end successively on PVC base plate (6)
Product pad is sample application zone, and described sample pad is sprayed with determinand antibody 1 coupling label and biotin coupling label；
There are three lines on described antibody carrier film, from the past absorbent paper direction near sample pad, are T1 line (4) successively, T2
Line and C line (5)；Or be T1 line (4) successively, C line (5) and T2 line；Described T1 line is detection line, is to be sprayed by determinand antibody 2
At T1 line position；Described T2 line is that the antigen of thing to be checked is sprayed on T2 line position；Described C line is that Streptavidin is sprayed on C
Line position.
Further, described sample pad is glass fibre membrane, polyester film or Whole Blood Filtration film.
Further, described sample pad being sprayed with pepsin antibody 1 coupling label and biotin coupling label, they are two years old
Person's volume ratio is 1:1, and is sprayed on sample after being diluted to working concentration with pH7.4, the 0.01M PBS containing the BSA that mass ratio is 1%
On pad.
Further, described determinand antibody 1 coupling label and biotin coupling label spray by drawing film metal spraying instrument
It is coated with.
Further, described label is that rare earth elements europium is rolled on silica dioxide granule or fluorescein, latex fluorescent microsphere
Or gold colloidal.6, for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that described antibody carrier film
For nitrocellulose membrane or nylon membrane.
Further, described determinand antibody 2 is sprayed on T1 line position by drawing film metal spraying instrument；Described determinand antigen leads to
Cross a stroke film metal spraying instrument and be sprayed on T2 line position；Described C line is that Streptavidin is drawn film metal spraying instrument and is sprayed on C line position.
Further, described determinand is hormone or high molecular weight protein.
The present invention also provides for a kind of test kit, it is characterised in that containing the described reagent card for accurately detecting determinand.
The present invention also provides for the described reagent card for accurately detection determinand or described test kit for accurately detection
The purposes of the reagent card of determinand.
The reagent card of the present invention is to have done technological improvement in the most traditional method, and former traditional method immunochromatography carries
Detection line T and C line it is coated on film, existing in order to improve upper limit of detection and avoid hook effect, it is coated to be checked after detection line T1
The antigen T2 of thing, and carry out subsection calibration in standard curve calibration.Accordingly even when in the case of high dose, also can have good
Linearity curve, makes upper limit of detection be improved, so that the false negative rate caused by high dose hook effect is substantially reduced, improves
The accuracy of Clinical detection, effectively solves the impact that HOOK produces.
The position of this T2 line of the method for the present invention can be (can be certain according to actual experiment) before C line or after C line.
The present invention solves hook effect immunochromatography and quantitatively calibrates and the method for result conversion: calibration, with the mark of determinand
Quasi-product are diluted to required linear concentration, and strong with the fluorescence of T1, T2 and C line after fluorescence immunity analyzer collection specific reaction
Degree.Concentration, T1 and T2 are taken the logarithm, and using log concentration as abscissa, respectively using T1 and T2 as vertical coordinate linear
Section matched curve, concentration-T1 double-log matched curve is curve 1, and concentration-T2 double-log matched curve is curve 2.Result is changed
Calculating, test sample, collect the fluorescent value of T1, T2, C line, take the logarithm T2 comparing with section, less than section, is brought into by logarithm T2
Y in curve 2, obtains X antilogarithm and is concentration；If logarithm T2 is more than section, brings logarithm T1 into Y in curve 1, obtain X
Antilogarithm is concentration.Described section is log concentration-logT1 and log concentration-logT2 linear dependence R2More than 0.95, dense
The intermediate value that degree occurs simultaneously, the logarithm of corresponding T2 fluorescence intensity.This section is as judging to bring curve 1 or curve 2 conversion into
Foundation.
The present invention solves the principle of hook effect: testing sample is resisted with coated determinand in sample pad by chromatography effect
The label of body 1 coupling combines, and under capillary action, passes sequentially through sample pad, nitrocellulose filter, and and nitrocellulose filter
Determinand antibody 2 specific reaction fixing on T 1 line, forms double antibody immuno-sandwich complex, and complex is with testing sample
Concentration raises and raises, when being increased to a certain degree start no longer to raise and slowly decline.Unnecessary do not react with sample to be tested
The label chromatography surveying thing antibody 1 coupling forms specific reaction, specific reaction to the determinand antigen on T2 line, with T2 line
Along with the rising of testing sample concentration is gradually reduced.In chromatography process, Quality Control biotin coupling label is also with sample
Moving and advance, and the Avidin fixed at nitrocellulose filter C line position captures, the capture of C line position is fixing, no
Impact with individual diversity XOR testing sample concentration.Unreacted microsphere continues to move to adsorptive pads position.Pass through fluorescence immunoassay
Analyser, to T1, T2, C line fluorescence signal collection, analyzes determined antigen concentration in testing sample.
Accompanying drawing explanation
Fig. 1 is the structure structural map of reagent card article of the present invention.
Fig. 2 is the HCG standard concentration canonical plotting at 10-40000mIU/mL.
Fig. 3 is the HCG standard concentration canonical plotting at 40000-200000mIU/mL.
Fig. 4 is the AFP standard concentration canonical plotting at 5-400ng/mL.
Fig. 5 is the AFP standard concentration canonical plotting at 400-2000ng/mL.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is shown in the drawings, the most from start to finish
Same or similar label represents same or similar element or has the element of same or like function.Below with reference to attached
The embodiment that figure describes is exemplary, it is intended to is used for explaining the present invention, and is not considered as limiting the invention.Embodiment
In unreceipted concrete technology or condition person, according to the technology described by the document in this area or condition or according to the description of product
Book is carried out.Agents useful for same or instrument unreceipted production firm person, be can by city available from conventional products.
The preparation of embodiment 1:HCG detection kit (fluorescence immune chromatography method) and detection
HCG antibody 1 coupling fluorescent microsphere and the preparation of biotin coupling fluorescent microsphere: choose 10mg fluorescent microsphere and join
1mL reacts 1h, 12000 turns of 5min and is centrifuged after removing supernatant and adds in the 0.01M pH7.4 PBS solution containing 5 mass % glutaraldehydes
1mgHCG antibody 1 uniformly mixes, in 4 DEG C react 12h, 12000 turns of 5min centrifugal after remove supernatant, and with 1mL 0.01M pH7.4
PBS is resuspended, forms HCG antibody 1 coupling fluorescent microsphere；In kind, selection takes 10mg fluorescent microsphere addition 1mL containing 5 matter
The 0.01M pH7.4PBS solution of amount % glutaraldehyde reacts 1h, 12000 turns of 5min be centrifuged after removing supernatant and add 1mg biotin
Mixing, reacts 12h in 4 DEG C, removes supernatant after 12000 turns of 5min are centrifugal, and resuspended with 1mL 0.01M pH7.4PBS, forms biology
Element coupling fluorescent microsphere.
The preparation of sample pad: HCG antibody 1 coupling fluorescent microsphere and biotin coupling fluorescent microsphere 1:1 by volume are mixed
Close, and with being that the pH7.4 of 1%BSA, 0.01M PBS is diluted to working concentration (100-5000 times dilutes, according to reality containing mass ratio
Fluorescence intensity and the linear trend of border experiment adjust) it is being coated in sample pad by a stroke film metal spraying instrument.The sample that will be coated
Product are padded on 45 DEG C of 2h and dry.Described sample pad is glass fibre membrane, polyester film or Whole Blood Filtration film.
T1 line is coated the preparation of liquid: with pH7.4,0.01M PBS diluent, HCG antibody 2 is diluted to 1.0mg/ml (0.5-
5mg/ml adjusts according to actual experiment), 2-8 DEG C is standby.
T2 line is coated the preparation of liquid: with pH7.4,0.01M PBS diluent by HCG antigen diluent to 1.0mg/ml (0.5-
5mg/ml adjusts according to actual experiment), 2-8 DEG C is standby.
C line is coated the preparation of liquid: with pH7.4,0.01M PBS diluent, Quality Control thing Avidin is diluted to 1.0mg/ml
(0.5-5mg/ml adjusts according to actual experiment), 2-8 DEG C standby.
Antibody carrier film is coated: by drawing, T1 line is coated liquid by film gold spraying instrument, T2 line is coated liquid and C line is coated liquid and wraps simultaneously
By on NC film, forming T1 line, T2 line and C line, the NC film after this being coated is dried in 45 DEG C of 2h.
Pasting board, cutting are assembled into detection card: according to shown in Fig. 1 by the NC film after being coated of glass fibre membrane and drying, suction
Assembling pasted successively by water paper, and antibody carrier film is positioned at centre, and sample pad and absorbent paper are overlapped in about nitrocellulose filter respectively
Two ends.It is cut into the strip of every width 4mm after being completed with cutting cutter, puts into strip and get stuck, pressure card, it is prepared as detection card.
Under dry environment, lucifuge is standby.
The most 1. it is sample pad；2. it is antibody carrier film；3. for absorbent paper；4. it is T1 line position；5. for T2 line institute
In position；6. it is C line position；7. it is the PVC base plate of band binding agent.
Sample buffer: containing mass ratio be 10% bovine serum albumin, volume ratio is 10%TWEEN-20 and mass ratio is
The 0.05M pH7.4PBS solution of 0.01% sodium azide.2-8 DEG C of preservation.
Detection sample: use human serum or blood plasma, detected in 24 hours, as do not detected in time, and whole blood 2-8 DEG C preservation
7 days, serum or blood plasma-20 DEG C preserved 1 month, and haemolysis and serious lipidemia sample are unavailable.
Testing conditions:
(1) instrument: immunofluorescence analysis instrument；
(2) working environment: room temperature, indoor humidity 45%-80%；
(3) test kit recovers room temperature, and reagent card now surveys existing unpacking, it is to avoid make moist.
Detection method: take out the 2-8 DEG C of sample buffer preserved, recover room temperature, draw 20ul standard substance or clinical sample
Be added in 180-580 μ l sample buffer (concrete buffer volume according to experiment sensitivity ＜ 0.05, testing standard product 5-
200000mIU/mL, linear dependence R2>=0.9801 determines), fully mix, draw 80ul mixed liquor, be added drop-wise to detection card
Adding mouth on, by detection card insert fluorescence immunoassay detecting instrument, show the standard detection on screen by instrument.
Calibration: with the fluorescence intensity of T1, T2 and C line after fluorescence immunity analyzer collection specific reaction.By standard substance
Concentration, the fluorescent value of T1 and T2 are taken the logarithm, and using log concentration as abscissa, respectively using logarithm T1 and logarithm T2 as vertical seat
Mark, concentration-T1 double-log matched curve is curve 1HCG concentration becomes good linear relationship (curve 1 at 10-40000mIU/mL
See Fig. 2), concentration-T2 double-log matched curve is that curve 2HCG concentration becomes good linearly closing at 40000-200000mIU/mL
System's (curve 2 is shown in Fig. 3).The results are shown in Table 1.
Wherein C line is for judging the foundation that this reagent card is the most invalid.If C line fluorescent value less than standard substance detection is
The 30-100% of C line value, then this reagent card is invalid；If C line fluorescent value when standard substance detect C line value ± 30%, then
This reagent card is effective, and result can use；
Result converts: test clinical sample, collects the fluorescent value of T1, T2, C line, and take the logarithm T2 comparing with section, is less than
Section, brings logarithm T2 into Y in curve 2, obtains X antilogarithm and is concentration；If logarithm T2 is more than section, logarithm T1 is brought into
Y in curve 1, obtains X antilogarithm and is concentration.
Figure it is seen that HCG concentration log concentration-logarithm T1 fluorescence in the range of 10-40000ng/mL presents well
Linear relationship, in Fig. 3, HCG concentration log concentration-logarithm T2 fluorescence in the range of 40000-200000ng/mL presents good
Linear relationship, occurs without HOOK effect more than 10000ng/mL concentration.Section seen from table 2 is 4.355.
The testing result table of table 1 standard substance
| Concentration | T1 | T2 | C | Logarithm (concentration) | Logarithm (T1) | Logarithm (T2) |
| 200000 | 20584 | 4102 | 22053 | 5.301 | 4.314 | 3.613 |
| 150000 | 28645 | 6254 | 21056 | 5.176 | 4.457 | 3.796 |
| 100000 | 34128 | 9347 | 22047 | 5.000 | 4.533 | 3.971 |
| 80000 | 35627 | 11234 | 20298 | 4.903 | 4.552 | 4.051 |
| 50000 | 34362 | 19325 | 21269 | 4.699 | 4.536 | 4.286 |
| 40000 | 30598 | 22656 | 22125 | 4.602 | 4.486 | 4.355 |
| 20000 | 22541 | 32056 | 21654 | 4.301 | 4.353 | 4.506 |
| 5000 | 14520 | 33055 | 20365 | 3.699 | 4.162 | 4.519 |
| 500 | 5202 | 34106 | 22546 | 2.699 | 3.716 | 4.533 |
| 50 | 2289 | 33451 | 19899 | 1.699 | 3.360 | 4.524 |
| 10 | 1014 | 30574 | 20953 | 1.000 | 3.006 | 4.485 |
Precision: withinrun precision, takes with batch of detection card and buffer, and the blood by same concentration known is parallel
Detect 20 detection cards, calculate CV=9.4%.Betweenrun precision, takes detection card and the buffer of 3 batches, with same
The blood Parallel testing of concentration known 60 detection card, calculates CV=10.8%.It can be seen that the reagent fixture of the present embodiment
Having preferable precision, error is little.
Accuracy: be separately added into HCG standard substance 50000mIU/mL, 100mIU/mL in the sample, the response rate is respectively
105.6% and 103.3%.Within the detection response rate falls into 85%-115%, illustrate that the reagent card of the present invention has good standard
Exactness.
Sensitivity: calculate by the average+2SD of 20 test 0 concentration samples gained log fluorescence values, minimum detection limit ＜
5.0mIU/mL.Illustrate that the reagent card of the present invention has high sensitivity.
Clinical trial: test 40 example clinical samples, Comparative result such as table 2 below:
Table 2 clinical test results table
As can be seen from Table 2, the reagent card of the present embodiment all has preferable accuracy, and high concentration does not haves hook effect.
Embodiment 2:AFP detection kit (fluorescence immune chromatography method)
Compared with Example 1, difference is to replace to HCG antibody 2 AFP antibody 2, and HCG antigen is substituted into AFP for another surreptitiously and resisted
Former, additive method and step are all with embodiment 1.
The testing result table of table 2 standard substance
From fig. 4, it can be seen that AFP concentration log concentration-logarithm T1 fluorescence in the range of 5-400ng/mL presents good
Linear relationship, in Fig. 5, AFP concentration log concentration-logarithm T2 fluorescence in the range of 400-2000ng/mL presents good linear pass
System, occurs without HOOK effect more than 2000ng/mL concentration.Section seen from table 2 is 4.186.
Precision test: withinrun precision, takes with batch of detection card and buffer, with the blood of same concentration known
Parallel testing 20 detection card, calculates CV=8.7%.Betweenrun precision, takes detection card and the buffer of 3 batches, with same
The blood Parallel testing of one concentration known 60 detection card, calculates CV=12.9%.It can be seen that the reagent of the present embodiment
Card has preferable precision, and error is little.
Accuracy testing: be separately added into AFP standard substance 20ng/mL, 500ng/mL in blood sample, the response rate is respectively
106.8% and 101.4%.Within the detection response rate falls into 85%-115%, illustrate that the reagent card of the present invention has good standard
Exactness.
Sensitivity test: calculate by the average+2SD of 20 test 0 concentration samples gained log fluorescence values, minimum detection limit
＜ 3ng/mL.Illustrate that the reagent card of the present embodiment has high sensitivity.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment is example
Property, it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art is without departing from the principle of the present invention and objective
In the case of above-described embodiment can be changed within the scope of the invention, revise, replace and modification.
Claims (10)
1. the reagent card for accurately detection determinand, it is characterised in that past another from one end successively on PVC base plate (6)
One end is bonding with sample pad (1), antibody carrier film (2) and absorbent paper (3), and wherein sample pad is sample application zone, in described sample pad
It is sprayed with determinand antibody 1 coupling label and biotin coupling label；
There are three lines on described antibody carrier film, from the past absorbent paper direction near sample pad, are T1 line (4) successively, T2 line and C
Line (5)；Or be T1 line (4) successively, C line (5) and T2 line；Described T1 line is detection line, is that determinand antibody 2 is sprayed on T1 line
Position；Described T2 line is that the antigen of thing to be checked is sprayed on T2 line position；Described C line is that Streptavidin is sprayed on C line position.
2. for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that described sample pad is glass fibre
Film, polyester film or Whole Blood Filtration film.
3. for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that be sprayed with stomach egg in described sample pad
White enzyme antibody 1 coupling label and biotin coupling label, both volume ratios are 1:1, and with being 1% containing mass ratio
The pH7.4 of BSA, 0.01M PBS is sprayed in sample pad after being diluted to working concentration.
4. for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that the coupling of described determinand antibody 1
Label and biotin coupling label spray by drawing film metal spraying instrument.
5. for accurately detecting the reagent card of determinand described in any one of claim 1-4, it is characterised in that described label is
Rare earth elements europium is rolled on silica dioxide granule or fluorescein, latex fluorescent microsphere or gold colloidal.
6. for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that described antibody carrier film is nitric acid
Fibrous membrane or nylon membrane.
7. for accurately detecting the reagent card of determinand described in claim 1, it is characterised in that described determinand antibody 2 passes through
Draw film metal spraying instrument and be sprayed on T1 line position；Described determinand antigen is sprayed on T2 line position by drawing film metal spraying instrument；Described C
Line is that Streptavidin is drawn film metal spraying instrument and is sprayed on C line position.
8. for accurately detecting the reagent card of determinand described in any one of claim 1-7, it is characterised in that described determinand is
Hormone or high molecular weight protein.
9. a test kit, it is characterised in that containing being used for accurately detecting the reagent card of determinand described in claim 1.
10. it is used for accurately for the test kit described in the accurate reagent card detecting determinand or claim 9 described in claim 1
The purposes of the reagent card of detection determinand.
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| CN201610503539.8A CN106053794A (en) | 2016-06-30 | 2016-06-30 | Reagent card for accurately detecting test object, kit and application |
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| CN109283343A (en) * | 2017-07-21 | 2019-01-29 | 上海吉宣生物科技有限公司 | A kind of near-infrared fluorescent chromatography immune quantitative detection reagent box of anti-Miao Le Shi pipe hormone |
| CN110596380A (en) * | 2019-09-20 | 2019-12-20 | 成都艾科斯伦医疗科技有限公司 | Method for eliminating HOOK effect and immunochromatography detection card thereof |
| CN113759129A (en) * | 2021-09-26 | 2021-12-07 | 北京倍肯恒业科技发展股份有限公司 | Rapid detection card for leucocyte-bound C-reactive protein and preparation method thereof |
| EP3904878A4 (en) * | 2018-12-28 | 2022-09-07 | Proteometech Inc. | Immunochromatography strip for pregnancy diagnosis with multiple test lines, and pregnancy diagnosis kit comprising same |
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