CN108593935A - A kind of inflammation quickly detects colloidal gold strip - Google Patents
A kind of inflammation quickly detects colloidal gold strip Download PDFInfo
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- CN108593935A CN108593935A CN201810435358.5A CN201810435358A CN108593935A CN 108593935 A CN108593935 A CN 108593935A CN 201810435358 A CN201810435358 A CN 201810435358A CN 108593935 A CN108593935 A CN 108593935A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
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Abstract
The invention discloses a kind of inflammation quickly to detect colloidal gold strip, including test strips bottom plate, and sequentially mutual overlapped sample pad, the gold pad of label, nitrocellulose filter and blotting paper on test strips bottom plate, the gold pad of the wherein described label is coated with the antibody that can be specifically bound with antigen PCT to be checked and IL 6, is equipped with the T1 lines of coating 6 antibody of IL on the nitrocellulose filter successively, is coated with the T2 lines of PCT antibody and is coated with the C lines of sheep anti-mouse antibody.Colloidal gold strip of the present invention can detect PCT and IL 6 simultaneously, and easily prepared, easy to operate, high sensitivity, efficient, accuracy is good.
Description
Technical field
The invention belongs to clinical medical inspection fields, and in particular to a kind of inflammation quickly detects colloidal gold strip.
Background technology
Interleukin-6 (IL-6) is mainly to be generated by macrophage, T lymphocytes, bone-marrow-derived lymphocyte, is had a variety of multiple
The polypeptide type cytokines of miscellaneous physiological function.When body is inflamed reaction or infection, virus, endotoxin, tumor necrosis factor
The cytokine profiles such as son can induce body and generate IL-6.IL-6 biological actions are mainly reflected in induction B cell differentiation and generate
Immunoglobulin promotes T cell proliferation growth, enhances differentiation and its Antineoplastic effect of haemocyte, promote marrow hemopoietic stem cells
Proliferation etc..IL-6 has been acknowledged as a kind of important proinflammatory cytokine at present, participates in body inflammatory reaction and anti-infectious function.
Studies have shown that IL-6 cooperates with the pathologic process for participating in wound and inflammation disease with cytokine profiles under infection conditions, grind
Study carefully personnel and clinician thinks the diagnosis index that in infectious diseases IL-6 is more more sensitive than c reactive protein.
Procalcitonin (PCT) is the propetide of calcitonin, and PCT is secreted by parafollicular cells of thyroid gland under physiological conditions, by cell
The active constituent formed after interior proteolytic enzymes hydrolize, stability is preferable, and content is micro in vivo, can be fast simple
Carry out biochemistry detection.And yield increases PCT in vivo under pathologic condition, research shows that in severe bacterial infections, carefully
Bacterium endotoxin can induce monocyte and macrophage, lung, the lymphocyte of enteron aisle and endocrine cell synthesis and secretion in liver
PCT, with deepening continuously for clinical research, the collection and analysis of various clinical datas, PCT as bacterial infection disease diagnosis and
The conventional index of antidiastole will be used widely.
IL-6 ratios PCT can be earlier be diagnosed to be infection, faster reflection antibiosis extract for treating effect.IL-6 when inflammatory reaction
Level it is rapid increase, but decline also very fast, the explanation of clinical effectiveness is relatively prudent.For Small Cell Lung Cancer, thyroid gland
The patients such as cephaloma, duration or severe cardiogenic shock, duration organ perfusion's exception will differentiate since its PCT can be increased
Diagnosis cell infect and when pyemia can joint-detection IL-6, improve accuracy rate, avoid false positive.PCT and IL-6 are as acute
Inflammation Testing index, since the two respectively has the characteristics that oneself, this two indexs of joint-detection in process of clinical application
Different effects can be played, medical diagnosis on disease, to judge that disease progression has the function of positive to assisting.In the disease of severe pneumonia
In diagnosis, the joint-detection of PCT and IL-6 has important clinical meaning, research shows that the two be sensibility and specificity compared with
Good inflammatory parameters, joint-detection can assess the severity of bacterium infection, judge that the handplay of disease, and evaluation antibiotic make
With effect and clinic diagnosis is instructed, while need to be analyzed in conjunction with the clinical data of patient.In conclusion examining in clinical disease
In disconnected therapeutic process, in the case that based on patients clinical data joint-detection PCT and IL-6, contribute to clinician for bacterium
The diagnosis of infectious diseases, treatment and the prediction for prognosis, but yet there are no currently on the market joint quickly detection PCT and
The immunochromatography diagnostic test product of IL-6 also lacks the patent of related research and development product.
Invention content
In view of the deficiencies of the prior art, PCT and IL-6, and easily prepared, behaviour can be detected simultaneously the present invention provides a kind of
Make simplicity, high sensitivity, inflammation efficient, that accuracy is good and quickly detects colloidal gold strip.
The present invention adopts the following technical scheme that.
A kind of inflammation quickly detects on colloidal gold strip, including test strips bottom plate and test strips bottom plate sequentially mutually
Sample pad that overlapped, the gold pad of label, nitrocellulose filter and blotting paper, it is characterised in that the gold pad of the label is coated with energy
The antibody specifically bound with antigen PCT to be checked and IL-6 is equipped with coating IL-6 antibody on the nitrocellulose filter successively
T1 lines, the T2 lines for being coated with PCT antibody and the C lines for being coated with sheep anti-mouse antibody.
As optimal technical scheme, the antibody specifically bound with antigen PCT to be checked and IL-6 is anti-for monoclonal
One kind in body, polyclonal antibody, antibody fragment or chimeric antibody.
As optimal technical scheme, the preparation method of the test strips includes the following steps：
(1) preparation of colloidal gold
It is that 0.04% aqueous solution of chloraurate is heated to boiling to take mass fraction, and the lower mass fraction that is added of agitation later is 2%
Trisodium citrate aqueous solution, continue to boil 5-15min, it is cooling after with distilled water constant volume, obtain colloid gold particle；
(2) colloid gold label
IL-6 antibody marks：The colloid gold particle in step (1) is taken, the potassium carbonate buffer of 0.2M is first added, adds
IL-6 monoclonal antibodies mix well reaction 10-30min, are eventually adding the BSA solution that mass fraction is 10% and are closed,
5-15min is reacted, supernatant is abandoned in centrifugation, and suspension is added in precipitation up to IL-6 antibody marking fluids；
PCT antibody marks：The colloid gold particle in step (1) is taken, the potassium carbonate buffer of 0.2M is first added, adds
PCT monoclonal antibodies mix well reaction 10-30min, are eventually adding the BSA solution that mass fraction is 10% and are closed, instead
It answers 5-15min, centrifugation to abandon supernatant, suspension is added in precipitation up to PCT antibody marking fluids；
It is 1 by volume by IL-6 antibody marking fluid and PCT antibody marking fluid:1 mixing is uniform, uniformly paving to glass fibre
On, up to the gold pad of label after drying；
(3) film is drawn
IL-6 draws film：Take IL-6 antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4,
It is later T1 lines with drawing film instrument and drawing on nitrocellulose filter；
PCT draws film：Take PCT antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4, it
It is afterwards T2 lines with drawing film instrument and drawing on nitrocellulose filter；
C lines：Take sheep anti-mouse antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4, later
It is C lines with drawing film instrument and drawing on nitrocellulose filter；
Above-mentioned IL-6 antibody, PCT antibody, sheep anti-mouse antibody are drawn with film instrument is drawn on same nitrocellulose filter, dry
Afterwards up to the nitrocellulose filter of change film process；
(4) it assembles
The sample pad handled well, the gold pad of label, nitrocellulose filter and blotting paper are assembled in successively on PVC bottom plates,
Colloidal gold strip is quickly detected up to inflammation of the present invention.
As optimal technical scheme, the configuration method of suspension is in the step (2)：Take Tris 0.6g, casein-sodium
0.5g, PVP 0.6g, sucrose 10g, PEG 0.2g, 200ml is settled to distilled water.
Description of the drawings
Fig. 1 is the structural schematic diagram that patent inflammation of the present invention quickly detects colloidal gold strip；
Yin and yang attribute result figure when Fig. 2 is using colloid gold test strip provided by the present invention detection IL-6/PCT.
Specific implementation mode
The present invention will be further described in the form of specific embodiment below in conjunction with the accompanying drawings, it is pointed out that following real
It is the indicative explaination done to the present invention in the form of enumerating to apply mode only, but protection scope of the present invention is not limited in
This, all those skilled in the art each fall within the guarantor of the present invention with the equivalent replacement that the spirit of the present invention is the present invention
Protect range.
Embodiment 1
A kind of inflammation quickly detects colloidal gold strip, referring to Fig. 1, including test strips bottom plate 1 and test strips bottom plate
On sequentially mutually overlapped sample pad 2, label gold pad 3, nitrocellulose filter 4 and blotting paper 5；Wherein, the gold pad 4 of label applies
It is covered with the antibody that can be specifically bound with antigen PCT to be checked and IL-6, coating IL-6 antibody is equipped with successively on nitrocellulose filter 4
T1 lines 8, be coated with PCT antibody T2 lines 6 and be coated with sheep anti-mouse antibody C lines 7；Further, with antigen PCT and IL-6 to be checked
The antibody of specific binding is one kind in monoclonal antibody, polyclonal antibody, antibody fragment or chimeric antibody.
Embodiment 2
The preparation method of colloidal gold strip of the present invention.
1) preparation of colloidal gold
It is that 0.04% gold chloride (sigma) aqueous solution 1L is heated to boiling with electric jacket to take mass fraction, after 1 minute,
The lower accurate trisodium citrate aqueous solution 26ml that mass fraction is added and is 2% is stirred, continues to boil 10 minutes, with distillation after cooling
Water constant volume obtains colloid gold particle to 1L；
2) colloid gold label
IL-6 antibody label takes the gold particle 1ml in step (1), and the potassium carbonate buffer of 12 μ l 0.2M is added, and 6- is added
14 μ g IL-6 monoclonal antibodies (Medix biologies), the dosage of antibody is debugged according to actual result, and the present embodiment is with 8 μ g
The condition of label mixes well reaction 20min, and the BSA solution that 40 μ l mass fractions are 10% is added and is closed, reacts
10min, 16000 turns of 4 degree of centrifugations abandon supernatant in 20 minutes, 300 μ l suspension are added in precipitation up to IL-6 antibody marking fluids；
PCT antibody label takes the gold particle 1ml in step (1), and the potassium carbonate buffer of 10 μ l 0.2M is added, and 6- is added
14 μ g PCT monoclonal antibodies (open safe biology in Hangzhou), and the dosage of antibody is debugged according to actual result, and the present embodiment is with 10 μ
G is the condition of label, mixes well reaction 20min, and the BSA solution that 40 μ l mass fractions are 10% is added and is closed, reacts
10min, 16000 turns of 4 degree of centrifugations abandon supernatant in 20 minutes, 300 μ l suspension are added in precipitation up to PCT antibody marking fluids；
Wherein, the configuration method of suspension is：Take Tris 0.6g, casein-sodium (places of production sigma New Zealand) 0.5g, PVP
(molecular weight 36) ten thousand 0.6g, sucrose 10g, PEG (molecular weight 20,000) 0.2g, 200ml is settled to distilled water；
It is 1 by volume by IL-6 antibody marking fluid and PCT antibody marking fluid:1 mixing is uniform, uniformly spread with pipette tips to
(size on 8950 glass fibres：0.5cm × 30cm), placement air dry oven is interior 2 hours dry in 42 DEG C, obtains the gold of label
Pad；
3) film is drawn
IL-6 draws film：IL-6 antibody (Medix biologies) 40 μ g are taken, the trehalose that 8 μ l mass fractions are 50% is added, uses
0.1M, pH's 7.4 is settled to 40 μ l, takes 35 μ l uniform and is on nitrocellulose filter (Sai Duolisi N95) with stroke film instrument stroke
T1 lines；
PCT draws film：It takes PCT to draw membrane antibody (opening safe biology in Hangzhou) 40 μ g, the seaweed that 8 μ l mass fractions are 50% is added
Sugar is settled to 40 μ l with 0.1M, pH 7.4, and 35 μ l is taken uniformly to be drawn in nitrocellulose filter (Sai Duolisi with stroke film instrument
N95 it is T2 lines on)；
C lines：Sheep anti-mouse antibody (the grand base biology in Hangzhou) 20 μ g are taken, the trehalose that 8 μ l mass fractions are 50% is added, uses
0.1M, pH's 7.4 is settled to 40 μ l, takes 35 μ l uniform and is on nitrocellulose filter (Sai Duolisi N95) with stroke film instrument stroke
C lines；
Above-mentioned IL-6 antibody, PCT antibody, sheep anti-mouse antibody are drawn with film instrument is drawn on same nitrocellulose filter, are placed
42 DEG C of dryings 2 hours, must change the nitrocellulose filter of film process in air dry oven；
4) it assembles
The sample pad handled well, the gold pad of label, nitrocellulose filter and blotting paper are assembled in successively on PVC bottom plates,
It is as shown in Figure 1 to obtain finally detection colloidal gold immunochromatographimethod strip, prepared colloidal gold strip structure.
Embodiment 3
The detection of antigen.
Made test strips are cut into 3mm every, it is spare that drier is added.
People's matrix serum is simulated with calf serum (GBICO), IL-6 and PCT antigens are diluted to 10ng/ml, 5ng/ respectively
Ml, 2.5ng/ml, 1.25ng/ml, 0.75ng/ml, 0.375ng/ml, 0.2ng/ml, 0.1ng/ml do the moon with calf serum
Property, 45 μ l samples are added and directly react 5min, naked eyes interpretation.
| C(ng/ml) | 10 | 5 | 2.5 | 1.25 | 0.75 | 0.375 | 0.2 | 0.1 | Cow's serum |
| IL-6 | ++++ | ++++ | +++ | ++ | ++ | + | ± | - | - |
| PCT | ++++ | +++ | ++ | ++ | + | + | ± | - | - |
With CRP, HBS, SAA, HCV antigens interfere.
People's matrix serum is simulated with calf serum (GBICO), by CRP, HBS, SAA, HCV antigens are diluted to 10 μ g/ respectively
Ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.75 μ g/ml are added 45 μ l samples and directly react 5min, naked eyes interpretation.
| C(μg/ml) | 10 | 5 | 2.5 | 1.25 | 0.75 |
| CRP | - | - | - | - | - |
| HBS | - | - | - | - | - |
| SAA | - | - | - | - | - |
| HCV | - | - | - | - | - |
For gained yin and yang attribute testing result as shown in Fig. 2, from left to right testing result is followed successively by the IL-6 positives, IL-6 is negative,
IL-6 and PCT is positive, and PCT is positive.The result of test illustrates that inflammation of the present invention quickly detects colloidal gold strip can be minimum
It detects 0.2ng/ml, substantially meets the two theoretically CUT OFF values demand, and other several antigens do not have cross reaction.
Claims (4)
1. a kind of inflammation quickly detects on colloidal gold strip, including test strips bottom plate and test strips bottom plate sequentially phase interconnection
Gold pad, nitrocellulose filter and the blotting paper of the sample pad, label that connect, it is characterised in that the gold pad of the label is coated with can be with
The antibody of antigen PCT and IL-6 to be checked specific binding is equipped with the T1 of coating IL-6 antibody on the nitrocellulose filter successively
Line, the T2 lines for being coated with PCT antibody and the C lines for being coated with sheep anti-mouse antibody.
2. a kind of inflammation according to claim 1 quickly detects colloidal gold strip, it is characterised in that it is described with it is to be checked
The antibody of antigen PCT and IL-6 specific binding is one in monoclonal antibody, polyclonal antibody, antibody fragment or chimeric antibody
Kind.
3. a kind of inflammation according to claim 1 quickly detects colloidal gold strip, it is characterised in that preparation method includes
Following steps：
(1) preparation of colloidal gold
It is that 0.04% aqueous solution of chloraurate is heated to boiling to take mass fraction, and agitation later is lower to be added the lemon that mass fraction is 2%
Lemon three sodium water solutions of acid, continue to boil 5-15min, with distilled water constant volume after cooling down, obtain colloid gold particle；
(2) colloid gold label
IL-6 antibody marks：The colloid gold particle in step (1) is taken, the potassium carbonate buffer of 0.2M is first added, adds IL-6
Monoclonal antibody mixes well reaction 10-30min, is eventually adding the BSA solution that mass fraction is 10% and is closed, is reacted
Supernatant is abandoned in 5-15min, centrifugation, and suspension is added in precipitation up to IL-6 antibody marking fluids；
PCT antibody marks：The colloid gold particle in step (1) is taken, the potassium carbonate buffer of 0.2M is first added, it is mono- to add PCT
Clonal antibody mixes well reaction 10-30min, is eventually adding the BSA solution that mass fraction is 10% and is closed, reacts 5-
Supernatant is abandoned in 15min, centrifugation, and suspension is added in precipitation up to PCT antibody marking fluids；
It is 1 by volume by IL-6 antibody marking fluid and PCT antibody marking fluid:1 mixing is uniform, uniformly on paving to glass fibre,
Up to the gold pad of label after drying；
(3) film is drawn
IL-6 draws film：Take IL-6 antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4, later
It is T1 lines with drawing film instrument and drawing on nitrocellulose filter；
PCT draws film：Take PCT antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4, Zhi Houyong
It is T2 lines to draw film instrument and draw on nitrocellulose filter；
C lines：It takes sheep anti-mouse antibody that the trehalose that mass fraction is 50% is added, with the PB constant volumes of 0.1M, pH 7.4, uses draw later
It is C lines that film instrument, which is drawn on nitrocellulose filter,；
Above-mentioned IL-6 antibody, PCT antibody, sheep anti-mouse antibody are drawn with film instrument is drawn on same nitrocellulose filter, are after dry
The nitrocellulose filter of film process must be changed；
(4) it assembles
By the sample pad handled well, the gold pad of label, nitrocellulose filter and blotting paper be assembled in successively on PVC bottom plates to get
Inflammation of the present invention quickly detects colloidal gold strip.
4. a kind of inflammation according to claim 3 quickly detects colloidal gold strip, it is characterised in that in the step (2)
The configuration method of suspension is：Tris 0.6g, casein-sodium 0.5g, PVP 0.6g, sucrose 10g, PEG 0.2g are taken, with distillation
Water is settled to 200ml.
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Cited By (3)
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| CN110780067A (en) * | 2019-11-04 | 2020-02-11 | 南京欧凯生物科技有限公司 | Fluorescence immunochromatographic test paper for detecting procalcitonin and preparation method thereof |
| CN111879924A (en) * | 2020-01-15 | 2020-11-03 | 杭州奥泰生物技术股份有限公司 | Colloidal gold immunochromatography test paper for rapidly diagnosing hemoglobin and combining globin-hemoglobin compound and preparation method thereof |
| CN115980341A (en) * | 2022-12-26 | 2023-04-18 | 南京珀尔泰生物技术有限公司 | Novel PCT immunochromatography test strip for quantitatively detecting procalcitonin by colloidal gold method and preparation method thereof |
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