CN106841606B - Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of PCT - Google Patents
Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of PCT Download PDFInfo
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Abstract
The invention discloses a kind of colloidal gold immuno-chromatography test paper strips for detecting PCT, kit and preparation method thereof, the test strips include bottom liner, and it is sequentially arranged at the sample pad on the bottom liner, gold-labelled pad, coated film and blotting paper, the anti-human PCT detection antibody and chicken IgY antibody of colloid gold label are coated in the gold-labelled pad, the coated film includes being arranged in parallel, and the detection zone being spaced apart from each other and check plot, the detection zone is coated with anti-PCT coated antibody, the check plot is coated with the anti-chicken IgY antibody of goat, the anti-PCT detection antibody has the epitope such as any shown amino acid sequence of SEQ ID No:1~SEQ ID No:5.Colloidal gold immuno-chromatography test paper strip of the invention has easy, quick, practical, sensitive, efficient, accurate, stable beneficial effect, realizes the half-quantitative detection to human serum PCT, has higher sensitivity and specificity.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to a kind of half-quantitative detection Procalcitonins (PCT)
Colloidal gold immuno-chromatography test paper strip, kit and preparation method thereof.
Background technique
Procalcitonin (procalcitonin, PCT) is the precursor substance of mankind's calcitonin of discovery in 1992, no hormone
Activity is made of 116 amino acid residues, and relative molecular weight is the glycoprotein of 13kD.Calcitonin under physiological condition, in blood
Original is mainly synthesized by the C cell of parathyroid gland.Intracellular, PCT can be become by gradually hydrolysis CT, anticalcium plain (katacalcin),
The end N- residue three parts are secreted extracellularly.Therefore the concentration of blood-serum P CT is extremely low, and without larger fluctuation (< 50ng/L), generally
It is difficult to detect.In the case where system inflammation reacts the severe bacterial infections disease conditions such as syndrome, dense toxaemia, septicemia, Huan Zheshen
The various kinds of cell at other positions of body can largely synthesize PCT, and be secreted in the form of proteinogen to extracellular, lead to PCT in serum
It is significant to increase, concentration up to several times even up to ten thousand times of normal level, research it has also been found that PCT concentration and infection severity at
It is positively correlated, thus, compared to Testing index such as traditional C reactive protein, cell factor, complement measurements, PCT albumen relies on it
Itself early stage property, stability, specificity, the main feature of positive correlation, have been developed as quick diagnosis severe bacterial infections,
Judge the fudiciary marker of the bacterial infection disease state of an illness and prognosis and its observation of curative effect.
Calcitonin (Calcitonin, CT) is a kind of polypeptide extracted from Thyroid Neoplasms culture solution at first
Hormone, therefore become the tumor serology marker.The precursor of PCT, CT, 116 acid glycoproteins, in people intracorporal half
The phase of declining is about 20-24 hours, and stability is good；Content is extremely low in normal human serum, in addition to thyroid gland wound or tumour, system
In the patients serums such as inflammatory reaction syndrome (SIRS), septicemia, acute and chronic pneumonia, acute pancreatitis, active hepatitis, wound
Significant to increase, especially to SIRS/ septicemia, PCT (compared with WBC, IL-6, TNF-2, CRP, soluble selection element etc.) is a kind of
Very sensitive special serologic marker.And low-level is then kept in sick element poison infection, tumour object art wound, PCT is serious
Bacterium infection (after 2-3 hours) can increase in early days, therefore have early diagnosis value；In local infection, virus infection, chronic
PCT concentration does not increase or gently when nonspecific inflammation, cancer fever, the reaction of graft host rejection or the diseases such as autoimmune
Micro- increase, and only just obviously increased when serious whole body system sexuality contaminates, this just determines the high degree of specificity of PCT, therefore
It can also be used for the antidiastole of various clinical settings；PCT concentration and severity of inflammation are positively correlated, and with the control of inflammation
Alleviation with the state of an illness and be reduced to normal level, thus PCT is but also as judging the reliable of the state of an illness and prognosis and observation of curative effect
Index
PCT clinically has extensive and important application value；And the death rate and hospital stays to patient ICU
Offer standard.PCT can be widely used for the ward ICU, hematology, oncology, paediatrics, premature and newborn intensive care unit, surgery, interior
Section, organ transplant section, emergency department and Experiment on therapy room etc..
PCT detection method has at present:
1, radioimmunology analytic approach --- this method can detect the blood-serum P CT of normal person, and reliable susceptibility is 4pg/ml,
But time-consuming (19~22h) for method detection, and has the pollution of radioactive element, so that using being restricted.
2, double antibodies sandwich immunochemiluminescence method --- the method is easy to operate, high specificity, and sensibility is high, the lower bound of measurement
Value is 0.1ng/ml, and 2h can go out result.
3, Gold standard --- the method has the characteristics of fast and convenient, easily to observe.
4, immune turbidimetry is transmitted --- the measuring method is easy, quick, can automate, be suitable for batch detection, still
Immune transmittance turbid methodology and clinical application are verified it is still necessary to further.
Currently, external, the PCT detection kit manufacturer for obtaining authentication code mainly has four, respectively Roche
The PCT detection kit of electrochemical luminescence, the sxemiquantitative immunochromatography PCT detection kit of B.R.A.H.M.S GmbH,
The chemoluminescence method PCT detection kit of B.R.A.H.M.S GmbH, the enzyme-linked fluorescence analysis method PCT of bioMerieux, sa
Detection kit.There has been the authentication code of 40 or so producer's acquisition PCT detection kits in the country, wherein chemoluminescence method
7, enzyme linked immunological 2, immunoturbidimetry 6, other more than 20 families are chromatography method, and can be carried out quantitative and carry out quick
The method of detection but only has several families of only a few.Since pyemic patient carries out quick diagnosis after illness, to instructing patient to use
Medical instrument has the meaning of larger guidance, therefore developing quick PCT detection kit is needed for market.On this basis, it opens at home
The quick PCT detection kit of hair quantitatively or semi-quantitatively has biggish market significance.
Summary of the invention
Based on this, in order to overcome the defects of the prior art described above, the present invention provides a kind of colloid gold immunes for detecting PCT
Chromatograph test strip, kit and preparation method thereof, the test strip are short, special with half-quantitative detection, detection time is able to achieve
The advantages such as the opposite sex is good, and economic cost is low.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes:
A kind of colloidal gold immuno-chromatography test paper strip detecting PCT, including bottom liner and the sample being sequentially arranged on the bottom liner
Product pad, gold-labelled pad, coated film and blotting paper are coated with the anti-human PCT detection antibody and glue of colloid gold label in the gold-labelled pad
The chicken IgY antibody of body gold label, the coated film include being arranged in parallel and the detection zone being spaced apart from each other and check plot, the inspection
It surveys area and is coated with anti-PCT coated antibody, the check plot is coated with the anti-chicken IgY antibody of goat, and the anti-PCT detection antibody is corresponding
Epitope with the amino acid sequence as shown in SEQ ID No:1~SEQ ID No:5.
In wherein some embodiments, the anti-human PCT detection antibody of the colloid gold label and the concentration of chicken IgY antibody are equal
For 0.2mg/ml and 0.2mg/ml, the package amount in the gold-labelled pad is 1.2ul/cm.
In wherein some embodiments, the colloid gold particle diameter in the gold-labelled pad is 35~45nm.
In wherein some embodiments, the detection zone close to the gold-labelled pad, the check plot close to the blotting paper,
0.4cm is divided between the detection zone and the check plot.
In wherein some embodiments, the concentration of the anti-PCT coated antibody is 2mg/ml, in the coating of the detection zone
Amount is 1.2ul/cm.
In wherein some embodiments, the concentration of the anti-chicken IgY antibody of goat is 0.7mg/ml, in the check plot
Package amount is 1.2ul/cm.
The present invention also provides the preparation methods of the colloidal gold immuno-chromatography test paper strip of above-mentioned detection PCT, including following step
It is rapid:
(1), anti-PCT coated antibody is prepared, anti-PCT coated antibody is fixed respectively on coated film and the anti-chicken IgY of goat is anti-
Body forms detection zone and check plot；
(2), anti-human PCT is detected antibody and chicken IgY antibody to be marked with colloidal gold, and be sprayed in gold-labelled pad；Institute
State the anti-corresponding epitope with the amino acid sequence as shown in SEQ ID No:1~SEQ ID No:5 of PCT detection antibody；
(3), on bottom liner overlap joint paste sample pad, gold-labelled pad, coated film and blotting paper to get.
In wherein some embodiments, the detection antibody of anti-PCT described in step (2) the preparation method comprises the following steps: according to PCT shell egg
White sequence design difference peptide fragment is immunized animal and obtains specific antibody；Specific antibody is matched, screening obtains sensitivity
Highest antibody, as anti-PCT coated antibody.
The present invention also provides a kind of colloidal gold immunochromatographykit kit for detecting PCT, the colloid including above-mentioned detection PCT
Golden immuno-chromatographic test paper strip.
Compared with prior art, the invention has the following advantages:
(1), test strips of the invention use colloidal gold as marker, the colour developing power and serum sample of detection zone sample
PCT concentration is directly proportional in this, and the concentration range of PCT in sample can be detected according to the colorimetric card interpretation of offer, is realized to human body
The half-quantitative detection of blood-serum P CT has higher sensitivity and specificity and is able to achieve half-quantitative detection, certain sample is added
This dilution reduces influence of non-spy's property the led reaction to result to a certain extent, and test method is simple, accuracy rate is high；And
Instrument is not needed, it is easy to operate, it is easy interpretation, detection speed is fast, it can be as a result easy observation in 20min or so interpretation result, and
And it is accurate and reliable, use is safe, shortens the sample turnaround time；
(2), test strips high specificity of the invention is not sent out using cross jamming substance testing test strips of the invention
Existing cross jamming phenomenon has carried out clinical research in 2 Grade A hospitals respectively, test strips of the invention and control stripes item have compared with
Good consistency, measurement result are more accurate, stable and reliable；Health especially suitable for base and rural area broad masses of the people
Demand has great economic benefit.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the colloidal gold immuno-chromatography test paper strip of the embodiment of the present invention 1；
Fig. 2 is the relevance verification result in test example 1 of the present invention using anti-PCT-5 antibody as detection antibody；
Fig. 3 is the HOOK effect test result of the colloidal gold immuno-chromatography test paper strip of three batches of embodiment of the present invention 1；
Appended drawing reference: 1, sample pad；2, gold-labelled pad；3, coated film；4, detection zone；5, check plot；6, blotting paper；7, bottom
Plate.
Specific embodiment
Below by way of the drawings and specific embodiments, the present invention will be described in detail.
Raw material used in following embodiment unless otherwise specified, derives from commercially available.
The colloidal gold immuno-chromatography test paper strip and preparation method thereof of the detection of embodiment 1 PCT
Referring to Fig. 1, the structural schematic diagram of the colloidal gold immuno-chromatography test paper strip for detection PCT of the invention, this implementation
The colloidal gold immuno-chromatography test paper strip of the detection PCT of example, including bottom liner 7 and the sample pad 1 being sequentially arranged on the bottom liner 7,
Gold-labelled pad 2, coated film 3 and blotting paper 6 are coated with the anti-human PCT detection antibody and colloid of colloid gold label in the gold-labelled pad 2
The chicken IgY antibody of gold label, the coated film 3 include being arranged in parallel and the detection zone 4 being spaced apart from each other and check plot 5, the inspection
It surveys area 4 and is coated with anti-PCT coated antibody, the check plot 5 is coated with the anti-chicken IgY antibody of goat, the anti-PCT detection antibody tool
The epitope of amino acid sequence just like SEQ ID No:5.
In the present embodiment, the anti-human PCT of the colloid gold label detects the chicken IgY antibody of antibody and colloid gold label
Concentration is 0.2mg/ml, and the package amount in the gold-labelled pad 2 is 1.2ul/cm.Colloidal gold in the gold-labelled pad 2
Grain diameter is about 40nm.The detection zone 4 is close to the gold-labelled pad 2, and the check plot 5 is close to the blotting paper 6, the detection
0.4cm is divided between area 4 and the check plot 5.The concentration of the anti-PCT coated antibody is 2mg/ml, in the detection zone 4
Package amount is 1.2ul/cm.The concentration of the anti-chicken IgY antibody of goat is 0.7mg/ml, and the package amount in the check plot 5 is
1.2ul/cm。
The preparation method of the colloidal gold immuno-chromatography test paper strip of the detection PCT of the present embodiment, comprising the following steps:
(1), prepared by colloidal gold
It takes 0.5-1.2% trisodium citrate aqueous solution 3ml in 297ml tri-distilled water, and shakes up the round bottom burning that 500ml is added
In bottle；Round-bottomed flask is placed in heating mantle to the stirrer stirring that suitable size is added；It is heated to boiling, and starts to stir, and
Timing at once；Boil 3 minutes, is rapidly added the 0.5-0.8% aqueous solution of chloraurate of 3ml, blender stir 10 minutes it
Mixing speed is turned down afterwards；Round-bottomed flask is taken out from heating mantle, is put to the station for being placed with hot pad；The colloid prepared
Gold solution is placed at room temperature for after temperature lowers, and sealing is kept in dark place in 4 DEG C of refrigerators.The colloidal gold that magnetic heating stirrer is fired
Grain size is uniform, and diameter is about 40nm；
(2), prepared by gold-labelled pad
Gold-labelled pad is layered in metal working tool, the gold-labelled pad treatment fluid of 12ml is added in the gold-labelled pad of 15*20cm；37 DEG C of drums
4 hours are dried in wind drying box to be completely dried up to gold-labelled pad；Dried gold-labelled pad will be handled to take out, it is close with aluminium foil bag
Envelope.Gold-labelled pad treatment fluid: 20-100mM Tris-Hcl, 5% sucrose, 5%BSA, 0.5%-1.0% trisodium citrate and
Logical -100 composition of 0.1%-0.15% Qula.
By the amount of package amount 1.2ul/cm, using spray film instrument by concentration be 0.2mg/ml colloid gold label it is anti-human
The chicken IgY antibody of PCT detection antibody and colloid gold label is sprayed in the gold-labelled pad handled well and 35-42 degrees Celsius of drying for standby.
(3), prepared by sample pad
Sample pad is lain in paving metal working tool；The sample pad treatment fluid of 25ml is added in the sample pad of 15*20cm；With paving
Gold coating is uniform；4 hours are dried in 37 DEG C of air dry ovens to be completely dried up to sample pad.The formula of sample pad treatment fluid
Are as follows: 20-100mM Tris-HCl, 5% sucrose, 5%BSA, 0.5%-1.0% trisodium citrate and 0.1%-0.15% tween-
20。
(4), the preparation of coated film
By the amount of package amount 1.2ul/cm, the anti-human PCT coated antibody that concentration is 2mg/ml is sprayed on packet using spray film instrument
The detection zone of envelope, by the amount of package amount 1.2ul/cm, the anti-chicken IgY antibody of goat for the use of spray film instrument being 0.7mg/ml by concentration
It is sprayed on the check plot of coated film, detection zone and check plot are arranged in parallel, are spaced apart from each other 0.4cm.
(5), the assembling of test strips
Blotting paper is pasted and pastes sample pad on the left of the right side of bottom plate, bottom plate, gold-labelled pad is pasted in sample pad and packet
Above envelope junction, film adhesive then is sticked on gold-labelled pad surface, is packed.
The preparation of the anti-PCT specific antibody of test example 1
According to PCT holoprotein sequence design difference peptide fragment, animal is immunized and obtains specific antibody, the specific steps are as follows:
(1), the preparation of PCT native protein
According to the DNA sequence dna of the PCT provided in Genbank, pair of primers is designed, ' end introduces respectively by the 5 of two primers
NdeI+XhoI restriction enzyme site obtains the target gene of PCT by PCR amplification, by carrier pET-28a and passes through Ago-Gel
The PCT target gene fragment of purifying, carries out double digestion processing with NdeI+XhoI, digestion will be produced after purification with T4DNA ligase
Object connection, obtains recombinant plasmid pET-28a-PCT, recombinant plasmid transformed is entered bacillus coli DH 5 alpha, is containing ammonia benzyl mould
Selected clone on the LB plate of element, prepares plasmid in a small amount, goes out positive colony, sequencing result table by double digestion/PCR evaluation and screening
The PCT segment of bright recombination and the sequence of design are completely the same.
For recombinant plasmid pET-28a-PCT after sequence verification, conversion enters Escherichia coli (BL21), is containing ammonia benzyl mould
It is cultivated in the LB culture medium of element, positive colony can be selected on LB plate and carry out plasmid enzyme restriction identification, prepare plasmid in a small amount, used
Double digestion PCR evaluation and screening goes out positive colony, final to obtain the recombinant plasmid engineering bacteria containing PCT.
In expression, the recombinant plasmid engineering bacteria of PCT is cultivated in the LB culture medium containing 100 μ g/ml ampicillins,
A600Reach between 0.5-0.6, the Isopropyl β-D-1- of final concentration of 0.5mM is then added
Thiogalactopyranoside (IPTG) is in 37 DEG C of induction 4h, bacterium solution 4 after the completion of inducing, and 000rpm is centrifuged 10min, receives
Collect thallus, and washs precipitating with PBS；PBS is resuspended precipitating and is placed in ice bath, and 12000rpm is centrifuged 20min after carrying out ultrasonic bacteria breaking, on
Cleer and peaceful precipitating carries out SDS-PAGE electrophoresis respectively, the results showed that the PCT recombinant protein of expression is the expression of endochylema insolubility, will
The recombinant protein is named as BL21 (DE3)-PCT.
The thallus that great expression is obtained, is centrifuged after ultrasonication, then carries out inclusion body washing, and GE is used after the completion of washing
The His Trap FF purification column of Healthcare company albumen is purified (according to product description carry out preparation of reagents and
Purifying).The albumen finally obtained is analyzed with SDS-PAGE electrophoresis, is measured its concentration with BCA protein quantification kit and is
0.25mg/ml。
(2), the preparation of PCT peptide fragment
PCT recombinant protein sequence is imported into online epitope design software first, amino outside protein conformation will be located at
Acid sequence comes out.Full PCT protein sequence is imported into DNASTAR software again, peptide fragment is counted by Epitope prediction tool
Combination.Two groups of data are compared and show that most possible is 5 groups of peptide fragments of epitope.It is public by the biochemical biology of Shanghai gill
Department's 5 groups of peptide fragments of synthesis, particular sequence are as follows:
Peptide fragment 1:NH2-CALESSPADPATLSEDEARL(SEQ ID No:1)
Peptide fragment 2:NH2-CELEQEQEREGSSLDSPRSKRC(SEQ ID No:2)
Peptide fragment 3:NH2-CIGVGAPGKKRDMSSDLERDH(SEQ ID No:3)
Peptide fragment 4:NH2-CSSDLERDHRPHVSMPQNANY(SEQ ID No:4)
Peptide fragment 5:NH2-CTQDFNKFHTFPQTAIGV(SEQ ID No:5)
(3), the preparation and purification of specific antibody
Female BAl BIc/c of 8 week old, weight 18g or so and health are immunized using PCT native protein and above-mentioned 5 groups of peptide fragments
After mouse each 2, adaptive feeding 1 week, acquires negative blood and used as control；Using intermediate range immunization protocol (0.3ml/ only, 2 weeks/
It is secondary), (50 μ g/ are only) is by immunogene and isometric Freund's complete adjuvant stirring and emulsifying, dorsal sc multiple spot note when first immunisation
It penetrates, hereafter carries out routine immunization by immunogene and isometric incomplete Freund's adjuvant stirring and emulsifying；3 times it is immune when, general 50 μ
G antigen+TiterMax mixed in equal amounts emulsifies back part multi-point injection, surveys potency after 7 days.Mouse titers obviously reach certain requirement
Booster immunization afterwards, booster immunization are not added adjuvant, and booster immunization dosage is 50 μ g, 3 days after booster immunization, pluck eyeball blood sampling, separation
Serum keeping.Spleen is taken to be merged simultaneously.When cell fusion, splenocyte is mixed with myeloma cell by 4:1 or so,
And it is merged in the case where the rush of polyethylene glycol (PEG, molecular weight 1450) melts effect, fused cell HAT selectivity culture solution again
In cultivated, the positive hybridoma cell that can be reacted with PCT native protein is filtered out by indirect ELISA method after 10 days,
And the positive hybridoma cell for obtaining primary dcreening operation expands culture, carries out label protein (His-tag) hybridoma two days later
It excludes, goes out to be directed to the hybridoma of PCT albumen rather than label with secondary screening；It is with limiting dilution assay that the positive hybridoma of acquisition is thin
Born of the same parents are continuously subcloned more than at least twice, and subclone is cultivated with HT selective medium every time, and subclone 8-10 days laggard
Row ELISA screening obtains energy stably excreting and is directed to PCT native protein and peptide fragment until detection cell positive rate is 100%
Specific antibody detection cell strain.
The female sex-health BALB/c mouse of 8-12 week old is selected, norphytane is injected intraperitoneally, 0.5ml/ is only；After 7-10 days, to every
Mouse peritoneal injection 1 × 106~5 × 106A detection hybridoma notices that PBS or nothing need to be used by blowing down cell or diluting cells
Blood serum medium；By ascites 10,000r/min is centrifuged 15min, removes cell component and other sediments, fat and oil reservoir
Deng, collect middle layer, measure antibody titer, packing, set -70 DEG C freeze it is spare.Saturated ammonium sulphate: it draws 5ml and handles well
Ascites move into small beaker in, under stiring, the PBS 5.0ml of 0.22 μm of filter membrane was added dropwise；After mixing, then dropwise
It is added 10ml saturated ammonium sulfate solution (pH7.4), continues to be slowly stirred 30min；10,000r/min is centrifuged 15 points after standing 2h
Clock discards supernatant, and the PBS of the used 0.22 μm of filter membrane of sediment is resuspended, and the re-suspension liquid is then crossed 0.22 μm of filter membrane again；According to anti-
Body different subtype selectes the purification column of different GE Healthcare companies, collects antibody peak；With PBS buffer solution by antibody into
Row dialysis with BCA protein quantification kit measurement antibody concentration, and antibody is dispensed and is saved.
(4) pairing experiment obtains and is directed to the natural highest antibody pair of PCT albumen potency
By natural PCT albumen as antigen, the antibody of native protein preparation, which is used as, captures antibody, and the antibody of peptide fragment preparation
As detection antibody.By matching experiment (the capture antibody of 100ng/100ul is coated in 96 orifice plates and is incubated for；It sequentially adds
By degree of passing diluted natural PCT protein 10 0ng, 50ng, 25ng, 12.5ng, 6.25ng, 3.125ng, 1.5625ng, Yi Jikong
White control PBS；5 groups of different detection antibody are added to verify its potency) detect the antibody pair matched.Experimental result such as table 1
It is shown.
Table 1 matches experimental result
| Detect antibody | Anti- PCT-1 antibody | Anti- PCT-2 antibody | Anti- PCT-3 antibody | Anti- PCT-4 antibody | Anti- PCT-5 antibody |
| PCT native antigen | Peptide fragment 1 | Peptide fragment 2 | Peptide fragment 3 | Peptide fragment 4 | Peptide fragment 5 |
| 100ng | 2.241 | 1.997 | 0.873 | 1.606 | 0.586 |
| 50ng | 1.713 | 1.511 | 0.651 | 1.1 | 0.49 |
| 25ng | 1.35 | 1.109 | 0.541 | 0.826 | 0.389 |
| 12.5ng | 0.951 | 0.857 | 0.453 | 0.615 | 0.351 |
| 6.25ng | 0.744 | 0.621 | 0.392 | 0.433 | 0.337 |
| 3.125ng | 0.533 | 0.424 | 0.302 | 0.386 | 0.265 |
| 1.5625ng | 0.34 | 0.31 | 0.241 | 0.276 | 0.222 |
| NC | 0.285 | 0.3 | 0.197 | 0.271 | 0.198 |
Table 1 the result shows that, by anti-PCT-1 antibody, anti-PCT-2 antibody, anti-PCT-4 antibody, (peptide fragment 1,2,4 is prepared anti-
Body) as detection antibody, anti-PCT antibody can get as capture antibody and be directed to the natural higher antibody pair of PCT albumen potency.?
Anti- PCT-1 antibody is used in subsequent serology confirmatory experiment, anti-PCT-2 antibody and anti-PCT-4 antibody are verified.
(5), serology verifies specific antibody pair
Using anti-PCT antibody as capture antibody, anti-PCT-1 antibody, anti-PCT-2 antibody, anti-PCT-3 antibody, anti-PCT-4
Antibody and anti-PCT-5 antibody determine 20 samples respectively and have done correlation analysis as detection antibody, as a result prove anti-
PCT-1 antibody, anti-PCT-2 antibody, anti-PCT-3 antibody are weak to the PCT identification in serum, and correlation is lower, general
All over more lower than sample true value of measured value；Into after crossing cross match, screening has obtained a pair of preferable to serum identification anti-
Body pair: therefore anti-PCT antibody and anti-PCT-5 antibody illustrate peptide fragment 5 (SEQ ID No:5) for the higher antigen table of specificity
Position.
Using anti-PCT-5 antibody as detection antibody relevance verification result as shown in Fig. 2, as can be seen from Figure 2, using anti-
The combine detection of PCT-5 antibody and anti-PCT antibody, R consistent with the correlation of the detected value of hospital2Reach 0.991.
The performance detection of the colloidal gold immuno-chromatography test paper strip of 2 embodiment 1 of test example
1, negative reference product coincidence rate
1.1 test method
The test strip of the embodiment of the present invention 1 of each batch is respectively taken into 5 person-portions respectively, each batch detects 5 parts of feminine genders
Reference material, each negative reference product sample (mixing Healthy Human Serum) measurement 1 time, guarantees the correct of result, sees in 20~25min
It examines as a result, counting the negative reference product coincidence rate of each test strip.
1.2 test results are as shown in table 2.
2 negative reference product coincidence rate test result of table
| Lot number | 5 parts of negative reference product colour developing situations | Negative reference product coincidence rate |
| 2013002 | Without colour developing | 100% |
| 2013003 | Without colour developing | 100% |
| 2013004 | Without colour developing | 100% |
1.3 conclusion (of pressure testing)
By 2 result of table as it can be seen that test strip negative reference product are without colour developing, coincidence rate 100%.
2, positive reference product coincidence rate
2.1 test method
The test strip of the embodiment of the present invention 1 of each batch is respectively taken into 3 person-portions respectively, it is (calibrated to positive reference product
PCT recombinant protein) detected, each positive reference product sample measures 1 time, guarantee the correct of result, seen in 20~25min
It examines as a result, each batch reference material colour developing situation is compared with reference to colorimetric card with respective batch, statistics colour developing result consistency,
To count the positive reference product coincidence rate of each test strip.
2.2 test results are as shown in table 3.
3 positive reference product coincidence rate test result of table
2.3 conclusion (of pressure testing)
By 3 result of table as it can be seen that the positive reference product of the test strip of the embodiment of the present invention 1 develop the color and refer to colorimetric card
Unanimously, coincidence rate 100%.
3, minimum detection limit
3.1 verifying minimum detection limit test methods
10 person-portion reagent strips are randomly selected from 2013002 products, take positive reference product (0.5ng/mL, calibrated PCT weight
Histone), minimum detection limit reference material (0.2ng/mL, calibrated PCT recombinant protein) is less than 0.05ng/ using PCT content
The negative serum (calibrated mixing Healthy Human Serum) of mL as dilution by minimum detection limit reference material (0.2ng/mL) by
Grade is diluted to 0.1ng/mL, 0.05ng/mL, 0 (dilution).With the test strip of embodiment 1 to the above sample (0.5ng/
ML, 0.2ng/mL, 0.1ng/mL, 0.05ng/mL, 0 (dilution)) it is detected, the above experiment is repeated 2 times, minimum detection limit
The PCT minimum concentration value of result can be shown with reagent detection detection line to indicate.
3.2 minimum detection limits repeat test method
The test strip of the embodiment of the present invention 1 of each batch takes 20 person-portions respectively, detects to minimum detection limit, 20
Observation in~25min should can develop the color, and it is consistent to develop the color as a result, colour developing situation is compared with respective batch with reference to colorimetric card.
3.3 test results are as shown in table 4 and table 5.
4 minimum detection limit testing result of table
5 minimum detection limit of table repeats testing result
3.4 conclusion (of pressure testing)
Minimum detection limit testing result through the above table 4 and table 5, the minimum inspection of the test strip of the embodiment of the present invention 1
Survey is limited to 0.2ng/mL.
4, interfering substance is tested
4.1 interfering substances detect test method
Negative serum using PCT content less than 0.05ng/mL is by three kinds of interfering substance bilirubin, hemoglobin and glycerol
Three esters are configured to the solution that concentration is 1mg/mL, are detected with the test strip of the embodiment of the present invention 1, and three of the above is observed
Interfering substance adds the influence in negative sample to negative sample testing result.
4.2 interfering substance checking test methods
200 μ g/mL of bilirubin concentration, 60 μ g/mL of hemoglobin concentration, 40 μ g/mL of triglyceride concentration are prepared, with three batches
Test strips detection, the interior observation colour developing situation of 20~25min.
4.3 test results are as shown in table 6~9.
6 bilirubin interference detection results of table
7 hemoglobin interference detection results of table
8 triglycerides interference detection results of table
The interference verifying testing result of table 9
4.4 conclusion (of pressure testing)
Above 6~9 interference testing inspection of table the result shows that, bilirubin be lower than 200 μ g/mL when do not influence testing result；
Hemoglobin concentration will not influence testing result when being lower than 50 μ g/mL；Triglyceride concentration will not influence inspection when being lower than 35 μ g/mL
Survey result.
5, HOOK effect
5.1 test method
PCT calibration antigen (Raybiotech company, the U.S.) is added separately to the feminine gender that PCT content is less than 0.05ng/mL
In serum, obtain the solution of following concentration: 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL,
640ng/mL, 1200ng/mL, 1500ng/mL, 1600ng/mL, 1800ng/mL, with the test strip of the embodiment of the present invention 1
It is detected, will test the concentration of calibration antigen as X-axis, numerical tabular (is used with the intensity that test strips develop the color in 20~25min
Show) as Y-axis, curve is done, observation generates the detectable concentration of high dose Hook effect.
5.2 test results are as shown in Figure 3.
5.3 conclusion (of pressure testing)
By Fig. 3 result as it can be seen that test strip of the invention would not observe that height when being no more than 1500ng/mL
Dosage Hook effect.
6 repeatability
6.1 test method
Random sampling 20 from every batch of test strips are separately added into high concentration accuracy reference material (10ng/mL), repeat to survey
It is 10 times fixed；It is added low concentration accuracy reference material (0.5ng/mL), replication 10 times, guarantees the correct of result, 20~25min
Interior observation result.The high and low concentration accuracy of the high and low concentration accuracy reference material colour developing situation of each batch and respective batch is joined
It examines colorimetric card to be compared, statistics colour developing result consistency is to count the repeatability of test strips in each batch.
6.2 test results are as shown in table 10.
The repeated testing result of table 10
6.3 conclusion (of pressure testing)
Low, the high concentration accuracy reference material of the test strip of 3 batches repeats detection 10 times, all with reference colorimetric card
The band colour developing that develops the color is consistent.
The clinical performance of the test strip of 3 embodiment of the present invention 1 of test example is verified
Clinic is carried out in 2 hospitals of Hospital of Southern Medical University and Sun Yat-sen Memorial Hospital respectively to grind
Study carefully.This research is detected sample 220 times altogether using the clinical trial design of blind control, 73 positive, 147 negative, mark
This type is serum.Research uses import test strips (the Procalcitonin Measurement test strips of B.R.A.H.M.S GmbH company) conduct
Control test method, 220 pattern detection results judge completely the same with results of comparison compared with the control, wherein 2 deutero-albumoses
This testing result judges that section is not inconsistent.The positive coincidence rate of statistical analysis, this kit is 100%, and negative match-rate is
100%, youden index 1, Kappa value is 1.The test strip and control stripes item of the embodiment of the present invention 1 have preferable one
Cause property.The measurement result of the test strip of the embodiment of the present invention 1 is more accurate, stable and reliable.
220 parts of serum samples are detected with control stripes item respectively with the test strip of the embodiment of the present invention 1, are converged
Total testing result.It is for statistical analysis to determination data, it calculates the coincidence rate of two kinds of kits and uses SPSS software statistics
Kappa value.As a result as shown in table 11.
11 clinical performance verification result of table
From table 11, calculated result is as follows:
Positive sample coincidence rate (true positive rate)=73/73 × 100%=100%
Negative sample coincidence rate (true negative rate)=147/147 × 100%=100%
False positive rate=0/147 × 100%=0
False negative rate=0/73 × 100%=0
Total coincidence rate=110/110 × 100%=100%
Youden index=73/73+147/147-1=1
Kappa inspection is carried out with SPSS 20, as a result as follows:
Value=1 kappa, > 0.75, illustrate that the test strip of the embodiment of the present invention 1 and control stripes consistency are fine.
The analysis of difference sample
By the test strip and the control stripes item (calcitonin of B.R.A.H.M.S GmbH company of the embodiment of the present invention 1
Original measurement test strips) result judge section, 220 parts are examined the testing result for sharing 2 samples in samples to judge that section is not inconsistent,
Difference sample situation is shown in Table 12.The sample is checked with Roche reagent, review measurement result and the embodiment of the present invention 1
Test strip testing result is consistent.Clinic can carry out repeating detection as needed or be judged in conjunction with other diagnostic methods.
12 difference sample situation of table
Discussion and conclusion
Procalcitonin (PCT) test strip (colloidal gold method) of the invention carries out clinical test in 2 hospitals, with import
Test strips detect clinical samples 220 (73 positive, 147 negative), positive coincidence rate is as control stripes item altogether
100%, negative match-rate 100%, total coincidence rate is 100%, youden index 1.Kappa value be 1 (Kappa examine, P <
0.05), the test strip of the embodiment of the present invention 1 and control stripes item have good consistency.The result of appraisal show the present invention
The test strip of embodiment 1 and control stripes detection performance are similar, and stability is good, as a result more accurate and reliable.Kit
It is simple to operate, and cost is compared with the Procalcitonin Measurement test strips (immune chromatograph of the B.R.A.H.M.S GmbH company of import
Detection method) kit is cheap, there is good market application value.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
110 > Guangzhou Ray Biotechnology Co., Ltd. of <
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Claims (9)
1. a kind of colloidal gold immuno-chromatography test paper strip for detecting PCT, which is characterized in that the test strips include bottom liner, Yi Jiyi
The secondary sample pad being located on the bottom liner, gold-labelled pad, coated film and blotting paper are coated with colloid gold label in the gold-labelled pad
Anti-human PCT detection antibody and chicken IgY antibody, the coated film include being arranged in parallel and the detection zone being spaced apart from each other and check plot,
The detection zone is coated with anti-PCT coated antibody, and the check plot is coated with the anti-chicken IgY antibody of goat, the anti-human PCT detection
The amino acid sequence for the epitope that antibody is directed to is as shown in SEQ ID No:5.
2. the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 1, which is characterized in that the colloidal gold
The anti-human PCT detection antibody of label and the concentration of chicken IgY antibody are 0.2-0.5mg/ml, the package amount in the gold-labelled pad
It is 1.2ul/cm.
3. the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 1, which is characterized in that the gold-labelled pad
On colloid gold particle diameter be 35~45nm.
4. the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 1, which is characterized in that the detection zone
Close to the gold-labelled pad, the check plot is divided into 0.4cm between the blotting paper, the detection zone and the check plot.
5. the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 1, which is characterized in that the anti-PCT packet
It is 2mg/ml by the concentration of antibody, is 1.2ul/cm in the package amount of the detection zone.
6. the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 1, which is characterized in that the goat is anti-
The concentration of chicken IgY antibody is 0.7mg/ml, and the package amount in the check plot is 1.2ul/cm.
7. the preparation method of the colloidal gold immuno-chromatography test paper strip of the described in any item detection PCT of claim 1~6, feature
It is, comprising the following steps:
(1), anti-PCT coated antibody is prepared, fixes anti-PCT coated antibody and the anti-chicken IgY antibody of goat, shape respectively on coated film
At detection zone and check plot；
(2), anti-human PCT is detected antibody and chicken IgY antibody to be marked with colloidal gold, and be sprayed in gold-labelled pad；It is described anti-
The amino acid sequence for the epitope that people PCT detection antibody is directed to is as shown in SEQ ID No:5；
(3), on bottom liner overlap joint paste sample pad, gold-labelled pad, coated film and blotting paper to get.
8. the preparation method of the colloidal gold immuno-chromatography test paper strip of detection PCT according to claim 7, which is characterized in that
The detection antibody of anti-human PCT described in step (2) the preparation method comprises the following steps: according to PCT holoprotein sequence design difference peptide fragment, be immunized dynamic
Object obtains specific antibody；Specific antibody is matched, screening obtains anti-human PCT detection antibody.
9. a kind of colloidal gold immunochromatographykit kit for detecting PCT, which is characterized in that the kit includes claim 1~6
The colloidal gold immuno-chromatography test paper strip of described in any item detection PCT.
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|---|---|---|---|---|
| CN102395887A (en) * | 2009-04-28 | 2012-03-28 | B.R.A.H.M.S有限公司 | Immunoassay for the detection of procalcitonin |
| CN103645321A (en) * | 2013-12-03 | 2014-03-19 | 张超 | Test paper for screening procalcitonin and preparation method of test paper |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
| CN105510577A (en) * | 2015-11-27 | 2016-04-20 | 上海艾瑞德生物科技有限公司 | Method for rapidly and quantitatively detecting multiple analytes in blood by adopting multi-point calibration |
| CN105974110A (en) * | 2016-07-06 | 2016-09-28 | 北京康思润业生物技术有限公司 | Immune lateral chromatographic detection system as well as preparation method and application thereof |
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2017
- 2017-03-28 CN CN201710193931.1A patent/CN106841606B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102395887A (en) * | 2009-04-28 | 2012-03-28 | B.R.A.H.M.S有限公司 | Immunoassay for the detection of procalcitonin |
| CN103645321A (en) * | 2013-12-03 | 2014-03-19 | 张超 | Test paper for screening procalcitonin and preparation method of test paper |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
| CN105510577A (en) * | 2015-11-27 | 2016-04-20 | 上海艾瑞德生物科技有限公司 | Method for rapidly and quantitatively detecting multiple analytes in blood by adopting multi-point calibration |
| CN105974110A (en) * | 2016-07-06 | 2016-09-28 | 北京康思润业生物技术有限公司 | Immune lateral chromatographic detection system as well as preparation method and application thereof |
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Address after: No.79, Ruihe Road, Science City, Guangzhou hi tech Industrial Development Zone, Guangdong 510000 Patentee after: Reboo (Guangzhou) Biotechnology Co.,Ltd. Address before: 510530 No. 79 Ruihe Road, Luogang District, Guangzhou City, Guangdong Province Patentee before: RAYBIOTECH, Inc. |