CN105987998A - ELISA quantitative detection kit for human tissue kallikrein 1 - Google Patents
ELISA quantitative detection kit for human tissue kallikrein 1 Download PDFInfo
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- CN105987998A CN105987998A CN201510051031.4A CN201510051031A CN105987998A CN 105987998 A CN105987998 A CN 105987998A CN 201510051031 A CN201510051031 A CN 201510051031A CN 105987998 A CN105987998 A CN 105987998A
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- antibody
- human tissue
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- tissue kallikrein
- enzyme
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Abstract
The invention relates to an ELISA quantitative detection kit for detecting human tissue kallikrein 1 (hK1) and related antibodies. According to the invention, a plurality of monoclonal antibodies are prepared and are paired and screened, so an antibody combination (A24 and A32) with sensitivity and specificity meeting demands is obtained; and the antibody combination can be conveniently produced in a large scale and can meet demands of large-scale clinical application in the future. The antibody combination is subjected to adjustment and optimization of a detection system so as to obtain the ELISA quantitative detection kit for human tissue kallikrein 1 with the advantages of simple operation and good sensitivity, specificity and related detection performance.
Description
Technical field
The invention belongs to immunochemical technique field, be specifically related to detect human tissue kallikrein 1 (hK1) ELISA quantitative
Detection kit and associated antibodies thereof.
Background technology
Cardiovascular and cerebrovascular disease is the major chronic noninfectious of serious harm human health.Wherein coronary heart disease and apoplexy are generation
The modal cause of death in boundary.In China, along with the aging of population, with coronary heart disease, apoplexy is the cardiovascular and cerebrovascular vessel of representative
The sickness rate of disease, fatality rate and disability rate are in the trend risen year by year.But, the apoplexy of 80% is preventable.Sugar
The sick nephropathy of urine is the modal complication of diabetes, the quality of life of serious harm diabetics and Medical Consumption quality.If no
Taking positive intervening measure from the height of evidence-based medicine EBM, diabetic nephropathy will develop into irreversible in the short period of time
End stagerenaldisease, the existence life-span of serious threat patient.Therefore, effective method, early diagnosis apoplexy, sugar are actively found
The sick nephropathy of urine quality of life and the existence life-span of patient effectively to protect direct relation.
Kallikrein kinin system (kallikrein-kinin system, KKS), also known as kinin system, is widely present in animal body
Interior multiple systems, are especially distributed the most intensive in cardiovascular system.This system has extensive biologic activity, and and solidifying
There is close contact and crosstalk in blood system, renin-angiotensin system and multiple vasoactive factors system etc., altogether
With safeguard the normal physiological function of human body multiple organ and participate in various complexity pathophysiological process, have regulation cardiovascular, kidney,
Nervous system and glucose metabolism, vasodilator, participation inflammatory reaction, pain stimulation and shock reaction.In recent years about kassinin kinin
The clinical research of system is concentrated mainly on the effect in cardiovascular, kidney, central nervous system disease.
Human tissue kallikrein is the most important ingredient of kinin system, is one group of secreting type serine protease, comprises 15
Individual member.In all known tissue kallikreins, only human tissue kallikrein 1 (pancreas/kidney kallikrein,
Human kallikrein 1, hK1, KLK1, also known as kininogenase, Kininogenase) can effectively hydrolyze low molecular weight kininogen
(LMWK), discharge active kassinin kinin, and then play cardiovascular system and the regulation effect of renal function.Basic research
Show, it has proven convenient that hK1 has reduces blood pressure in multiple hypertension animal model, alleviate kidney and cardiac hypertrophy and fibrosis
Effect；It is carrying out heart reconstruction, alleviates kidney damage, reduces cerebral infarction incidence rate and reduces the sides such as nerve injury harm
The effect in face.Meanwhile, by the method for exogenous administration, also demonstrate hK1 and preventing apoplexy, cardiovascular and cerebrovascular vessel and kidney disease
Effect in terms of Bing.In the last few years, further investigations have shown that, hK1 level people cardiovascular and cerebrovascular disease occur development
During there is important clinical meaning, possibly as the index of predicting of stroke rate.The measurable apoplexy of hK1 level
Morbidity and 5 years, without event survival rate, can allow patient's early prevention taking appropriate measures, thus reduce brain to a certain extent
The probability of happening of apoplexy.It addition, hK1 can diagnosis early diabetic nephropathy earlier compared with Urinary Microalbumin Excretion rate.To sum up may be used
Seeing, the level of hK1 has important predictive value for cardiovascular and cerebrovascular disease and the diabetic kidney disease of people.Therefore, accurately
Measure the level of hK1, in clinic and scientific research, be respectively provided with important meaning.
In view of hK1 treatment in cardiovascular and cerebrovascular disease and diabetic kidney disease and predicting function, preparation hK1 detection by quantitative examination
Agent box has the using value of important clinical.Have hK1 scientific research immue quantitative detection reagent box both at home and abroad: wherein part kit is adopted
With competition law, antibody is polyclonal antibody, its trivial operations, calculates loaded down with trivial details and easily occurs that non-specific binding causes detection knot
Really error is bigger；Part kit uses the sandwich assay detection pattern of polyclonal antibody, also there is the impact of non-specific binding；
Other test kit uses the pattern of the double-antibody sandwich of polyclonal antibody and monoclonal antibody, and its detection performance needs further
Checking.The detection pattern of double antibody sandwich method can be prevented effectively from the problems referred to above.Therefore, multi-epitope, high sensitivity, specific
The preparation of antibody is one of key factor of high-quality human tissue kallikrein 1 immue quantitative detection reagent box.Natural hK1 exists
Multiple glycosylation sites, thus recombinant expressed hK1 may exist bigger with native protein in terms of other conformations such as glycosylation site
Difference.Use restructuring hK1 to carry out screening and arise that the specific antibody of screening can not effectively be known when specificity antibody screening
The problem of the most natural hK1.The present invention uses naive hK1 to carry out the screening operation of antibody, can be prevented effectively from sending out of above-mentioned situation
Raw, and have proven to the adhesion of itself and natural hK1.
Meanwhile, for meeting the different application demand in market, the present invention is prepared for three kinds of hK1 immue quantitative detection reagent boxes: people organizes sharp
Peptide release enzyme 1ELISA immue quantitative detection reagent box, can meet the application demand of general small comprehensive hospital, and can develop liter in the future
Level is chemoluminescence method detection by quantitative (automatically), meets the application demand of the fully-automated synthesis of general hospital.People organizes sharp
Peptide release enzyme 1 gold colloidal quantitative testing test paper card and human tissue kallikrein 1 fluorescent quantitation Test paper card, all can meet fast
Speed detection and the demand of bedside detection.Though wherein the sensitivity of gold colloidal quantitative assay and accuracy are not as fluorescent quantitation detection method,
But it is easy to use easy to spread and cost is relatively low, the detection by quantitative of disease indicators can be substantially met, be suitable for primary care system
In instant detection.Fluorescent quantitation possesses higher detection sensitivity, it is adaptable to inspection center that testing requirement is higher or large-scale doctor
The application demand of the bedside diagnosis of institute clinical department.
Summary of the invention
The technical problem to be solved in the present invention is to provide the antibody of energy human tissue kallikrein 1 effective, specific binding.More
Specifically:
The first object of the present invention is to provide two kinds of anti-human tissue kallikrein 1 antibody.
The first anti-human tissue kallikrein 1 antibody (A24),
Following complementary determining region is contained in its variable region of heavy chain: aminoacid sequence HCDR1 as shown in sequence SEQ ID NO:1,
HCDR2 as shown in sequence SEQ ID NO:2 and/or the HCDR3 as shown in sequence SEQ ID NO:3；
And its light-chain variable sequence contains following complementary determining region: aminoacid sequence is as shown in sequence SEQ ID NO:4
LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and/or the LCDR3 as shown in sequence SEQ ID NO:6.
Preferably the aminoacid sequence of the variable region of heavy chain of the antibody A 24 in the present invention is as shown in SEQ ID NO:7, and light chain can
Become the aminoacid sequence in district as shown in SEQ ID NO:8.
The second anti-human tissue kallikrein 1 antibody (A32),
Following complementary determining region is contained in its variable region of heavy chain: aminoacid sequence HCDR1 as shown in sequence SEQ ID NO:9,
HCDR2 as shown in sequence SEQ ID NO:10 and/or the HCDR3 as shown in sequence SEQ ID NO:11；
And its light-chain variable sequence contains following complementary determining region: aminoacid sequence is as shown in sequence SEQ ID NO:12
LCDR1, the LCDR2 as shown in sequence SEQ ID NO:13 and/or the LCDR3 as shown in sequence SEQ ID NO:14.
Preferably the aminoacid sequence of the variable region of heavy chain of the antibody A 32 in the present invention is as shown in SEQ ID NO:15, light chain
The aminoacid sequence of variable region is as shown in SEQ ID NO:16.
Second purpose of the present invention is to provide two kinds of single-chain antibodies, the aminoacid sequence such as SEQ ID NO of described single-chain antibody A24:
Shown in 17；The aminoacid sequence of described single-chain antibody A32 is as shown in SEQ ID NO:18.Preferably, described single-chain antibody
Without the HIS label being made up of six histidine in A24, A32.
The 3rd purpose of the present invention is to provide two kinds of nucleotide sequences encoding above-mentioned single-chain antibody, the core of coding single-chain antibody A24
Nucleotide sequence is as shown in SEQ ID NO:19, and the nucleotide sequence of coding single-chain antibody A32 is as shown in SEQ ID NO:20.
The 4th purpose of the present invention is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
The 5th purpose of the present invention is to provide a kind of recombinant host cell containing above-mentioned expression vector.Affiliated host cell can be
Escherichia coli, yeast or mammalian cell, preferably Pichia sp..
The 6th purpose of the present invention is to provide a kind of method producing above-mentioned single-chain antibody, including:
1) cultivate above-mentioned recombinant host cell under suitable conditions and express antibody；
2) then from host cell purification, collect antibody.
7th purpose of the present invention is to provide above-mentioned anti-human tissue kallikrein 1 antibody at detection human tissue kallikrein
Application in 1 content.
8th purpose of the present invention is to provide one group can carry out matching and detect the antibody of human tissue kallikrein 1 to group
Close；This antibody is high to the detection sensitivity of combination, and specificity is good.
9th purpose of the present invention is to provide one to utilize described anti-human tissue kallikrein 1 antibody test people to organize sharp
Peptide release enzyme 1 enzyme-linked immune quantitative detection reagent box, including be coated the ELISA Plate of A24 or A32 antibody, sample diluting liquid,
Standard substance, detection liquid, cleaning mixture, nitrite ion and stop buffer containing enzyme labelled antibody A32 or A24.Its detecting step mainly wraps
Include:
(1), after adding Sample dilution in the ELISA Plate being coated A24 or A32 antibody, Sample dilution dilution is added
Standard substance, negative control and serum or blood plasma wait sample this；
(2) the detection liquid of Sample dilution dilution is added；
(3) nitrite ion is added
(4) add stop buffer and read OD value.
Described enzyme labelled antibody A32 or A24 be horseradish peroxidase-labeled antibody A 32 or A24 (A32-HRP or
Or the antibody A 32 or A24 (A32-AP or A24-AP) of alkali phosphatase enzyme mark A24-HRP)；During described standard substance are
State's hamster ovary cell (CHO) expresses the purifying protein of recombined human tissue kallikrein 1.
Tenth purpose of the present invention is to provide one to utilize described anti-human tissue kallikrein 1 antibody test people to organize sharp
The colloidal gold immunochromatographimethod quantitative test card of peptide release enzyme 1, including sample absorption pad, gold mark pad, reaction film and adsorptive pads；Institute
State gold mark pad and be coated with the antibody A 32 or A24 of colloid gold particle labelling, described reaction film has detection band and quality control band, detection
Band position is coated with antibody A 24 or A32, and quality control band position is coated anti-His tag antibody or Protein L.
The 11st purpose of the present invention is to provide one to utilize described anti-human tissue kallikrein 1 antibody test people to organize kassinin kinin
The time resolution immunofluorescence chromatography quantitative test card of release enzyme 1, including sample absorption pad, fluorescent microsphere pad, reaction film and suction
Water cushion；Described fluorescent microsphere pad is coated with the antibody A described above 32 or A24 of fluorescent microsphere labelling, and described reaction film has inspection
Measuring tape and quality control band, detection band position is coated with antibody A 24 or A32 described above, and quality control band position is coated anti-His label and resists
Body or Protein L.
The preferred nitrocellulose filter of described reaction film.Described anti-His tag antibody preferred mouse-anti His antibody.
The present invention is prepared for Multiple Antibodies, and carries out pairing screening, it is thus achieved that sensitivity and specificity all can meet the antibody group of demand
Close (A24 and A32)；Its convenient a large amount of productions, can meet the demand of larger scale clinical application in the future simultaneously.To above-mentioned antibody group
Close the debugging Optimization Work carrying out detection system, it is thus achieved that easy and simple to handle, sensitivity, specificity and coherent detection performance can meet faces
The bed enzyme-linked immune quantitative detection reagent box of human tissue kallikrein 1 of pattern detection, the colloid of human tissue kallikrein 1
The time resolution immunofluorescence chromatography quantitative test card of gold immunochromatography quantitative test card and human tissue kallikrein 1.
Accompanying drawing explanation
Fig. 1. heavy chain of antibody and chain variable region gene electrophoretogram.Lane 1 is standard DNA, and Lane 2 is antibody A 24 weight chain variable
District DNA, Lane 3 be antibody A 24 variable region of light chain DNA, Lane 4 be antibody A 32 variable region of heavy chain DNA, Lane 5
For antibody A 32 variable region of light chain DNA.
Fig. 2. single-chain antibody structural representation.VHRepresent weight chain variabl area sequence, VLRepresenting light-chain variable sequence, His label is six
Individual histidine.
Fig. 3. single-chain antibody expresses the agarose gel electrophoresis figure of PCR primer.Figure (a) is A24 gene PCR product；Figure (b)
For A32 gene PCR product.
Fig. 4. recombinant pichia yeast strain abduction delivering supernatant culture fluid identifies figure.Fig. 4 (a) is antibody A 24 recombinant yeast pichia pastoris bacterium
Strain abduction delivering supernatant culture fluid identifies figure；Fig. 4 (b) is antibody A 32 recombinant pichia yeast strain abduction delivering supernatant culture fluid
Identify figure.Above-mentioned left figure be SDS-PAGE electroresis appraisal figure, right figure be Western blot qualification figure.
Fig. 5. single-chain antibody purification effect figure (SDS-PAGE).Figure (a) is antibody A 24, and figure (b) is antibody A 32.
Fig. 6. the Western Blot of antibody A 24 and A32 identifies figure.Swimming lane 1 is Urinary kallidinogenase (UK)；Swimming lane 2 is yeast expression
hK1；Swimming lane 3 is escherichia coli expression hK1；Swimming lane 4 expresses hK1 for CHO.Figure (a) is A32 antibody Western Blot
Result；Figure (b) is A24 antibody Western Blot result.
Fig. 7. enzyme-linked immunologic detecting kit standard curve of the present invention.Wherein abscissa is protein concentration (ng/mL)；Vertical coordinate is inspection
Survey OD450；R represents detection correlation coefficient, is 0.99980322.
Fig. 8. enzyme-linked immunologic detecting kit of the present invention detection specificity.Abscissa is detectable substance concentration (ng/mL)；Vertical coordinate is inspection
Survey OD value.UK represents Urinary kallidinogenase, is the human tissue kallikrein 1 extracted from human urine；KLK2 represents that people organizes
Kallikrein 2；KLK3 represents human tissue kallikrein 3.
Fig. 9. colloidal gold immunochromatographimethod quantitative test card of the present invention and time resolved immuno fluorometric chromatography quantitative test card structural representation.1
For sample pad, 2 be reaction film, 3 for absorption pad, 4 for nature controlling line (C line), 5 for detection line (T line), 6 for gold mark pad or
Fluorescence pad, 7 it is PVC sheet.
Figure 10. colloidal gold immunochromatographimethod quantitative test card standard curve of the present invention.Wherein abscissa is protein concentration (ng/mL)；Vertical seat
It is designated as T/C value；r2It is 0.992.
Figure 11. time resolution immunofluorescence of the present invention chromatography quantitative test card standard curve.Wherein abscissa is protein concentration (ng/mL)；
Vertical coordinate is detected value；r2It is 0.9952.
Figure 12. time resolution immunofluorescence chromatography detection by quantitative and enzyme linked immunosorbent detection results relevance
Detailed description of the invention
Definition
" antibody " also known as immunoglobulin, is the large-scale Y shape protein secreted by bone-marrow-derived lymphocyte of a class, it is possible to by Y shape
The immunoglobulin molecules of complementary site (antigen knot bound site) the specific binding target antigen on two of which bifurcated top, described
Target antigen such as protein, sugar, polynucleotide, fat, polypeptide, micromolecular compound etc..
" single-chain antibody " (scFv) refers to the variable region of heavy chain (V of antibodyH) and variable region of light chain (VL) by 15～20
Individual amino acid short peptide (linker) connects the single chain fusion protein formed, and is generally rich in glycine and silk for the linker connected
Propylhomoserin, is beneficial to stability and the pliability of single-chain antibody.Connected mode can be by VLN end be connected to VHC-terminal, or
Person is contrary.Although eliminating constant region and introducing linker, single-chain antibody still remains the antibody specificity to antigen, and its tool
Have that molecular weight is little, penetration power is strong and the feature such as antigenicity is weak.
Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Take shape in antibody list
The amino acid whose end of body, is the most critical zone of target antigen and antibodies, in Artificial Immune Network Theory, and the complementation of each antibody
Determine that district is otherwise known as idiotype or genotype.
Standard substance used by following example are CHO system expression restructuring hK1, and (1mg/mL, preparation method is shown in patent
201310746269.X)
The preparation of embodiment 1. anti-human tissue kallikrein 1 hybridoma cell strain
1. animal immune
With recombined human tissue kallikrein 1 (Chinese hamster ovary cell expression, preparation method is shown in patent 201310746269.X)
According to general immune programme for children immunity BALB/c female mice (purchased from this laboratory animal company limited of Cavan, Changzhou).Concrete immunity feelings
Condition sees " antibody preparation and use experiment guide ".Use indirect elisa method to follow the tracks of immune serum titre, choose serum
The immune mouse that titer is the highest, carries out fusion experiment by mouse boosting cell and murine myeloma cell.
2. cell merges
(1). the preparation of spleen cell
By immune mouse, pluck eyeball and take blood, put to death to be placed in the ethanol of 75% (v/v) through disconnected cervical vertebra and soak 10 minutes, in aseptic
Operating board takes out its spleen, is placed in cell screen cloth, be fully ground cell, cross screen cloth, (be purchased from by aseptic 1640 culture medium
Gibco company) centrifuge washing for several times after, re-suspended cell is to make single cell suspension, and counts, standby.
(2). the preparation of feeder cells
Take the female BAl BIc/c mice one of 8～10 week old, pluck eyeball and obtain negative serum, put to death rearmounted 75% (v/v) through disconnected cervical vertebra
Ethanol soaks 10 minutes；Aseptic skin of abdomen of opening, exposes peritoneum, about 10mL 1640HT culture medium (is purchased with syringe
From SIGMA company) inject mouse peritoneal, abdomen massage piping and druming are for several times gently.Draw the culture medium containing macrophage to inject
In 20%1640HAT culture medium standby；
Take the female BAl BIc/c mice one of 2～3 week old, put to death through disconnected cervical vertebra and be placed in 75% (v/v) ethanol immersion 10 points
Clock；The aseptic thymus that takes, in cell screen cloth, grinds, cross screen cloth, it is thus achieved that thymocyte cell is placed in above-mentioned containing macrophage
In 20%1640HAT culture medium, standby.
(3). cell merges
Select to be in the murine myeloma cell strain SP2/0 of exponential phase, collect and count.Take about 108Individual above-mentioned splenocyte with
2×107Individual above-mentioned SP2/0 cell strain adds in fusion pipe and mixes, and 1000rpm abandons supernatant (as far as possible abandoning clean) after being centrifuged 10 minutes,
Fusion pipe is put and rubs so that precipitating loose the most gently on palm.The PEG of 1mL preheating is added soon after elder generation is slow in 60 seconds
1450 (Polyethylene Glycol 1450, purchased from SIGMA company), add 1640HT culture medium 30mL and terminate, and 1000rpm is centrifuged
10 minutes, remove supernatant, gently friction make precipitation loose, add step 2 obtained 20% 1640HAT culture medium in.
After above-mentioned HAT culture medium is fully mixed, in 200 μ L/ hole subpackages to 96 porocyte culture plates, to put 37 DEG C, 5%CO2
Cell culture incubator in cultivate.Replace 20%1640HAT culture medium by 10%1640HT culture medium after one week, take after 3 days
Detect clearly.
3. anti-human tissue kallikrein 1 specific hybrid tumor cell strain screening
(1). the preparation of detection plate: be coated liquid dilution Urinary kallidinogenase (UK buys the general biochemical pharmaceuticals in sky, Guangdong) with CB
To 1 μ g/mL, being coated 96 hole ELISA ELISA Plate, 100 μ L/ holes, 2～8 DEG C are coated overnight, washed once and pat dry；Containing 2% cheese
The PBST buffer blind (200uL/ hole) of albumen, closes 2 hours for 37 DEG C；Pat dry, standby.
(2). the screening of positive colony: cells and supernatant 100 μ L/ hole to be checked is added in above-mentioned detection plate, act on 30 in 37 DEG C
Wash after minute and pat dry, adding the sheep anti-mouse igg of the HRP labelling in 100 μ L/ holes, washing also after acting on 30 minutes in 37 DEG C
Patting dry, add the TMB nitrite ion in 100 μ L/ holes, develop the color 15 minutes in 37 DEG C of lucifuges, every hole adds the 2M H of 50 μ L2SO4
Terminate reaction, and reading numerical values at OD450.Positive hole determines principle: OD450 value/negative control value >=2.1.Choose sun
Sex clone strain carries out Cell-cloned screening.After the cloning of three to four-wheel is screened, monoclonal cell strain positive rate 100%
I.e. it is defined as stable cell line, carries out determining strain to cell strain.Hybridoma cell strain C24 and C32 is respectively provided with higher titer, then
Follow-up further above-mentioned hybridoma cell strain is carried out antibody variable sequences sequencing analysis.
The mensuration of embodiment 2. hybridoma cell strain antibody variable sequences
Above-mentioned hybridoma cell strain C24 and C32 antibody variable sequences are measured.
The extraction of a.RNA: with reference to cell total rna extraction agent box (purchased from Roche company) description to above-mentioned hybridoma
Cell strain C24 and C32 carries out Total RNAs extraction and carries out reverse transcription immediately；
B.RNA reverse transcription becomes DNA: with reference to Thermo Scientific Reverted First strand cDNA Synthesis
Kit (purchased from Thermo company) carries out reverse transcription to the total serum IgE extracted in previous step, prepares cDNA, frozen in-20 DEG C
Standby；
C. the PCR of variable region sequences expands and reclaims: in previous step, gained cDNA is as template, with Mus IgG hypotype Dan Ke
Grand antibody variable sequences universal primer is primer, and the variable region sequences of heavy chain and light chain is carried out PCR amplification, is produced by PCR
Thing reclaims test kit (purchased from TIANGEN company) through DNA glue and reclaims, and sees accompanying drawing 1；
D. the clone of variable region sequences and sequencing: according to cloning vehicle pMD18-T kit (purchased from Takara company) explanation
Book, is attached with pMD18-T carrier respectively by heavy chain and chain variable region gene, converts bacillus coli DH 5 alpha, picking sun
Sex clone, transfers to InvitrogenTMCompany checks order.
Order-checking obtain the antibody heavy chain variable region aminoacid sequence of hybridoma cell strain C24 as shown in SEQ ID NO:7, light chain can
Become region amino acid sequence as shown in SEQ ID NO:8.The above-mentioned sequence of Vbase2 database analysis, its variable region of heavy chain each mutually
Mend and determine the aminoacid sequence in district respectively: HCDR1 as shown in sequence SEQ ID NO:1, such as sequence SEQ ID NO:
HCDR2 shown in the 2 and/or HCDR3 as shown in sequence SEQ ID NO:3；Each complementary determining region of its variable region of light chain
Aminoacid sequence be: the LCDR1 as shown in sequence SEQ ID NO:4, the LCDR2 as shown in sequence SEQ ID NO:5
And/or the LCDR3 as shown in sequence SEQ ID NO:6.
Order-checking obtains the antibody heavy chain variable region aminoacid sequence of hybridoma cell strain C32 as shown in SEQ ID NO:15, light chain
Variable region amino acid sequence is as shown in SEQ ID NO:16.The above-mentioned sequence of Vbase2 database analysis, its variable region of heavy chain each
The aminoacid sequence of complementary determining region is respectively: HCDR1 as shown in sequence SEQ ID NO:9, such as sequence SEQ ID NO:
HCDR2 shown in the 10 and/or HCDR3 as shown in sequence SEQ ID NO:11；The each complementary of its variable region of light chain determines
The aminoacid sequence in district is: LCDR1 as shown in sequence SEQ ID NO:12, as shown in sequence SEQ ID NO:13
LCDR2 and/or the LCDR3 as shown in sequence SEQ ID NO:14.
Recombinant expressed and the purification of embodiment 3. single-chain antibody
According to sequencing result in embodiment 2, respectively by the heavy chain of antibody of hybridoma cell strain C24 and C32 and variable region of light chain it
Between add connection peptides (GGGGS)3, introduce six histidine, and its full genome entered according to the preferences of pichia yeast expression system
The method that row is codon optimized, carries out the recombinant expressed of single-chain antibody.Expressed by the antibody that obtains be respectively designated as antibody A 24 He
Antibody A 32, its structure forms as shown in Figure 2.Above-mentioned single-chain antibody recombinant expressed has as follows:
1. the expression plasmid of antigen-4 fusion protein gene builds
The gene order of the antibody A 24 after codon optimized is as shown in SEQ ID NO:19, aminoacid sequence such as SEQ ID NO:
Shown in 17；The nucleotide sequence of the antibody A 32 after codon optimized is as shown in SEQ ID NO:20, aminoacid sequence such as SEQ
Shown in ID NO:18.The fragment upstream of the antibody A 24 and A32 full genome synthesis after optimizing introduces in pPICZ α A carrier
DNA sequence after XhoI sequence, downstream introduces XbaI enzyme cutting site, is building up to pMD19-T Simple Vector plasmid and (purchases
From Invitrogen company) in, obtaining one and preserve plasmid for a long time, plasmid is designated as pMD19-A24, pMD19-A32.Carry out
PCR expands, and wherein forward primer P1 is CGCCAGGGTTTTCCCAGTCAC GAC；Downstream primer P2 is:
AGCGGATAACAATTTCACACAGGA.After Standard PCR program, agarose gel electrophoresis analysis (accompanying drawing 3),
Show that two kinds of product sizes are consistent with expection size (790bp, 800bp).PCR obtains gene outcome reclaim after purification, adopt
With XhoI (#R0146S, purchased from New England Biolabs company) and XbaI, (#R0145V, purchased from New England
Biolabs company) double digestion, it is connected in pPICZ α A (V19520, purchased from Invitrogen) plasmid with T4 ligase, turns
Change in DH5 α competent cell, in the LB flat board containing Zeocin (R250-01, purchased from Invitrogen company) 37 DEG C
Overnight incubation.The order-checking of second day screening positive clone bacterium, comparison, completely the same with expected sequence, i.e. obtain antibody A 24 and
The expression plasmid of A32, is designated as pPICZ α-A24, pPICZ α-A24 respectively.
2. antigen-4 fusion protein gene Pichia sp. host's engineered strain structure, screen and express
Pichia sp. competent cell, YPDS solid medium, BMGY culture medium, BMMY culture medium: be purchased from Invitrogen
Company.
By pPICZ α-A24 and pPICZ α-A32 plasmid, with SacI digestion with restriction enzyme linearisation.Will after ethanol precipitation
Linearized vector, electricity converts and enters into X-33 competence yeast cells respectively, is respectively coated the YPDS containing Zeocin solid
Body culture medium, cultivates 3-5 days, just has positive colony to produce for 30 DEG C.
The monoclonal of the above-mentioned acquisition of picking, in 5mL BMGY culture medium, is cultivated to OD for 30 DEG C600When=2.0～6.0, take 1mL
Preserve strain, and by remain bacterium solution resuspended after transfer to BMMY Small Amount abduction delivering, add methanol to final concentration every 24h
It is 1% (v/v).After one week, centrifugal collection bacterium solution supernatant, by PAGE gel electrophoresis and Western blot analysis
(Western blot), object observing protein expression situation (accompanying drawing 4).In Western blot, one resists for anti-HIS-Tag antibody
(His-Tag (2A8) Mouse mAb, M20001 are purchased from Ai Bimate biological medicine (Shanghai) Co., Ltd.).
A24 and the A32 recombination fusion protein engineering strain of above-mentioned acquisition is inoculated in BMGY culture medium respectively,
30 DEG C, 220rpm cultivation to cell density to OD600=2.0～6.0, methanol extremely final concentration of 1.0% (v/v) was added every 24 hours.
After one week, collect fermentation culture.
3. fusion protein purification
Histidine-tagged affinity column antibody purification A24 and antibody A 32 fusion protein, prepackage pillar is used to be chosen as HisTrap HP,
Specifically comprise the following steps that
(1) the remove impurity pretreatment of fermentation liquid: above-mentioned expression is obtained antibody A 24 and A32 fusion protein fermented liquid supernatant, centrifugal collection
Supernatant, and add combine buffer so that supernatant final concentration of 300mM NaCl, 20mM NaH2PO4, 10mM
Imidazole, adjusts pH7.5,0.45 μm membrane filtration.
(2) HisTrap HP affinity column purification: (AKTA avant150, purchased from GE healcare to use fully-automatic intelligent protein purification system
Company) to pretreatment obtain antibody A 24 and antibody A 32 fusion protein fermentation liquid carry out affinity purification, pillar is HisTrap
HP (17-5248-02, purchased from GE healcare company).It is 300mM NaCl in conjunction with buffer, 20mM NaH2PO4,
10mM Imidazole, pH7.5, elution buffer is 300mM NaCl, 20mM NaH2PO4, 500mM Imidazole,
pH7.5.Carry out linear elution during eluting, and collect each eluting peak.By SDS-PAGE electroresis appraisal purity, by attached
Fig. 5 understands, and two kinds of purity of protein after purification all reach more than 95%；Merge satisfactory collecting pipe, change buffering
Liquid is PBS solution and (1mg/ml) is concentrated by ultrafiltration, and filtration sterilization saves backup in-20 DEG C.
Those skilled in the art know, and recombiant protein can not tape label, it is possible to carry other labels, it is possible to add other forms
Connection peptides.Regardless of whether the label of tape label or band multi-form all can use Capto L purification.
The performance evaluation of embodiment 4. antibody
1. the Western blot of antibody A 24 and A32 identifies
A. polyacrylamide gel electrophoresis: configure 12% separation gel, 5% concentration glue, respectively loading standard protein, You Ruike
Woods (UK), Pichia anomala expression hK1, escherichia coli expression hK1 and CHO system expression hK1, electrophoresis 1 under constant voltage
Hour；
B. transferring film: transferring film 1 hour under the conditions of constant current (35mA/ film), by the protein in two pieces of polyacrylamide gel respectively
It is transferred on two nitrocellulose filters.The SDS-PAGE glue completing transferring film is dyeed by Coomassie brilliant blue G250, observes
The residual condition of albumen；
C. close: the TBST buffer blind (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning mixture after closing (TBST,
Refer to TaKaRa company's T BST buffer) washed once, 10 minutes；
D. antigen antibody reaction: confining liquid dilution (by 1:400 volume ratio) horseradish peroxidase-labeled A24 (A24-HRP,
1mg/mL, our company uses classical Over-voltage protection labelling, lower same) and horseradish peroxidase-labeled A32 (A32-HRP,
1mg/mL, our company uses classical Over-voltage protection labelling, lower same), it is separately added in above-mentioned two nitrocellulose filters, room
Temperature reaction 1 hour；TBST washing 5 times, each 10 minutes；
E. developing the color and take pictures: blotting residual liquid on nitrocellulose filter, every nitrocellulose filter is separately added into 2mL and stablizes
Type Peroxidase Solution (1mL) and the mixed liquor (buying in Thermo company) of luminol/reinforcing agent solution (1mL),
The surface of uniform wet nitrocellulose filter, room temperature lucifuge is reacted after one minute in gel imaging system (buying in GE company)
Take pictures, leave and take result.
Experimental result (accompanying drawing 6) shows, the present invention two kinds of antibody all can react with the hK1 in four kinds of sources, it was demonstrated that the present invention
The reactivity of two kinds of antibody, and prove that itself and natural hK1 all have preferably reaction.It addition, experimental result is visible, four kinds of sources
The molecular weight of hK1 is all consistent with theoretical value: UK is natural hK1, and degree of glycosylation is the highest, therefore its molecular weight is maximum；Large intestine bar
It is minimum that bacterium expresses hK1 degree of glycosylation, therefore molecular weight is minimum；CHO system expression hK1 degree of glycosylation is slightly less than natural egg
White but higher than yeast expression hK1, therefore its molecular weight is between UK and yeast expression hK1.
2. antibody A 24 and A32 is in the performance evaluation of ELISA detection platform
Antibody in above-mentioned preparation example is carried out combinations of pairs, carries out pairing examination criteria product respectively as coated antibody or traget antibody,
Detecting step is as follows:
1) the PB buffer of employing 20mM PH7.4 by Y0, (irrelevant single-chain antibody, as negative control, join by preparation method
Add applicant's earlier application: 201410020156.6), A24 or A32 (as coated antibody) be diluted to 8ug/mL respectively, point
It is filled in ELISA Plate in (100uL/ hole), 4 DEG C, overnight (16 hours)；
2) PBST (the 20mM PB7.4 containing 0.05%Tween-20, lower same) pats dry after washed once (200uL/ hole)；
3) confining liquid (being the PBST of 2%BSA containing mass volume ratio) closes (200uL/ hole), 37 DEG C, 2 hours；Discard
Pat dry after confining liquid；
4) Sample dilution dilution standard product, are 200,100 and 0 to concentration (ng/mL).Respectively above-mentioned strength solution is added
Entering to step 3) in the ELISA Plate being coated with A24 and A32 that obtains (100uL/ hole), 37 DEG C are reacted 1 hour；PBST washs
Pat dry after 5 times (200uL/ holes)；
5) HRP labelling A24 (A24-HRP) or A32 (A32-HRP) are diluted (as labelling according to 1:2000 volume ratio
Antibody)；To step 4) obtained ELISA Plate is separately added into A32-HRP or A24-HRP of dilution, 37 DEG C are reacted 30 minutes；
PBST pats dry after washing 5 times；
6) adding TMB nitrite ion (buying in Huzhou Ying Chuan company), 100uL/ hole, 37 DEG C are reacted 15 minutes；
7) 2M H is added2SO4Stop buffer, 50uL/ hole, read OD450 in microplate reader (buying in Thermo company) immediately.
Result such as following table:
From the above results, the double-antibody sandwich detecting system of A32, A24 composition can be applicable to enzyme linked immunosorbent detection platform,
And A24 (being coated)-A32 (labelling), A32 (being coated)-A24 (labelling) two kinds pairing all has preferable Detection results.With
Irrelevant antibodies is without nonspecific reaction.
3. antibody A 24 and A32 is in the evaluation of gold colloidal detection platform
It is 200ng/ml, 100ng/ml with Sample dilution dilution standard product to concentration, by the two concentration and 0.025mol/L
The PBS of pH7.5 add respectively 50uL in colloidal-gold detecting-card (A24 is coated-A32 labelling or A32 is coated-A24 labelling,
Concrete preparation sees embodiment 6), in 10-15min, detection card is placed on readout instrument and detects.Testing result is as follows:
The above results is visible, and the double-antibody sandwich detecting system of A32, A24 composition can be applicable to gold colloidal detection platform, and A24
(being coated)-A32 (labelling), A32 (being coated)-A24 (labelling) two kinds pairing all has preferable Detection results.
4. antibody A 24 and A32 is in the evaluation of time-resolved fluorescence detection platform
With the PBS of 0.025mol/L pH7.5, A24 or A32 is diluted to 1mg/ml, lines on nitrocellulose filter；
With the borate buffer of 0.05mol/L pH8.0, A32 or A24 of time-resolved fluorescence microsphere labelling is diluted 20 times, specking
On pad；As shown in accompanying drawing 9 pad pasting, cutting, be installed (concrete preparation see embodiment 7).Detection card is detected dense respectively
Degree content is 200, the standard substance of 100ng/ml and the PBS of 0.025mol/L pH7.5, and testing result is as follows:
From the above results, it is flat that the double-antibody sandwich detecting system that A32, A24 form can be applicable to time-resolved fluorescence detection
Platform, and A24 (being coated)-A32 (labelling), A32 (being coated)-A24 (labelling) two kinds pairing all has preferable Detection results
Embodiment 5. antibody A 24 and A32 application in enzyme-linked immune quantitative detection reagent box
This detection method uses the principle of double antibody sandwich method.
1. material:
It is coated buffer: the PB buffer of 0.02M PH7.4；Confining liquid: containing 2%BSA's (mass volume ratio, lower same)
PBST buffer；Cleaning mixture: PBST buffer；Sample dilution: containing 1%BSA, 0.5% casein, 5% lowlenthal serum,
The phosphate buffer (PH7.4) of 0.02%Procline 300 and 0.05%Tween-20；Standard substance: CHO system expression is recombinated
HK1 (1mg/mL, preparation method is shown in patent 201310746269.X)；Nitrite ion: TMB (buys in Huzhou Ying Chuan company)；
Stop buffer: 2M H2SO4。
2. instrument:
Microplate reader (is bought in Thermo company), and universal microbiological incubator (is bought in Thermo company), automatic washing
Trigger (is bought in Shenzhen Hui Song company)
3. method:
(1) preparation of plate is detected: be coated buffer and antibody A 24 is diluted to 8ug/mL, 100uL/ hole adds in ELISA Plate, 4 DEG C
Stand overnight (16 hours)；PBST pats dry after washed once；200uL/ hole confining liquid is closed, 37 DEG C place 2 hours after pat dry,
Draining overnight (16 hours), aluminium foil bag is vacuum-packed, and 4 DEG C save backup；
(2) preparation of Sample dilution: containing 1%BSA, 0.5% casein, 5% lowlenthal serum, 0.02%Procline 300 and
The phosphate buffer (20mM, PH7.4) of 0.05%Tween-20, according to mentioned component proportional arrangement Sample dilution, in 4 DEG C
Save backup；
(3) preparation of standard substance: added to 19.99mL Sample dilution by 10uL standard substance, fully in 4 DEG C of guarantors after mixing
Deposit standby；
(4) preparation of liquid is detected: horseradish peroxidase (HRP) is coupled to antibody A 32 (A32-HRP, 1mg/ml)；
5uL A32-HRP is added to the Sample dilution of 9.995mL, fully saves backup in 4 DEG C after mixing.
4. the using method of Human kallikrein 1 enzyme-linked immunologic detecting kit
(1) in 4 DEG C take out vacuum-packed detection ELISA Plate and related reagent after equilibrium at room temperature standby；
(2) dilution of standard substance: added to 490uL Sample dilution by 10uL standard substance, fully i.e. obtains 10ng/mL after mixing
Standard solution；By 10ng/mL standard solution doubling dilution, obtain respectively final concentration (ng/mL) be 5,2.5,1.25,
0.625, the standard solution of 0.3125,0.15625 and 0.078125；With Sample dilution as negative control, i.e. 0ng/mL；
(3) ELISA Plate is separately added into the Sample dilution in 50uL/ hole；Again in ELISA Plate add standard substance, negative control with
And sample (50ul/ hole) to be checked；Place 60 minutes for 37 DEG C；PBST pats dry after washing 5 times；
(4) detection hole adds detection liquid, 100uL/ hole, place 30 minutes for 37 DEG C；PBST pats dry after washing 5 times；
(5) detection hole adds nitrite ion, 100uL/ hole, place 15 minutes for 37 DEG C；
(6) detection hole adds stop buffer, 50uL/ hole, read OD450 in microplate reader immediately.
According to the OD450 readings that each concentration of standard substance is corresponding, use CurveExpert 1.3 software with concentration as abscissa, OD
Value carries out the drafting of standard curve for vertical coordinate, it is thus achieved that standard curve equation；The OD value of detection sample is substituted into standard curve side
The concentration (ng/mL) of sample to be checked is calculated after journey.
5. detection performance evaluation:
The performance evaluation of detection method of the present invention is carried out according to above-mentioned testing conditions.
(1) TIANZHU XINGNAO Capsul:
The response rate is the index of checking detection accuracy, and detection kit based on A24 and A32 is added back by the present invention then
The checking of yield.
Obtaining pooled plasma after 20 parts of blood plasma mixing, the hK1 content using the method for the present invention to measure in pooled plasma B is
C0；The standard substance (A liquid) that concentration is respectively 5ng/mL, 2.5ng/mL, 1.25ng/mL and 0.625ng/mL join
In pooled plasma B, the volume ratio between added A liquid and blood plasma B is 1:9, calculates each addition according to formula (1)
The response rate.
In formula: R is the response rate；
V is the volume adding A liquid；
V0Volume for pooled plasma sample B；
C is the detectable concentration after pooled plasma sample B adds A liquid；
C0Detectable concentration for plasma sample B；
CSConcentration for A liquid.
Examination criteria curve of the present invention (accompanying drawing 7) is visible, and this detection has good linear relationship (r=0.99) and has Gao Ling
Sensitivity (156pg/mL) and negative background values (OD=0.0525).According to above-mentioned TIANZHU XINGNAO Capsul computational methods, result of calculation
See table:
In table visible, aforementioned four concentration TIANZHU XINGNAO Capsul meansigma methods is 101.17%, and each concentration TIANZHU XINGNAO Capsul is equal with the response rate
Value difference is less than or equal to 8.29%, it was demonstrated that this detection method accuracy is high.
(2) specificity:
Visible through protein sequence comparison, human tissue kallikrein 1 organizes kassinin kinin to release with the people in human tissue kallikrein family
The homology putting enzyme 2 (KLK2) and human tissue kallikrein 3 (KLK3) respectively reaches 66% and 60%, this experiment then
The detection method that the main investigation present invention the is set up detection specificity to KLK2 and KLK3.Detection method is as it has been described above, divide
Not Jia Ru Urinary kallidinogenase (UK) after Sample dilution gradient dilution, KLK2 (buying in R&D company) and KLK3 (purchases
Buy in R&D company), the final specificity detecting this detection method.Testing result (accompanying drawing 8) is visible, of the present invention
Human tissue kallikrein 1 enzyme linked immunological quantitative assay specificity is good.
(3) repeatability
By duplicate detection 10 times respectively of three parts of representative plasma samples, calculate meansigma methods M and the mark of 10 measurement results
According to formula CV=SD/M × 100%, quasi-difference SD, show that coefficient of variation CV result is as follows:
The above results is visible, and three parts of blood plasma detection CV < 10%, ELISA detection kit of the present invention has preferable repeatability.
6. the present invention attempts plurality of reagents box preparation method, following table show several different preparation methods (step unlisted in table,
Parameter is equal to described in the present embodiment) and the detection performance of corresponding reagent box.
The preparation of the colloid gold immune detection card of embodiment 6. anti-human tissue kallikrein 1
1. the labelling of gold colloidal
The colloid gold label of antibody A 32: use K2CO3Regulation gold colloidal pH value (adds 5uL 0.2M in every 1ml gold colloidal
K2CO3), the antibody A 32 being slowly added to dilute with the PBS of 0.2M in colloidal gold solution (adds 30ug in every 1ml gold colloidal
Antibody A 32), stirring at low speed 30 minutes；Add confining liquid (1%BSA) to its final concentration of 10% (mass percent),
Stir 20 minutes；After standing 30min, 12000rpm is centrifuged 30min；Remove supernatant gold labeling antibody redissolution liquid 0.01M PB
PH7.4+1%BSA+0.025%Tween 20+5% sucrose) redissolve and i.e. obtain the A32 antibody of colloid gold label.
2. prepared by gold mark pad and reaction film
After A32 gold labeling antibody being diluted well, it is sprayed on gold mark pad 6, drying for standby；Antibody A 24 and anti-His label are resisted
Body with after coated antibody diluent (3% methanol+25mM PBS (pH7.5)) dilution well, is coated on anti-respectively respectively
Answer the T line 5 of film 2 (nitrocellulose filter), C line 4 position, the most standby.
3. pad pasting, cut film, assembling
By sample pad 1, gold mark pad 6, be coated with the nitrocellulose filter 2 of antibody, adsorptive pads 3 set gradually from left to right (as
Shown in Fig. 9), and should contact a little between any two, described in be coated with the T line 5 of nitrocellulose filter of antibody at left, C line 4
On the right side, and cut according to shell size, load shell, complete detection card preparation.
4. test kit assembles
The detection card that will assemble, desiccant, dropper loads in aluminium foil bag, and heat sealing machine seals, and labels.
5. the using method of the colloid gold immune detection card of anti-human tissue kallikrein 1
1) Sample dilution (the PB buffer of 10mM PH7.4) recovers to room temperature, and concussion mixing is standby；
2) detection Sample Dilution: add 1mL Sample dilution at clean centrifuge tube, more accurately draw 10 μ L serum/plasma
Sample, joins in centrifuge tube, vibration fully mixing.
3) sample-adding and interpretation: the sample after drawing 50 μ L dilutions with liquid-transfering gun is slowly added in well, starts timing, 10～15
Being placed on readout instrument by detection card in minute and detect, detection card will be scanned by instrument, and show result at reading
On the screen of instrument.Judging more than 15min clock, result is invalid.
6. the colloid gold immune detection card Detection results assessment of anti-human tissue kallikrein 1
1) range of linearity detection
Measure the standard substance (0.125,0.25,0.5,1,2,4,8,16ng/ml) of variable concentrations, draw standard curve, line
Property detection range, result is as shown in Figure 10.Detection card sensitivity is 0.125ng/ml.
2) repeatability detection
Same batch detection card carries out duplicate detection 10 times to 1ng/ml, 4ng/ml sample, calculates coefficient of variation CV,
CV=standard deviation SD/ average M × 100%
| Concentration (ng/ml) | 1 | 4 |
| CV | 5.4% | 8.9% |
3) accuracy detection (TIANZHU XINGNAO Capsul)
The ability of pure analyte is added for assessing this detection kit Accurate Determining.Pooled serum is divided into that volume is identical 3 parts,
2 parts add standard substance wherein, make the recovery sample of 5ng/ml, 1.25ng/ml concentration, add in another part of sample with
The PB buffer of the PH7.4 without measured object of sample amount, makes basis sample.
| Concentration (ng/ml) | 1.25 | 5 |
| The response rate | 92.3% | 89% |
7., in addition to above-mentioned optimum preparation method, the present invention have also been attempted multiple preparation scheme, 4 kinds of preparation examples of such as following table:
The preparation of the time-resolved fluoroimmunoassay detection card of embodiment 7. human tissue kallikrein 1
This detection method uses the principle of double-antibody sandwich immunochromatographic method.
1, solution preparation
Prepared by the borate buffer of 0.05mol/L pH8.0: take the H of 0.1mol/L3BO370ml, with 0.025mol/L's
Na2B4O7·10H2O regulates pH to 8.0, and is settled to 100ml, be placed in 4 DEG C standby, 3 months effect duration.
Prepared by 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC, purchased from SIGMA company) solution: 1.5gEDC
Add 100ml deionized water, be made into aqueous solution be placed in 4 DEG C standby, 3 months effect duration.
The preparation of confining liquid: containing 10%BSA (described percentage ratio is mass volume ratio), the boric acid of 0.05mol/L pH8.0 delays
Rush liquid, use 0.22um membrane filtration, be placed in 4 DEG C standby, 7 days effect duration.
Prepared by fluorescent antibody diluent: containing 1%BSA, 10% sucrose, 0.025% tween 20, the boric acid of 0.05mol/L pH8.0
Buffer, uses 0.22um membrane filtration, be placed in 4 DEG C standby, 7 days effect duration.
Prepared by coated antibody diluent: containing 1% sucrose, the PBS of 3% methanol 0.025mol/L pH7.5, use 0.22U membrane filtration,
Be placed in 4 DEG C standby, 7 days effect duration.
Sample dilution: containing 0.1%BSA, 0.025% tween 20,0.01% phenazone, 0.02%proclin300,0.05mol/L
The borate buffer of pH8.0,10-30 DEG C preserves 1 year effect duration.
2, prepared by human tissue kallikrein 1 fluoroscopic examination card
1) time-resolved fluorescence microsphere labelling
A32 antibody labeling method is following (as a example by 500uL reaction system): adds 400uL borate buffer solution and is centrifuged in 2ml
Guan Zhong, adds the unloaded fluorescent microsphere (purchased from Thermo company) of 100uL concentration 1% particle diameter 200nm, and vortex oscillation mixes.
Add 10uLEDC solution, shaken at room temperature 15min.10 DEG C of centrifugal 10min of 14000rpm, go supernatant, precipitation 0.5ml
Borate buffer dissolves, ultrasonic disperse, power 100W, time 1min (ultrasonic 3s is spaced 3s).
Microsphere after activation adds the A32 antibody 50uL of concentration 1mg/ml, 250r/min20 DEG C of isothermal vibration reaction 2h；Add
Enter 55uL confining liquid, isothermal vibration reaction 4h.10 DEG C of centrifugal 15min of 14000rpm, wash 2 times, go supernatant, precipitation to use
0.5ml borate buffer dissolves, and last centrifuged deposit fluorescent antibody diluent dissolves and ultrasonic disperse, is placed in 4 DEG C
Constant temperature preserves.
2) specking of fluorescence pad
A32 antibody fluorescence pad specking method is as follows: the A32 fluorescent antibody of above-mentioned preparation diluted with fluorescent antibody diluent
20 times, specking is on whole piece pad.
3) being coated of nitrocellulose filter
Nitrocellulose filter method for coating is as follows: takes the A24 antibody of 0.5ml concentration 4mg/ml, is added to 5ml graduated centrifuge tube
In, add coated antibody diluent to 1ml, be coated in T line 5 position of nitrocellulose filter 2.Taking 0.5ml concentration is 4mg/ml
Anti-HIS antibody, is added in centrifuge tube, adds coated antibody diluent to 1ml, is coated in 4, the C line of nitrocellulose filter 2
Put.
4) pad pasting, cutting, be installed
Sample pad 1, fluorescence pad 6, be coated with A24 antibody nitrocellulose filter (reaction film) 2, by adsorptive pads 3 from
Left-to-right sets gradually, and contacts the most a little, the T line 5 of described nitrocellulose filter left, C line 4 right (as
Shown in accompanying drawing 9), and cut according to the size that gets stuck, loading is got stuck, and completes detection card preparation.
5) test kit assembles
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；Detection blocked, desiccant loads in aluminium foil bag；Seal with heat sealing machine
Good aluminium foil bag；Labelled.
Meanwhile, applicant be also adopted by A24 antibody do fluorescent labeling, use A32 antibody be coated on nitrocellulose filter (reaction film)
At 2T line, remaining step, parameter constant, carry out fluoroscopic examination card and prepare.
3, the using method of human tissue kallikrein 1 fluoroscopic examination card
1) dilution sample to be tested: add 1mL Sample dilution at clean centrifuge tube, more accurately draw 10 μ L serum/plasma
Sample, joins in centrifuge tube, vibration fully mixing.
2) sample-adding and interpretation: the sample after drawing 50 μ L dilutions with liquid-transfering gun is slowly added in well, starts timing, 10～15
Result is quantitatively judged with fluorescence immune chromatography instrument in minute.Judging more than 15 minutes, result is invalid.
4, human tissue kallikrein 1 fluoroscopic examination card Detection results assessment
1) elaboration: by A32 (being coated)-A24 (labelling) detection card detection 0.25,1, the standard substance of 4ng/ml repeat for each 25 times
Measure, after rejecting outlier, calculate detection card precision.Experimental result shows three Concentration Testing result coefficient of variation CV < 15%.
| Concentration point (ng/ml) | 0.25 | 1 | 4 |
| CV | 14% | 13% | 9% |
A24 (being coated)-A32 (labelling) detection card is repeated above-mentioned experiment, result such as following table,
| Concentration point (ng/ml) | 0.25 | 1 | 4 |
| CV | 18% | 16% | 14% |
0.0625,0.125 2) detection is linear: by the standard substance of A32 (being coated)-A24 (labelling) detection card detection variable concentrations:,
0.25,0.5,1,2,4,8,16,32ng/ml, standard curve and linear relationship (such as accompanying drawing 11).Detection card detection is sensitive
Degree is 0.0625ng/ml.
A24 (being coated)-A32 (labelling) detection card sensitivity is 0.0625ng/ml.
3) the accuracy response rate: A32 (being coated)-A24 (labelling) detection card detection addition is respectively 1,5,20ng/ml
Standard substance, testing result such as following table.
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The response rate | 112% | 94% | 104% |
A24 (being coated)-A32 (labelling) detection card is repeated above-mentioned experiment, result such as following table,
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The response rate | 116% | 108% | 89% |
5, accuracy methodology comparison:
The detection card performance of the above results display A32 (being coated)-A24 (labelling) is more excellent, is therefore made methodology comparison.Choosing
Select 20 parts of clinical patient specimen, by the serial number of 1 to 20, same with ELISA (prepared by embodiment 5) and fluorescence detection
Shi Jinhang tests, and according to 1,2,3......18, the sample order of 19,20,20,19,18......3,2,1 is measured.ELISA
Coefficient R with fluorescence detection testing result2=0.98, illustrate that fluorescence detection has preferable phase with ELISA testing result
Closing property (such as accompanying drawing 12).
6, the contrast of formula:
In addition to above-mentioned optimum preparation example 1, applicant also attempts multiple preparation scheme, such as below 5 groups of detection card preparations and application
Result such as following table:
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still may be made that various amendment
With conversion without departing from the spirit and scope of the present invention.Therefore, specification and drawings is considered as illustrative and non-limiting
's.
Claims (8)
1. the enzyme-linked immune quantitative detection reagent box of human tissue kallikrein 1, including the enzyme being coated antibody A 24 or A32
Target, sample diluting liquid, standard substance, detection liquid, cleaning mixture, nitrite ion and stop buffer containing enzyme labelled antibody A32 or A24；
Described antibody A 24 is anti-human tissue kallikrein 1 antibody, including:
Variable region of heavy chain, its aminoacid sequence contains following complementary determining region: HCDR1 as shown in sequence SEQ ID NO:1,
HCDR2 as shown in sequence SEQ ID NO:2 and/or the HCDR3 as shown in sequence SEQ ID NO:3；
And its variable region of light chain, its aminoacid sequence contains following complementary determining region: as shown in sequence SEQ ID NO:4
LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and/or the LCDR3 as shown in sequence SEQ ID NO:6；
Described antibody A 32 is anti-human tissue kallikrein 1 antibody, including:
Variable region of heavy chain, its aminoacid sequence contains following complementary determining region: HCDR1 as shown in sequence SEQ ID NO:9,
HCDR2 as shown in sequence SEQ ID NO:10 and/or the HCDR3 as shown in sequence SEQ ID NO:11；
And its variable region of light chain, its aminoacid sequence contains following complementary determining region: as shown in sequence SEQ ID NO:12
LCDR1, the LCDR2 as shown in sequence SEQ ID NO:13 and/or the LCDR3 as shown in sequence SEQ ID NO:14.
The enzyme-linked immune quantitative detection reagent box of human tissue kallikrein 1 the most according to claim 1, it is characterised in that
The aminoacid sequence of described antibody A 24 variable region of heavy chain is as shown in SEQ ID NO:7, and the aminoacid sequence of variable region of light chain is such as
Shown in SEQ ID NO:8.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 1, is characterized in that
Described antibody A 24 is single-chain antibody, and its aminoacid sequence is as shown in SEQ ID NO:17.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 3, is characterized in that
Without the HIS label being made up of six histidine in described antibody A 24.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 1, is characterized in that
The aminoacid sequence of described antibody A 32 variable region of heavy chain is as shown in SEQ ID NO:15, and the aminoacid sequence of variable region of light chain is such as
Shown in SEQ ID NO:16.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 1, is characterized in that
Described antibody A 32 is single-chain antibody, and its aminoacid sequence is as shown in SEQ ID NO:18.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 6, is characterized in that
Without the HIS label being made up of six histidine in described antibody A 32.
The enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 the most according to claim 1, is characterized in that
It is coated the PB buffer that buffer is 0.02M PH7.4；Confining liquid is the PBST buffer containing 2%BSA；Cleaning mixture is PBST
Buffer；Sample dilution be containing 1%BSA, 0.5% casein, 5% lowlenthal serum, 0.02%Procline 300 and
The phosphate buffer of 0.05%Tween-20, PH7.4；Standard substance are CHO system expression restructuring hK1；Nitrite ion is TMB；
Stop buffer: 2M H2SO4。
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