CN105987998B - Human tissue kallikrein 1ELISA immue quantitative detection reagent boxes - Google Patents
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- CN105987998B CN105987998B CN201510051031.4A CN201510051031A CN105987998B CN 105987998 B CN105987998 B CN 105987998B CN 201510051031 A CN201510051031 A CN 201510051031A CN 105987998 B CN105987998 B CN 105987998B
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Abstract
The present invention relates to detection (hK1) the ELISA immue quantitative detection reagent boxes of human tissue kallikrein 1 and its associated antibodies.The present invention is prepared for a variety of monoclonal antibodies, and carries out pairing screening, obtains the Antibody Combination (A24 and A32) of sensitivity and specificity energy meet demand；Its convenient a large amount of production simultaneously, can meet the needs of larger scale clinical application in the future.The debugging Optimization Work of detection architecture is carried out to above-mentioned Antibody Combination, obtains the enzyme-linked immune quantitative detection reagent box of easy to operate, sensitivity, specificity and all preferable human tissue kallikrein 1 of coherent detection performance.
Description
Technical field
The invention belongs to immunochemical technique field, is specifically related to detect human tissue kallikrein 1 (hK1) ELISA
Immue quantitative detection reagent box and its associated antibodies.
Background technology
Cardiovascular and cerebrovascular disease is the major chronic NCD for seriously endangering human health.Wherein coronary heart disease and brain soldier
In be the most common cause of death in the world.In China, with the aging of population, with coronary heart disease, cerebral apoplexy is the heart of representative
The incidence of disease of cranial vascular disease, fatal rate and disability rate are in the trend risen year by year.But 80% cerebral apoplexy is to prevent
's.Diabetic nephropathy is the most common complication of diabetes, seriously endangers the quality of life and Medical Consumption matter of diabetic
Amount.If not taking positive intervening measure from the height of evidence-based medicine EBM, diabetic nephropathy will develop into the short period of time
Irreversible ESRD, the existence life-span of serious threat patient.Therefore, effective method is actively found, is early diagnosed
Cerebral apoplexy, diabetic nephropathy are to carry out quality of life and the existence life-span that effective protection is directly related to patient.
Kallikrein kinin system (kallikrein-kinin system, KKS) is also known as kinin system, deposits extensively
The multiple systems being in animal body, especially it is distributed in cardiovascular system more intensive.The system has extensive biology to live
Property, and close contact be present with blood coagulation system, renin-angiotensin system and a variety of vasoactive factors systems etc.
And crosstalk, the normal physiological function of human body multiple organ is safeguarded jointly and participates in various complicated pathophysiological processes, is had
Angiocarpy, kidney, nervous system and glucose metabolism are adjusted, vasodilator, participates in inflammatory reaction, pain stimulation and shock instead
Should.The clinical research on kinin system is concentrated mainly on the work in angiocarpy, kidney, central nervous system disease in recent years
With.
Human tissue kallikrein is the most important part of kinin system, is one group of secreting type serine protease,
Include 15 members.In all known tissue kallikreins, only (pancreas/kidney kassinin kinin discharges human tissue kallikrein 1
Enzyme, also known as Human kallikrein 1, hK1, KLK1, kininogenase, Kininogenase) it can effectively hydrolyze low molecule amount and swash
Peptide former (LMWK), active kassinin kinin is discharged, and then play the adjustment effect of cardiovascular system and renal function.Basic research
Show, it has proven convenient that hK1 mitigates kidney and cardiomegaly and fibrosis with reducing blood pressure in a variety of hypertension animal models
Effect；It is carrying out heart reconstruction, mitigates kidney damage, reduces cerebral infarction incidence and reduces the sides such as neurotrosis harm
The effect in face.Meanwhile by the method for exogenous administration, also demonstrating hK1 is preventing apoplexy, cardiovascular and cerebrovascular and kidney trouble
The effect of aspect.In the last few years, further investigations have shown that, mistake of the hK1 levels in the occurrence and development of the cardiovascular and cerebrovascular disease of people
There is important clinical meaning, possibly as the index of predicting of stroke rate in journey.The hair of the horizontal predictable cerebral apoplexies of hK1
Disease and 5 years can allow patient's early prevention and taking appropriate measures, so as to reduce brain to a certain extent without event survival rate
The probability of happening of palsy.In addition, hK1 can be earlier compared with Urinary Microalbumin Excretion rate diagnosis early diabetic nephropathy.To sum up may be used
See, hK1 horizontal cardiovascular and cerebrovascular disease and diabetic kidney disease for people has important predictive value.Therefore, accurately
HK1 level is determined, important meaning is respectively provided with clinical and scientific research.
In view of treatments and predicting function of the hK1 in cardiovascular and cerebrovascular disease and diabetic kidney disease, prepare hK1 and quantitatively examine
Test agent box has the application value of important clinical.Have hK1 scientific research immue quantitative detection reagent boxes both at home and abroad：Which part reagent
Box uses competition law, and antibody is polyclonal antibody, its trivial operations, calculates cumbersome and easily non-specific binding occurs and cause to examine
It is larger to survey resultant error；Part kit uses the sandwich method detection pattern of polyclonal antibody, non-specific binding also be present
Influence；Other kit uses the pattern of polyclonal antibody and the double-antibody sandwich of monoclonal antibody, and its detection performance has
Treat further to verify.The detection pattern of double antibody sandwich method can effectively avoid above mentioned problem.Therefore, multi-epitope, high sensitivity are special
The preparation of the antibody of the opposite sex is one of key factor of the immue quantitative detection reagent box of human tissue kallikrein 1 of high quality.Naturally
Multiple glycosylation sites be present in hK1, thus recombinantly express hK1 may be with native protein in terms of other conformations such as glycosylation site
Larger difference be present.The specific antibody that screening just occurs is screened using restructuring hK1 in specificity antibody screening
The problem of natural hK1 can not effectively be identified.The present invention carries out the screening operation of antibody using naive hK1, can effectively avoid
The occurrence of stating, and have proven to its adhesion with natural hK1.
Meanwhile to meet the different application demand in market, the present invention is prepared for three kinds of hK1 immue quantitative detection reagent boxes：People's group
Kallikrein 1ELISA immue quantitative detection reagent boxes are knitted, the application demand of general small comprehensive hospital can be met, and can be sent out in the future
Exhibition upgrades to chemiluminescence standard measure detection (full-automatic), meets the application demand of the full-automatic detection of general hospital.People
The collaurum quantitative testing test paper card of tissue kallikrein 1 and the fluorogenic quantitative detection test card of human tissue kallikrein 1,
Meets the needs of quick detection and bedside detect.Wherein though the sensitivity of collaurum quantitative assay and the degree of accuracy are not so good as fluorescent quantitation
Detection method, but its easy to use easy to spread and cost is relatively low, can substantially meet the quantitative detection of disease indicators, be suitable for basic unit
Instant detection in medical system.Fluorescent quantitation possesses higher detection sensitivity, the inspection higher suitable for testing requirements
The heart or the application demand of the bedside of large hospital clinical department diagnosis.
The content of the invention
The technical problem to be solved in the present invention is to provide can be effective, specific binding human tissue kallikrein 1 anti-
Body.More specifically：
The first object of the present invention is to provide two kinds of antibody of anti-human tissue kallikrein 1.
The first antibody of anti-human tissue kallikrein 1 (A24),
Its weight chain variable district contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：Shown in 1
HCDR1, such as sequence SEQ ID NO：HCDR2 and/or such as sequence SEQ ID NO shown in 2：HCDR3 shown in 3；
And its light-chain variable sequence contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：4
Shown LCDR1, such as sequence SEQ ID NO：LCDR2 and/or such as sequence SEQ ID NO shown in 5：LCDR3 shown in 6.
The amino acid sequence such as SEQ ID NO of the weight chain variable district of antibody A 24 preferably in the present invention:Shown in 7, gently
The amino acid sequence of chain variable region such as SEQ ID NO:Shown in 8.
Second of antibody of anti-human tissue kallikrein 1 (A32),
Its weight chain variable district contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：Shown in 9
HCDR1, such as sequence SEQ ID NO：HCDR2 and/or such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；
And its light-chain variable sequence contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：12
Shown LCDR1, such as sequence SEQ ID NO：LCDR2 and/or such as sequence SEQ ID NO shown in 13：LCDR3 shown in 14.
The amino acid sequence such as SEQ ID NO of the weight chain variable district of antibody A 32 preferably in the present invention:Shown in 15,
The amino acid sequence of light chain variable district such as SEQ ID NO:Shown in 16.
Second purpose of the invention is to provide two kinds of single-chain antibodies, the amino acid sequence such as SEQ of the single-chain antibody A24
ID NO:Shown in 17；The amino acid sequence of the single-chain antibody A32 such as SEQ ID NO:Shown in 18.Preferably, it is described single-stranded anti-
Without the HIS labels being made up of six histidines in body A24, A32.
3rd purpose of the invention is to provide the nucleotide sequence of two kinds of above-mentioned single-chain antibodies of coding, encodes single-chain antibody
A24 nucleotide sequence such as SEQ ID NO:Shown in 19, and coding single-chain antibody A32 nucleotide sequence such as SEQ ID NO:20
It is shown.
4th purpose of the invention is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
5th purpose of the 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 pastoris.
6th purpose of the invention is to provide a kind of method for producing above-mentioned single-chain antibody, including：
1) above-mentioned recombinant host cell expression antibody is cultivated under suitable conditions；
2) and then from host cell purify, collect antibody.
The 7th purpose of the present invention is that provide the above-mentioned antibody of anti-human tissue kallikrein 1 organizes kassinin kinin in detection people
Discharge the application in the content of enzyme 1.
The 8th purpose of the present invention is to provide one group of antibody that can be matched and detect human tissue kallikrein 1
To combination；The antibody is high to the detection sensitivity of combination, and specificity is good.
The 9th purpose of the present invention is that providing one kind utilizes the antibody test people's group of anti-human tissue kallikrein 1
The enzyme-linked immune quantitative detection reagent box of kallikrein 1 is knitted, including has been coated with the ELISA Plate of A24 or A32 antibody, sample dilution
Liquid, standard items, detection liquid, cleaning solution, nitrite ion and terminate liquid containing enzyme labelled antibody A32 or A24.Its detecting step mainly wraps
Include:
(1) after adding Sample dilution into the ELISA Plate of coating A24 or A32 antibody, Sample dilution dilution is added
Standard items, negative control and serum or blood plasma wait sample sheet；
(2) the detection liquid of Sample dilution dilution is added；
(3) nitrite ion is added
(4) add terminate liquid and read OD values.
The enzyme labelled antibody A32 or A24 is the antibody A 32 or A24 (A32-HRP or A24- of horseradish peroxidase-labeled
HRP) or alkali phosphatase enzyme mark antibody A 32 or A24 (A32-AP or A24-AP)；The standard items are Chinese hamster ovary
The purifying protein of cell (CHO) expression restructuring human tissue kallikrein 1.
The tenth purpose of the present invention is that providing one kind utilizes the antibody test people's group of anti-human tissue kallikrein 1
Knit the colloidal gold immunochromatographimethod quantitative test card of kallikrein 1, including sample absorption pad, gold standard pad, reaction film and adsorptive pads；
The gold standard pad is coated with the antibody A 32 or A24 of colloid gold particle mark, has detection band and quality control band on the reaction film, examines
Measuring tape position is coated with antibody A 24 or A32, and quality control band position is coated with anti-His tag antibodies or Protein L.
11st purpose of the invention is that providing one kind utilizes the antibody test people's group of anti-human tissue kallikrein 1
Knit the time resolution immunofluorescence chromatography quantitative test card of kallikrein 1, including sample absorption pad, fluorescent microsphere pad, reaction
Film and adsorptive pads；The fluorescent microsphere pad is coated with the antibody A described above 32 or A24 of fluorescent microsphere mark, the reaction film
On have a detection band and quality control band, detection band position is coated with antibody A 24 described above or A32, and quality control band position is coated with anti-His marks
Sign antibody or Protein L.
The preferred nitrocellulose filter of reaction film.The anti-anti- His antibody of the preferred mouse of His tag antibodies.
The present invention is prepared for Multiple Antibodies, and carries out pairing screening, obtains sensitivity and specificity energy meet demand
Antibody Combination (A24 and A32)；Its convenient a large amount of production simultaneously, can meet the needs of larger scale clinical application in the future.To above-mentioned anti-
Body combination carries out the debugging Optimization Work of detection architecture, obtains easy to operate, sensitivity, and specificity and coherent detection performance can expire
The enzyme-linked immune quantitative detection reagent box of the human tissue kallikrein 1 of sufficient clinical sample detection, human tissue kallikrein 1
The time resolution immunofluorescence of colloidal gold immunochromatographimethod quantitative test card and human tissue kallikrein 1 chromatographs quantitative test card.
Brief description of the drawings
Fig. 1 heavy chain of antibody and chain variable region gene electrophoretogram.Lane 1 is standard DNA, and Lane 2 is the weight of antibody A 24
Chain variable region DNA, Lane 3 is that the light chain variable district DNA, Lane 4 of antibody A 24 is the weight chain variable district DNA, Lane 5 of antibody A 32
For the light chain variable district DNA of antibody A 32.
Fig. 2 single-chain antibody structural representations.VHRepresent weight chain variabl area sequence, VLRepresent light-chain variable sequence, His marks
Sign as six histidines.
Fig. 3 single-chain antibodies express the agarose gel electrophoresis figure of PCR primer.It is A24 gene PCR products to scheme (a)；Scheme (b)
For A32 gene PCR products.
Fig. 4 recombinant pichia yeast strain induced expression supernatant nutrient solution qualification figures.Fig. 4 (a) is that the restructuring of antibody A 24 is complete red
Yeast strain induced expression supernatant nutrient solution qualification figure；Fig. 4 (b) is the recombinant pichia yeast strain induced expression supernatant of antibody A 32
Nutrient solution qualification figure.Above-mentioned left figure is SDS-PAGE electroresis appraisals figure, right figure is Western blot qualification figures.
Fig. 5 single-chain antibody purification effect figures (SDS-PAGE).It is antibody A 24 to scheme (a), and figure (b) is antibody A 32.
The Western Blot qualification figures of Fig. 6 antibody As 24 and A32.Swimming lane 1 is Urinary kallidinogenase (UK)；Swimming lane 2 is yeast
Express hK1；Swimming lane 3 is Bacillus coli expression hK1；Swimming lane 4 is that CHO expresses hK1.Scheme (a) to tie for A32 antibody Western Blot
Fruit；It is A24 antibody Western Blot results to scheme (b).
Fig. 7 enzyme-linked immunologic detecting kit standard curves of the present invention.Wherein abscissa is protein concentration (ng/mL)；It is vertical to sit
It is designated as detecting OD450；R represents detection coefficient correlation, is 0.99980322.
Fig. 8 enzyme-linked immunologic detecting kit detection specificity of the present invention.Abscissa is detectable substance concentration (ng/mL)；It is vertical to sit
It is designated as detecting OD values.UK represents Urinary kallidinogenase, is the human tissue kallikrein 1 extracted from human urine；KLK2 represents people's group
Knit kallikrein 2；KLK3 represents human tissue kallikrein 3.
Fig. 9 colloidal gold immunochromatographimethod quantitative test cards of the present invention and time resolved immuno fluorometric chromatography quantitative test card knot
Structure schematic diagram.1 be sample pad, 2 be reaction film, 3 be absorption pad, 4 be nature controlling line (C lines), 5 be detection line (T lines), 6 be gold mark
Pad or fluorescence pad, 7 it is PVC sheet.
Figure 10 colloidal gold immunochromatographimethod quantitative test card standard curves of the present invention.Wherein abscissa is protein concentration (ng/
mL)；Ordinate is T/C values；r2For 0.992.
Figure 11 time resolution immunofluorescence chromatography quantitative test card standard curves of the present invention.Wherein abscissa is that albumen is dense
Spend (ng/mL)；Ordinate is detected value；r2For 0.9952.
The quantitative detection of Figure 12 time resolutions immunofluorescence chromatography and enzyme linked immunosorbent detection results relevance
Embodiment
Definition
" antibody " is also known as immunoglobulin, is a kind of large-scale Y shape protein secreted by bone-marrow-derived lymphocyte, can pass through Y shape
Two of which bifurcated top complementary site (antigen knot bound site) specific binding target antigen immunoglobulin molecules, institute
State target antigen such as protein, sugar, polynucleotides, fat, polypeptide, micromolecular compound etc..
" single-chain antibody " (scFv) refers to the weight chain variable district (V of antibodyH) and light chain variable district (VL) pass through 15~20
The single chain fusion protein that amino acid short peptide (linker) connection is formed, the linker for connection are generally rich in glycine and silk
Propylhomoserin, in favor of the stability and pliability of single-chain antibody.Connected mode can be by VLN-terminal be connected to VHC-terminal, Huo Zhexiang
Instead.Although eliminating constant region and introducing linker, single-chain antibody still remains specificity of the antibody to antigen, and it has
Molecular weight is small, penetration power is strong and it is antigenic weak the features such as.
Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Shaping
It is the most critical zone of target antigen and antibody binding in the end of antibody monomer amino acid, in Artificial Immune Network Theory, Mei Gekang
The complementary determining region of body is otherwise known as idiotype or genotype.
Standard items used are that (1mg/mL, preparation method are shown in patent to CHO system expressions restructuring hK1 in following examples
201310746269.X)
The preparation of the hybridoma cell strain of 1. anti-human tissue kallikrein of embodiment 1
1. animal immune
To recombinate human tissue kallikrein 1, (Chinese hamster ovary cell expression, preparation method are shown in patent
BALB/c female mices 201310746269.X) are immunized according to general immune programme for children and (it is limited to be purchased from this experimental animal of Changzhou Cavan
Company).Specific Immunity referring to《Antibody preparation is with using experiment guide》.Immune mouse blood is tracked using indirect elisa method
Clear titre, chooses serum titer highest and mouse is immunized, and mouse boosting cell and murine myeloma cell are carried out into fusion experiment.
2. cell fusion
(1) preparation of spleen cells
By immune mouse, pluck eyeball and take blood, be placed in 75% (v/v) alcohol and soak 10 minutes after disconnected cervical vertebra is put to death,
Its spleen is taken out in aseptic operating platform, is placed in cell screen clothes, is fully ground cell, screen cloth is crossed, with sterile 1640 culture medium
(being purchased from Gibco companies) centrifuge washing for several times after, cell is resuspended so that single cell suspension is made, and counts, it is standby.
(2) preparation of feeder cells
Female BAl BIc/c mouse one of 8~10 week old are taken, eyeball is plucked and obtains negative serum, are put to death through disconnected cervical vertebra rearmounted
Soaked 10 minutes in 75% (v/v) alcohol；It is sterile to open skin of abdomen, exposure peritonaeum, about 10mL 1640HT are trained with syringe
Support base (be purchased from SIGMA companies) injection mouse peritoneal, gently abdomen massage and piping and druming for several times.Draw the culture containing macrophage
It is standby in base injection 20%1640HAT culture mediums；
Female BAl BIc/c mouse one of 2~3 week old are taken, is placed in 75% (v/v) alcohol and soaks after disconnected cervical vertebra is put to death
10 minutes；It is sterile to take thymus gland in cell screen clothes, grinding, cross screen cloth, obtain thymocyte be placed in it is above-mentioned containing macrophage
It is standby in 20%1640HAT culture mediums.
(3) cell fusions
Murine myeloma cell strain SP2/0 of the selection in exponential phase, collects and counts.Take about 108Individual above-mentioned spleen
Cell and 2 × 107Individual above-mentioned SP2/0 cell lines, which are added in fusion pipe, to be mixed, and 1000rpm abandons supernatant (as far as possible after centrifuging 10 minutes
Abandon net), fusion pipe is put and gently rubbed back and forth on palm so that precipitation is loose.1mL preheatings are added after elder generation is slow in 60 seconds soon
PEG1450 (polyethylene glycol 1450, purchased from SIGMA companies), add 1640HT culture mediums 30mL and terminate, 1000rpm centrifuges 10 points
Clock, supernatant is removed, gently friction makes precipitation loose, adds in the 20% 1640HAT culture mediums that step 2 is obtained.
After above-mentioned HAT culture mediums are fully mixed, dispensed with 200 μ L/ holes into 96 porocyte culture plates, put 37 DEG C, 5%
Cultivated in CO2 cell culture incubator.20%1640HAT culture mediums are replaced with 10%1640HT culture mediums after one week, are taken after 3 days
Detected clearly.
3. the specific hybrid tumor cell strain of anti-human tissue kallikrein 1 screens
(1) preparation of detection plates：(UK, bought public in the general biochemical medicine in Guangdong day with CB coating buffers dilution Urinary kallidinogenase
Department) to 1 μ g/mL, 96 hole ELISA ELISA Plates, 100 μ L/ holes are coated with, 2~8 DEG C of coatings overnight, washed once and pat dry；Containing 2% junket
The PBST buffer blinds (200uL/ holes) of albumen, 37 DEG C are closed 2 hours；Pat dry, it is standby.
(2) screening of positive colonies：The μ L/ holes of cells and supernatant 100 to be checked are added in above-mentioned detection plate, in 37 DEG C
Effect was washed and patted dry after 30 minutes, is added the sheep anti-mouse igg of the HRP marks in 100 μ L/ holes, is washed after being acted on 30 minutes in 37 DEG C
Wash and pat dry, add the TMB nitrite ions in 100 μ L/ holes, developed the color 15 minutes in 37 DEG C of lucifuges, 50 μ L 2M H are added per hole2SO4Eventually
Only react, and the reading numerical values at OD450.Positive hole determines principle：OD450 values/negative control value >=2.1.Choose positive gram
Grand strain carries out Cell-cloned screening.After three to four-wheel cloning screening, monoclonal cell strain positive rate 100% is i.e. true
It is set to stable cell line, singling is carried out to cell line.Hybridoma cell strain C24 and C32 are respectively provided with higher potency, subsequently enter then
One step carries out antibody variable sequences sequencing analysis to above-mentioned hybridoma cell strain.
The measure of the hybridoma cell strain antibody variable sequences of embodiment 2.
Above-mentioned hybridoma cell strain C24 and C32 antibody variable sequences are measured.
A.RNA extraction：Reference cell total serum IgE extraction agent box (being purchased from Roche companies) specification is to above-mentioned hybridoma
Cell line C24 and C32 carry out Total RNAs extraction and carry out reverse transcription immediately；
B.RNA reverse transcriptions turn into DNA：With reference to Thermo Scientific Reverted First strand cDNA
Synthesis Kit (being purchased from Thermo companies) carry out reverse transcription to the total serum IgE extracted in previous step, and cDNA is made, and freeze
Be stored in -20 DEG C it is standby；
C. the PCR amplifications and recovery of variable region sequences：So that gained cDNA is template in previous step, with mouse IgG hypotype lists
Clonal antibody variable region sequences universal primer is primer, enters performing PCR amplification to the variable region sequences of heavy chain and light chain, PCR is produced
Thing is reclaimed through DNA glue reclaims kit (being purchased from TIANGEN companies), sees accompanying drawing 1；
D. the clone of variable region sequences and sequencing：According to cloning vector pMD18-T kit (being purchased from Takara companies)
Specification, heavy chain and chain variable region gene are attached with pMD18-T carriers respectively, convert bacillus coli DH 5 alpha, picking
Positive colony, transfer to InvitrogenTMCompany is sequenced.
Sequencing obtains hybridoma cell strain C24 heavy chain of antibody variable region amino acid sequence such as SEQ ID NO:Shown in 7, gently
Chain variable region amino acid sequence such as SEQ ID NO：Shown in 8.The above-mentioned sequence of Vbase2 database analysises, its weight chain variable district it is each
The amino acid sequence of complementary determining region is respectively：Such as sequence SEQ ID NO：HCDR1, such as sequence SEQ ID NO shown in 1：2 institutes
The HCDR2 and/or such as sequence SEQ ID NO shown：HCDR3 shown in 3；The amino acid of each complementary determining region of its light chain variable district
Sequence is：Such as sequence SEQ ID NO：LCDR1, such as sequence SEQ ID NO shown in 4：LCDR2 and/or such as sequence shown in 5
SEQ ID NO：LCDR3 shown in 6.
Sequencing obtains hybridoma cell strain C32 heavy chain of antibody variable region amino acid sequence such as SEQ ID NO:Shown in 15,
Chain variable region amino acid sequence such as SEQ ID NO：Shown in 16.The above-mentioned sequence of Vbase2 database analysises, its weight chain variable district
The amino acid sequence of each complementary determining region be respectively：Such as sequence SEQ ID NO：HCDR1, such as sequence SEQ ID shown in 9
NO：HCDR2 and/or such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；Each complementary determining region of its light chain variable district
Amino acid sequence be：Such as sequence SEQ ID NO：LCDR1, such as sequence SEQ ID NO shown in 12：LCDR2 shown in 13 and/
Or such as sequence SEQ ID NO：LCDR3 shown in 14.
The recombination expression of the single-chain antibody of embodiment 3. and purifying
According to sequencing result in embodiment 2, respectively by hybridoma cell strain C24 and C32 heavy chain of antibody and light chain variable
Connection peptide (GGGGS) is added between area3, six histidines are introduced, and by its full genome according to the inclined of pichia yeast expression system
The method that love property carries out codon optimization, carry out the recombination expression of single-chain antibody.Expressed obtained antibody is respectively designated as resisting
Body A24 and antibody A 32, its structure composition is as shown in Figure 2.The recombination expression of above-mentioned single-chain antibody has as follows：
1. the expression plasmid structure of antigen-4 fusion protein gene
The gene order of antibody A 24 after codon optimization such as SEQ ID NO:19 is shown, amino acid sequence such as SEQ ID
NO:Shown in 17；The nucleotide sequence of antibody A 32 after codon optimization such as SEQ ID NO:20 is shown, amino acid sequence such as SEQ
ID NO:Shown in 18.The fragment upstream that antibody A 24 after optimization and A32 full genomes synthesize is introduced into XhoI in pPICZ α A carriers
DNA sequence dna after sequence, downstream introduce XbaI enzyme cutting site, are building up to pMD19-T Simple Vector plasmids and (are purchased from
Invitrogen companies) in, a kind of long-term preservation plasmid is obtained, plasmid is designated as pMD19-A24, pMD19-A32.Enter performing PCR expansion
Increase, wherein sense primer P1 is CGCCAGGGTTTTCCCAGTCAC GAC；Anti-sense primer P2 is:
AGCGGATAACAATTTCACACAGGA.After Standard PCR program, agarose gel electrophoresis analysis (accompanying drawing 3), two kinds of products are shown
Size is consistent with expected size (790bp, 800bp).By PCR obtain gene outcome recovery purifying after, using XhoI (#R0146S,
Purchased from New England Biolabs companies) and XbaI (#R0145V, purchased from New England Biolabs companies) double enzymes
Cut, be connected to T4 ligases in pPICZ α A (V19520, purchased from Invitrogen) plasmid, be transformed into DH5 α competent cells
In, 37 DEG C of overnight incubations in the LB flat boards containing Zeocin (R250-01, purchased from Invitrogen companies).Screen within second day
Positive colony bacterium is sequenced, and compares, completely the same with expected sequence, that is, obtains antibody A 24 and A32 expression plasmid, be designated as respectively
pPICZα-A24、pPICZα-A24。
2. antigen-4 fusion protein gene is in the structure of Pichia pastoris host's engineered strain, screening and expression
Pichia pastoris competent cell, YPDS solid mediums, BMGY culture mediums, BMMY culture mediums：It is purchased from
Invitrogen companies.
By pPICZ α-A24 and pPICZ α-A32 plasmids, linearized with SacI digestion with restriction enzyme.After ethanol precipitation
By linearized vector, electricity conversion respectively enters X-33 competence yeast cells, is respectively coated the YPDS containing Zeocin and consolidates
Body culture medium, 30 DEG C are cultivated 3-5 days, just there is positive colony generation.
The monoclonal of the above-mentioned acquisition of picking is in 5mL BMGY culture mediums, 30 DEG C of cultures to OD600When=2.0~6.0, take
1mL preserves strain, and will be transferred to BMMY Small Amount induced expressions after the resuspension of remaining bacterium solution, and it is dense to end to add methanol every 24h
Spend for 1% (v/v).After one week, bacterium solution supernatant is collected by centrifugation, passes through PAGE gel electrophoresis and Western blot analysis
(Western blot), object observing protein expression situation (accompanying drawing 4).Primary antibody is anti-HIS-Tag antibody in Western blot
(His-Tag (2A8) Mouse mAb, M20001, be purchased from Ai Bimate biological medicines (Shanghai) Co., Ltd.).
A24 the and A32 recombination fusion proteins engineering strain of above-mentioned acquisition is inoculated in BMGY culture mediums respectively, 30
DEG C, 220rpm is cultivated to cell density to OD600=2.0~6.0, methanol was added every 24 hours to final concentration of 1.0% (v/
v).After one week, fermentation culture is collected.
3. fusion protein purification
Using histidine-tagged affinity column antibody purification A24 and the fusion protein of antibody A 32, prepackage pillar selection is
HisTrap HP, are comprised the following steps that：
(1) the removal of impurities pretreatment of zymotic fluid：Above-mentioned expression is obtained into antibody A 24 and A32 fusion protein fermented liquid supernatants, from
The heart collects supernatant, and adds combination buffer so that supernatant final concentration of 300mM NaCl, 20mM NaH2PO4, 10mM
Imidazole, adjust pH7.5,0.45 μm of membrane filtration.
(2) HisTrap HP affinity columns purify：With fully-automatic intelligent protein purification system, (AKTA avant150, are purchased from
GE healcare companies) to the antibody A 24 and the fusion protein zymotic fluid of antibody A 32 progress affinity purification of pretreatment acquisition, pillar
For HisTrap HP (17-5248-02, purchased from GE healcare companies).Combination buffer is 300mM NaCl, 20mM
NaH2PO4, 10mM Imidazole, pH7.5, elution buffer is 300mM NaCl, 20mM NaH2PO4, 500mM
Imidazole, pH7.5.Linear elution is carried out during elution, and collects each eluting peak.By SDS-PAGE electroresis appraisal purity,
From accompanying drawing 5, two kinds of purity of protein after purification reach more than 95%；Merge satisfactory collecting pipe, change buffering
Liquid is PBS solution and is concentrated by ultrafiltration (1mg/ml) that filtration sterilization saves backup in -20 DEG C.
Those skilled in the art know, recombinant protein can not tape label, also can also add other shapes with other labels
The connection peptide of formula.Regardless of whether tape label or with various forms of labels all can use Capto L purify.
The performance evaluation of the antibody of embodiment 4.
1. antibody A 24 and A32 Western blot are identified
A. polyacrylamide gel electrophoresis：12% separation gel, 5% concentration glue are configured, respectively loading standard protein, You Rui
Crin (UK), Pichia anomala expression hK1, Bacillus coli expression hK1 and CHO system expression hK1, electrophoresis 1 hour under constant pressure；
B. transferring film：Transferring film 1 hour under the conditions of constant current (35mA/ films), by the protein on two blocks of polyacrylamide gels point
It is not transferred on two nitrocellulose filters.Coomassie brilliant blue G250 dyes to the SDS-PAGE glue for completing transferring film, observes
The residual condition of albumen；
C. close：TBST buffer blinds (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning solution after closing (TBST,
Refer to TaKaRa company's T BST buffer) it washed once, 10 minutes；
D. antigen-antibody reaction：Confining liquid dilution (presses 1:400 volume ratios) horseradish peroxidase-labeled A24 (A24-
HRP, 1mg/mL, our company are marked using classical Over-voltage protection, similarly hereinafter) and horseradish peroxidase-labeled A32 (A32-HRP,
1mg/mL, our company are marked using classical Over-voltage protection, similarly hereinafter), it is separately added into above-mentioned two nitrocellulose filters, room temperature
Reaction 1 hour；TBST is washed 5 times, every time 10 minutes；
E. develop the color and take pictures：Residual liquid on nitrocellulose filter is blotted, it is steady that every nitrocellulose filter is separately added into 2mL
The mixed liquor (buying in Thermo companies) of sizing Peroxidase Solution (1mL) and luminol/enhancing agent solution (1mL),
The surface of even wetting nitrocellulose filter, room temperature lucifuge is reacted one minute claps after gel imaging system (buying in GE companies)
According to leaving and taking result.
Experimental result (accompanying drawing 6) shows, two kinds of antibody of the invention can react with the hK1 in four kinds of sources, it was demonstrated that this hair
The reactivity of bright two kinds of antibody, and prove that it has preferable reaction with natural hK1.In addition, experimental result is visible, four kinds of sources
HK1 molecular weight is consistent with theoretical value：UK is natural hK1, degree of glycosylation highest, therefore its molecular weight is maximum；Escherichia coli
It is minimum to express hK1 degree of glycosylation, therefore molecular weight is minimum；CHO system expression hK1 degree of glycosylation is slightly less than native protein but height
In Yeast expression hK1, therefore its molecular weight is between UK and Yeast expression hK1.
2. antibody A 24 and A32 are in the performance evaluation of ELISA detection platforms
Antibody in above-mentioned preparation example is subjected to combinations of pairs, pairing detection is carried out respectively as coated antibody or labelled antibody
Standard items, detecting step are as follows：
1) using 20mM PH7.4 PB buffer solutions by Y0 (irrelevant single-chain antibody, as negative control, preparation method ginseng
Add applicant's earlier application：201410020156.6), A24 or A32 is diluted to 8ug/mL (as coated antibody) respectively, packing
Into ELISA Plate (100uL/ holes), 4 DEG C, overnight (16 hours)；
2) PBST (the 20mM PB7.4 containing 0.05%Tween-20, similarly hereinafter) is patted dry after washed once (200uL/ holes)；
3) confining liquid (containing the PBST that mass volume ratio is 2%BSA) closing (200uL/ holes), 37 DEG C, 2 hours；Discard envelope
Patted dry after closing liquid；
4) Sample dilution dilution standard product, it is 200,100 and 0 to concentration (ng/mL).Above-mentioned strength solution is added respectively
Enter to what step 3) obtained and be coated with A24 and A32 ELISA Plate in (100uL/ holes), 37 DEG C are reacted 1 hour；PBST is washed 5 times
Patted dry after (200uL/ holes)；
5) according to 1:2000 volume ratios dilution HRP mark A24 (A24-HRP) or A32 (A32-HRP) are (anti-as mark
Body)；The A32-HRP or A24-HRP that dilution is separately added into ELISA Plate are obtained to step 4), 37 DEG C are reacted 30 minutes；PBST is washed
Patted dry after washing 5 times；
6) TMB nitrite ions (buying in Huzhou Ying Chuan companies) are added, 100uL/ holes, 37 DEG C are reacted 15 minutes；
7) 2M H are added2SO4Terminate liquid, 50uL/ holes, read OD450 in ELIASA (buying in Thermo companies) immediately.
As a result such as following table：
From the above results, the double-antibody sandwich detecting system that A32, A24 are formed can be applied to enzyme linked immunosorbent detection and put down
Platform, and two kinds of pairings of A24 (coating)-A32 (mark), A32 (coating)-A24 (mark) have preferable Detection results.With non-phase
Antibody is closed without nonspecific reaction.
3. antibody A 24 and A32 are in the evaluation of collaurum detection platform
Be 200ng/ml, 100ng/ml with Sample dilution dilution standard product to concentration, by the two concentration and
0.025mol/LpH7.5 PBS adds 50uL respectively, and into colloidal-gold detecting-card, (A24 coatings-A32 is marked or A32 coatings-A24
Mark, is specifically prepared referring to embodiment 6), interior detection card is placed on readout instrument of 10-15min is detected.Testing result is such as
Under：
The above results are visible, and the double-antibody sandwich detecting system of A32, A24 composition can be applied to collaurum detection platform, and
Two kinds of pairings of A24 (coating)-A32 (mark), A32 (coating)-A24 (mark) have preferable Detection results.
4. antibody A 24 and A32 are in the evaluation of time-resolved fluorescence detection platform
A24 or A32 are diluted to 1mg/ml with 0.025mol/L pH7.5 PBS, lined on nitrocellulose filter；With
The A32 or A24 that 0.05mol/L pH8.0 borate buffer marks time-resolved fluorescence microballoon dilute 20 times, and specking is in knot
Close on pad；As shown in accompanying drawing 9 pad pasting, cut, be loaded and (specifically prepare referring to embodiment 7).By detection card, detectable concentration contains respectively
The PBS of the standard items and 0.025mol/L pH7.5 for 200,100ng/ml is measured, testing result is as follows：
From the above results, the double-antibody sandwich detecting system of A32, A24 composition can be applied to time-resolved fluorescence inspection
Platform is surveyed, and two kinds of pairings of A24 (coating)-A32 (mark), A32 (coating)-A24 (mark) there are preferable Detection results
The application of the antibody A 24 of embodiment 5. and A32 in enzyme-linked immune quantitative detection reagent box
This detection method uses the principle of double antibody sandwich method.
1. material：
It is coated with buffer solution：0.02M PH7.4 PB buffer solutions；Confining liquid：Containing 2%BSA's (mass volume ratio, similarly hereinafter)
PBST buffer solutions；Cleaning solution：PBST buffer solutions；Sample dilution：Containing 1%BSA, 0.5% casein, 5% lowlenthal serum,
0.02%Procline 300 and 0.05%Tween-20 phosphate buffer (PH7.4)；Standard items：CHO system expression weights
Group hK1 (1mg/mL, preparation method are shown in patent 201310746269.X)；Nitrite ion：TMB (is bought in Huzhou Ying Chuan companies)；Eventually
Only liquid：2M H2SO4。
2. instrument：
ELIASA (is bought in Thermo companies), universal microbiological incubator (buying in Thermo companies), automatically
Board-washing machine (is bought in Shenzhen Hui Song companies)
3. method：
(1) preparation of detection plate：Antibody A 24 is diluted to 8ug/mL by coating buffer solution, and 100uL/ holes are added to ELISA Plate
In, 4 DEG C stand overnight (16 hours)；PBST is patted dry after washed once；200uL/ holes confining liquid closing, after 37 DEG C are placed 2 hours
Pat dry, drain overnight (16 hours), aluminium foil bag vacuum packaging, 4 DEG C save backup；
(2) preparation of Sample dilution：Containing 1%BSA, 0.5% casein, 5% lowlenthal serum, 0.02%Procline
300 and 0.05%Tween-20 phosphate buffer (20mM, PH7.4), according to mentioned component proportional arrangement Sample dilution,
Saved backup in 4 DEG C；
(3) preparation of standard items：10uL standard items are added into 19.99mL Sample dilutions, fully mixed after 4 DEG C
Save backup；
(4) preparation of liquid is detected：Horseradish peroxidase (HRP) is coupled to antibody A 32 (A32-HRP, 1mg/ml)；Will
5uL A32-HRP are added into 9.995mL Sample dilution, are fully mixed and are saved backup after 4 DEG C
4. the application method of the enzyme-linked immunologic detecting kit of Human kallikrein 1
(1) it is standby after equilibrium at room temperature that vacuum-packed detection ELISA Plate and related reagent are taken out in 4 DEG C；
(2) dilution of standard items：10uL standard items are added into 490uL Sample dilutions, produced after fully mixing
10ng/mL standard solutions；By 10ng/mL standard solution doubling dilutions, obtain respectively final concentration (ng/mL) be 5,2.5,
1.25th, 0.625,0.3125,0.15625 and 0.078125 standard solution；Using Sample dilution as negative control, i.e. 0ng/
mL；
(3) Sample dilution in 50uL/ holes is separately added into ELISA Plate；It is right that standard items, feminine gender are added into ELISA Plate again
According to and sample to be checked (50ul/ holes)；37 DEG C are placed 60 minutes；PBST is patted dry after washing 5 times；
(4) detection liquid is added in detection hole, 100uL/ holes, 37 DEG C are placed 30 minutes；PBST is patted dry after washing 5 times；
(5) nitrite ion is added in detection hole, 100uL/ holes, 37 DEG C are placed 15 minutes；
(6) terminate liquid is added in detection hole, 50uL/ holes, reads OD450 in ELIASA immediately.
According to OD450 readings corresponding to each concentration of standard items, the softwares of CurveExpert 1.3 are used using concentration as horizontal seat
Mark, OD values are the drafting that ordinate carries out standard curve, obtain calibration curve equation；The OD value substitutions standard for detecting sample is bent
The concentration (ng/mL) of sample to be checked is calculated after line equation.
5. detection performance is evaluated：
The performance evaluation of detection method of the present invention is carried out according to above-mentioned testing conditions.
(1) TIANZHU XINGNAO Capsul:
The rate of recovery is to verify the index of detection accuracy, and the present invention adds to the detection kit based on A24 and A32 then
The checking of add-back yield.
Pooled plasma is obtained after 20 parts of blood plasma are mixed, the hK1 contents in pooled plasma B are measured using the method for the present invention
For C0；The standard items (A liquid) that concentration is respectively 5ng/mL, 2.5ng/mL, 1.25ng/mL and 0.625ng/mL are added to mixed
Close in blood plasma B, the volume ratio added between A liquid and blood plasma B is 1:9, the recovery of each addition is calculated according to formula (1)
Rate.
In formula：R is the rate of recovery；
V is the volume for adding A liquid；
V0For the volume of pooled plasma sample B；
C is the detectable concentration that pooled plasma sample B is added after A liquid；
C0For plasma sample B detectable concentration；
CSFor the concentration of A liquid.
Examination criteria curve (accompanying drawing 7) of the present invention is visible, the detection with good linear relationship (r=0.99) and with
High sensitivity (156pg/mL) and negative background values (OD=0.0525).According to above-mentioned TIANZHU XINGNAO Capsul computational methods, knot is calculated
Fruit see the table below：
Visible in table, aforementioned four concentration TIANZHU XINGNAO Capsul average value is 101.17%, and each concentration TIANZHU XINGNAO Capsul is with returning
Yield average difference is less than or equal to 8.29%, it was demonstrated that this detection method accuracy is high.
(2) it is specific:
Visible, human tissue kallikrein 1 and people's tissue in human tissue kallikrein family are compared through protein sequence
The homology of kallikrein 2 (KLK2) and human tissue kallikrein 3 (KLK3) respectively reaches 66% and 60%, this experiment then
The main detection specificity for investigating the detection method of the invention established to KLK2 and KLK3.Detection method is as described above, respectively
The Urinary kallidinogenase (UK) added after Sample dilution gradient dilution, KLK2 (buying in R＆D companies) and KLK3 (are bought public in R＆D
Department), finally detect the specificity of the detection method.Testing result (accompanying drawing 8) is visible, and people of the present invention organizes kassinin kinin to discharge
The enzyme linked immunological quantitative assay specificity of enzyme 1 is good.
(3) it is repeated
Three parts of representative plasma samples are repeated into detection 10 times respectively, calculate the average value M of 10 measurement results
With standard deviation SD, show that coefficient of variation CV results are as follows according to formula CV=SD/M × 100%：
The above results are visible, and three parts of blood plasma detect CV<10%, ELISA detection kit of the present invention, which has, preferably to be repeated
Property.
6. the present invention attempts plurality of reagents box preparation method, following table shows that several different preparation methods are (unlisted in table
Described in the equivalent the present embodiment of step, parameter) and corresponding reagent box detection performance.
The preparation of the colloid gold immune detection card of the anti-human tissue kallikrein 1 of embodiment 6.
1. the mark of collaurum
The colloid gold label of antibody A 32：Use K2CO3Regulation collaurum pH value (adds 5uL in per 1ml collaurums
0.2MK2CO3), it is slowly added to (add in per 1ml collaurums with the antibody A 32 of 0.2M PBS dilutions into colloidal gold solution
30ug antibody As 32), stirring at low speed 30 minutes；Confining liquid (1%BSA) is added to its final concentration of 10% (mass percent), is stirred
Mix 20 minutes；12000rpm centrifuges 30min after standing 30min；Supernatant gold labeling antibody is gone to redissolve liquid 0.01M PBPH7.4+1%
BSA+0.025%Tween 20+5% sucrose) redissolve the A32 antibody for producing colloid gold label.
2. gold standard pad and reaction film preparation
After A32 gold labeling antibodies are diluted well, it is sprayed in gold standard pad 6, drying for standby；Antibody A 24 and anti-His labels are resisted
Body with after coated antibody dilution (3% methanol+25mM PBSs (pH7.5)) dilution well, is coated on reaction respectively respectively
The T lines 5 of film 2 (nitrocellulose filter), the position of C lines 4 are standby after drying.
3. pad pasting, cut film, assembling
Sample pad 1, gold standard pad 6, the nitrocellulose filter 2 for being coated with antibody, adsorptive pads 3 are set gradually from left to right
(as shown in Figure 9), and should contact a little between any two, the T lines 5 of the nitrocellulose filter for being coated with antibody are in left, C lines 4
Cut on the right side, and according to shell size, load shell, it is standby to complete detection blocking.
4. kit assembles
The detection card that will be assembled, drier, dropper are fitted into aluminium foil bag, heat sealing machine sealing, labelling.
5. the application method of the colloid gold immune detection card of anti-human tissue kallikrein 1
1) Sample dilution (10mM PH7.4 PB buffer solutions) recovers to mix standby to room temperature, concussion；
2) Sample Dilution is detected：1mL Sample dilutions, then accurate absorption 10 μ L serum/blood are added in the centrifuge tube of cleaning
Sample is starched, is added in centrifuge tube, vibrates and fully mixes.
3) sample-adding and interpretation：Sample after drawing 50 μ L dilutions with liquid-transfering gun is slowly added into well, beginning timing, and 10
Detection card is placed on readout instrument in~15 minutes and detected, instrument will be scanned to detection card, and result is shown
On the screen of readout instrument.Judge more than 15min clocks, it is as a result invalid.
6. the colloid gold immune detection card Detection results of anti-human tissue kallikrein 1 are assessed
1) range of linearity detects
The standard items (0.125,0.25,0.5,1,2,4,8,16ng/ml) of various concentrations are determined, draw standard curve, line
Property detection range, as a result as shown in Figure 10.It is 0.125ng/ml to detect card sensitivity.
2) repeatability detection
Same batch detection card carries out repeating detection 10 times to 1ng/ml, 4ng/ml sample, calculates the coefficient of variation CV, CV=
Standard deviation SD/ average M × 100%
| Concentration (ng/ml) | 1 | 4 |
| CV | 5.4% | 8.9% |
3) degree of accuracy detection (TIANZHU XINGNAO Capsul)
The ability for adding pure analyte for assessing the detection kit Accurate Determining.It is identical that pooled serum is divided into volume
3 parts, add standard items in wherein 2 parts, 5ng/ml, the recovery sample of 1.25ng/ml concentration, in another sample be made
The PH7.4 without measured object of same amount PB buffer solutions are added, basic sample is made.
| Concentration (ng/ml) | 1.25 | 5 |
| The rate of recovery | 92.3% | 89% |
7. in addition to above-mentioned optimal preparation method, the present invention have also been attempted a variety of preparation schemes, such as prepared by 4 kinds of following table
Example：
The preparation of the time-resolved fluoroimmunoassay detection card of the human tissue kallikrein 1 of embodiment 7.
This detection method uses the principle of double-antibody sandwich immunochromatographic method.
1st, solution is prepared
It is prepared by 0.05mol/L pH8.0 borate buffer：Take 0.1mol/L H3BO370ml, with 0.025mol/L's
Na2B4O7·10H2O adjusts pH to 8.0, and is settled to 100ml, is placed in 4 DEG C of standby, terms of validity 3 months.
It is prepared by 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC, purchased from SIGMA companies) solution：
1.5gEDC adds 100ml deionized waters, is made into the aqueous solution and is placed in 4 DEG C of standby, terms of validity 3 months.
The preparation of confining liquid：Containing 10%BSA (percentage is mass volume ratio), 0.05mol/L pH8.0 boron
Acid buffer, with 0.22um membrane filtrations, it is placed in 4 DEG C of standby, terms of validity 7 days.
It is prepared by fluorescence antibody dilution：Containing 1%BSA, 10% sucrose, 0.025% Tween-20,0.05mol/L pH8.0's
Borate buffer, with 0.22um membrane filtrations, it is placed in 4 DEG C of standby, terms of validity 7 days.
It is prepared by coated antibody dilution：Containing 1% sucrose, 3% methanol 0.025mol/L pH7.5 PBS, with 0.22U film mistakes
Filter, is placed in 4 DEG C of standby, terms of validity 7 days.
Sample dilution：Containing 0.1%BSA, 0.025% Tween-20,0.01% antipyrine, 0.02%proclin300,
0.05mol/LpH8.0 borate buffer, 10-30 DEG C preserves the term of validity 1 year.
2nd, the fluoroscopic examination blocking of human tissue kallikrein 1 is standby
1) time-resolved fluorescence microballoon marks
A32 antibody labeling methods are following (by taking 500uL reaction systems as an example)：400uL borate buffer solutions are added to be centrifuged in 2ml
Guan Zhong, adds the particle diameter 200nm of 100uL concentration 1% unloaded fluorescent microsphere (being purchased from Thermo companies), and vortex oscillation mixes.Again
Add 10uLEDC solution, shaken at room temperature 15min.10 DEG C of centrifugation 10min of 14000rpm, remove supernatant, precipitate and delayed with 0.5ml boric acid
Fliud flushing dissolves, ultrasonic disperse, power 100W, time 1min (ultrasonic 3s interval 3s).
Concentration 1mg/ml A32 antibody 50uL is added in microballoon after activation, 250r/min20 DEG C of isothermal vibration reacts 2h；
Add 55uL confining liquids, isothermal vibration reaction 4h.10 DEG C of centrifugation 15min of 14000rpm, wash 2 times, remove supernatant, precipitation is used
0.5ml borate buffers dissolve, and last time the centrifuged deposit dissolving of fluorescence antibody dilution and ultrasonic disperse, are placed in 4 DEG C
Constant temperature preserves.
2) specking of fluorescence pad
A32 antibody fluorescence pad specking methods are as follows：With fluorescence antibody dilution by the A32 fluorescence antibodies of above-mentioned preparation
20 times of dilution, specking is on whole piece pad.
3) coating of nitrocellulose filter
Nitrocellulose filter method for coating is as follows：0.5ml concentration 4mg/ml A24 antibody is taken, is added to 5ml graduated centrifuge tubes
In, add coated antibody dilution to 1ml, be coated in the position of T lines 5 of nitrocellulose filter 2.0.5ml concentration is taken to resist for 4mg/ml
HIS antibody, is added in centrifuge tube, adds coated antibody dilution to 1ml, is coated in the position of C lines 4 of nitrocellulose filter 2.
4) pad pasting, cut, be loaded
Sample pad 1, fluorescence pad 6, the nitrocellulose filter (reaction film) 2 for being coated with A24 antibody, by adsorptive pads 3 from
Left-to-right is set gradually, and a little contact between any two, and the T lines 5 of the nitrocellulose filter are in left, C lines 4 in right (such as accompanying drawing 9
It is shown), and cut according to the size that gets stuck, loading is got stuck, and it is standby to complete detection blocking.
5) kit assembles
Take aluminium foil bag and drier；Open heat sealing machine, preheating；Detection card, drier are fitted into aluminium foil bag；Use heat sealing machine
Seal aluminium foil bag；It is labelled.
Meanwhile applicant is also done fluorescence labeling using A24 antibody, nitrocellulose filter (reaction is coated on using A32 antibody
Film) at 2T lines, remaining step, parameter constant, it is standby to carry out fluoroscopic examination blocking.
3rd, the application method of the fluoroscopic examination card of human tissue kallikrein 1
1) sample to be tested is diluted：1mL Sample dilutions, then accurate absorption 10 μ L serum/blood are added in the centrifuge tube of cleaning
Sample is starched, is added in centrifuge tube, vibrates and fully mixes.
2) sample-adding and interpretation：Sample after drawing 50 μ L dilutions with liquid-transfering gun is slowly added into well, beginning timing, and 10
In~15 minutes result is quantitatively judged with fluorescence immune chromatography instrument.Judged more than 15 minutes, it is as a result invalid.
4th, the fluoroscopic examination card Detection results of human tissue kallikrein 1 are assessed
1) accuracy：A32 (coating)-A24 (mark) is detected and blocks detection 0.25,1,4ng/ml each 25 weights of standard items
Repetition measurement is determined, and detection card precision is calculated after rejecting outlier.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 (coating)-A32 (mark) is detected into card and repeats above-mentioned experiment, as a result such as following table,
| Concentration point (ng/ml) | 0.25 | 1 | 4 |
| CV | 18% | 16% | 14% |
2) detection is linear：A32 (coating)-A24 (mark) is detected to the standard items of card detection various concentrations：0.0625、
0.125th, 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
Spend for 0.0625ng/ml.
A24 (coating)-A32 (mark) detection card sensitivity is 0.0625ng/ml.
3) degree of accuracy-rate of recovery：By A32 (coating)-A24 (mark) detect card detection addition be respectively 1,5,20ng/
Ml standard items, testing result such as following table.
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The rate of recovery | 112% | 94% | 104% |
A24 (coating)-A32 (mark) is detected into card and repeats above-mentioned experiment, as a result such as following table,
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The rate of recovery | 116% | 108% | 89% |
5th, degree of accuracy methodology compares：
The above results show that A32 (coating)-A24 (mark) detection card performance is more excellent, therefore are made methodology comparison
Checking.20 parts of clinical patient samples are selected, by 1 to 20 serial number, with ELISA (prepared by embodiment 5) and fluorescence detection
Tested, be measured according to 1,2,3......18,19,20,20,19,18......3,2,1 sample order simultaneously.
ELISA and fluorescence detection testing result coefficient R2=0.98, illustrate fluorescence detection and ELISA testing results have compared with
Good correlation (such as accompanying drawing 12).
6th, the contrast of formula：
In addition to above-mentioned optimal preparation example 1, applicant also attempts a variety of preparation schemes, for example, 5 groups of detection blockings below it is standby and
Using result such as following table：
In this description, the present invention is described with reference to its specific embodiment.But it is clear that it can still make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings are considered as illustrative
It is and nonrestrictive.
Claims (8)
1. the enzyme-linked immune quantitative detection reagent box of human tissue kallikrein 1, including it has been coated with antibody A 24 or A32 enzyme mark
Plate, sample diluting liquid, standard items, detection liquid, cleaning solution, nitrite ion and terminate liquid containing enzyme labelled antibody A32 or A24；
As ELISA Plate coated antibody A24, detection liquid contains enzyme labelled antibody A32；As ELISA Plate coated antibody A32, liquid is detected
Contain enzyme labelled antibody A32；
The antibody A 24 is the single-chain antibody of anti-human tissue kallikrein 1, including：
Weight chain variable district, its amino acid sequence contain following complementary determining region：Such as sequence SEQ ID NO：HCDR1 shown in 1,
Such as sequence SEQ ID NO：HCDR2 and such as sequence SEQ ID NO shown in 2：HCDR3 shown in 3；
And its light chain variable district, its amino acid sequence contain following complementary determining region：Such as sequence SEQ ID NO：Shown in 4
LCDR1, such as sequence SEQ ID NO：LCDR2 and such as sequence SEQ ID NO shown in 5：LCDR3 shown in 6；
The antibody A 32 is the single-chain antibody of anti-human tissue kallikrein 1, including：
Weight chain variable district, its amino acid sequence contain following complementary determining region：Such as sequence SEQ ID NO：HCDR1 shown in 9,
Such as sequence SEQ ID NO：HCDR2 and such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；
And its light chain variable district, its amino acid sequence contain following complementary determining region：Such as sequence SEQ ID NO：Shown in 12
LCDR1, such as sequence SEQ ID NO：LCDR2 and such as sequence SEQ ID NO shown in 13：LCDR3 shown in 14.
2. the enzyme-linked immune quantitative detection reagent box of human tissue kallikrein 1 according to claim 1, it is characterised in that
The amino acid sequence such as SEQ ID NO of the weight chain variable district of antibody A 24:Shown in 7, the amino acid sequence of light chain variable district is such as
SEQ ID NO:Shown in 8.
3. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 1, it is characterized in that
The antibody A 24 is single-chain antibody, its amino acid sequence such as SEQ ID NO:Shown in 17.
4. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 3, it is characterized in that
Without the HIS labels being made up of six histidines in the antibody A 24.
5. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 1, it is characterized in that
The amino acid sequence such as SEQ ID NO of the weight chain variable district of antibody A 32:Shown in 15, the amino acid sequence of light chain variable district is such as
SEQ ID NO:Shown in 16.
6. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 1, it is characterized in that
The antibody A 32 is single-chain antibody, its amino acid sequence such as SEQ ID NO:Shown in 18.
7. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 6, it is characterized in that
Without the HIS labels being made up of six histidines in the antibody A 32.
8. the enzyme-linked immune quantitative detection reagent box of anti-human tissue kallikrein 1 according to claim 1, it is characterized in that
Coating buffer solution used is 0.02M PH7.4 PB buffer solutions when preparing ELISA Plate；Prepare confining liquid used during ELISA Plate
It is the PBST buffer solutions containing 2%BSA；Cleaning solution is PBST buffer solutions；Sample dilution be containing 1%BSA, 0.5% casein,
The phosphate buffer of 5% lowlenthal serum, 0.02%Procline 300 and 0.05%Tween-20, PH7.4；Standard items are
CHO system expressions recombinate human tissue kallikrein 1；Nitrite ion is TMB；Terminate liquid is 2M H2SO4。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
| CN101928346A (en) * | 2009-07-31 | 2010-12-29 | 华中科技大学同济医学院附属同济医院 | Monoclonal antibody of anti-human tissue kallikrein and preparation thereof |
| CN103710368A (en) * | 2013-12-30 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Human kallikrein of mammalian cell expression as well as coding gene and application thereof |
| CN104049082A (en) * | 2014-05-30 | 2014-09-17 | 华中科技大学同济医学院附属同济医院 | Human tissue kallikrein activity detection kit and application thereof |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
| CN101672851A (en) * | 2009-07-31 | 2010-03-17 | 华中科技大学同济医学院附属同济医院 | Preparation of Human Tissue Kallikrein ELISA Kit |
| CN101928346A (en) * | 2009-07-31 | 2010-12-29 | 华中科技大学同济医学院附属同济医院 | Monoclonal antibody of anti-human tissue kallikrein and preparation thereof |
| CN103710368A (en) * | 2013-12-30 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Human kallikrein of mammalian cell expression as well as coding gene and application thereof |
| CN104049082A (en) * | 2014-05-30 | 2014-09-17 | 华中科技大学同济医学院附属同济医院 | Human tissue kallikrein activity detection kit and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 人组织激肽释放酶单克隆抗体的制备及其ELISA检测试剂盒的建立;冉晓等;《中华微生物学和免疫学杂志》;20110228;第31卷(第2期);179-180页 * |
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