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CN107817347A - A kind of kit of homogeneous luminescent method detection animal Procalcitonin - Google Patents

A kind of kit of homogeneous luminescent method detection animal Procalcitonin Download PDF

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Publication number
CN107817347A
CN107817347A CN201710873498.6A CN201710873498A CN107817347A CN 107817347 A CN107817347 A CN 107817347A CN 201710873498 A CN201710873498 A CN 201710873498A CN 107817347 A CN107817347 A CN 107817347A
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kit
pct
cell
procalcitonin
antibody
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刘秀峰
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Nanjing Puguang Biotechnology Co., Ltd
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Nanjing Ideal Biotechnology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

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Abstract

The invention belongs to kit technical field, especially a kind of kit of homogeneous luminescent method detection animal Procalcitonin；The composition in testing tube in the kit is 10mMPBS buffer solutions, and PH=7.0, it is 170.uL volumes often to test part, wherein containing 20ug/mL acridinium ester label PCT antibody and 20ug/mLHRP mark PCT antibody, in addition also containing 0.01% Proclin300；1st, the luminous separation of solid and liquid saved required for out-phase chemiluminescence of homogeneous chemistry and cleaning step, reactions steps are simple, and detection is quick, device simple, realize that chemiluminescence POCT is detected；2nd, testing result is accurate, continues the advantage of chemiluminescence platform, high sensitivity, the range of linearity is wide, and stability is good；3rd, animal doctor's infectious disease Testing index is increased, for PCT with respect to CRP and blood routine, diagnostic value is higher, veterinary antibiotic can be instructed to use.

Description

A kind of kit of homogeneous luminescent method detection animal Procalcitonin
Technical field
The invention belongs to kit technical field, especially a kind of reagent of homogeneous luminescent method detection animal Procalcitonin Box.
Background technology
Chemoluminescence method is current state-of-the-art immunologic detection method, is divided into direct chemiluminescence by label difference Method, enzyme-catalyzed chemical luminescence method and Electrochemiluminescince；By with the presence or absence of separation cleaning step, being divided into out-phase chemoluminescence method and Phase chemoluminescence method.At present, in-vitro diagnosis context of detection is cured in people, both at home and abroad using direct chemoluminescence method and enzymatic chemistry hair The detection product of light method is more, belongs to out-phase chemoluminescence method.External producer include Abbott Laboratories, Siemens, Suo Lin, Beckman and Wish this Meikang etc., domestic manufacturer includes NPD projects, Antu, mikey, Mai Rui, pool into, long brilliance doctor etc..Each producer's instrument platform is big Same small difference, especially domestic manufacturer, apparatus structure is complicated, fault rate is higher, and quality is uneven, have impact on client to product Trust.Meanwhile domestic manufacturer does not carry out bold innovation in varieties of reagent, the kind that others has, the original used are simply repeated Material（Antigen-antibody）Same source, cause homogeneous serious, Market competition.Only have Siemens one at this stage and use pure state The homogeneous chemistry luminescence method of oxygen mediation（Light-induced chemiluminescent method）Launch, detection need special LOCI modules.LOCI skills Art is single step chemiluminescence sandwich immune detecting method, containing two kinds of synthesis pearl reagents and a kind of biotin chemistry in reagent Monoclonal antibody.The first pearl reagent（Sensitive pearl）Streptavidin is coated with, and contains photaesthesia dyestuff；Second of pearl reagent （Chemical pearl）Another antibody is coated with, and contains chemiluminescence dye；Sample and chemical pearl and biotinylated antibody are incubated After form sandwich complex；Then sensitive pearl is added, the immune complex of aggregation is formed after being combined with biotin；Compound exists Sensitive pearl therein can produce singlet oxygen under 680nm light irradiations, and singlet oxygen can trigger chemiluminescence after permeating into chemical pearl Reaction, the chemiluminescence signal caused by measurement reaction under 612nm wavelength.This method needs exciting light, high to equipment requirement, Marker material is special, is not easy to obtain.
A kind of homogeneous chemistry luminescence method has been invented by Lumigen companies1,2, use chemiluminescent labels labelled antigen or anti- Body, while horseradish peroxidase（HRP）Another plant of antigen or antibody are marked, goes to detect the analyte in sample together, pure Excited species is directly injected under liquid-phase condition and produces chemiluminescence, reaches quantitative testing goal.This method not only ensure that chemistry The advantage of luminescence method detection performance, while detection device is required to reduce, device miniaturization, summary.We use this method In to animal（Including dog and pig）Procalcitonin（PCT）Detection.
Procalcitonin, a kind of 14KD protein, for the precursor of calcitonin.Meeting up-regulated expression during bacterium infection, is cured in people It is middle to be used as pyemic mark, instruct antibiotic usage.Document report3Dog occurs to infect and inflammation, in PCTmRNA expression Adjust.It is another to have document report, LPS induction pigs PCT expression4, and the developing sepsis that immune neutralization PCT can suppress pig enters Journey5。
At present, it is more for the Procalcitonin detection platform of people, including immunochromatography, chemiluminescence etc., but do not occur The PCT that phase chemiluminescence is used for people is detected.It is even more not have in veterinary applications, does not occur this detection project of PCT also.We pass through PCT Protein homology analysis, it is found that PCT sequences are highly conserved between some species, including pig, dog and cat etc..We pass through system Standby polyclonal antibody detects the PCT of each species by antibody sandwich homogeneous chemistry luminescence method, finds in pig and dog bacterium infection Diagnostic value in property disease is higher, therefore develops this kit.The detection means master of the existing diagnosis bacterial infection disease of animal doctor To be blood routine and CRP, specificity is not high, and antibiotic usage is more chaotic, and there is an urgent need to the appearance of PCT projects.
The content of the invention
The purpose of the present invention is：Overcome deficiency of the prior art, there is provided a kind of homogeneous chemistry luminescence method detection animal drop The former kit of calcium element.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of kit of homogeneous chemistry luminescence method detection animal Procalcitonin,
The composition in testing tube in the kit is 10mMPBS buffer solutions, and PH=7.0, it is 170.uL volumes often to test part, Wherein containing 20ug/mL acridinium ester label PCT antibody and 20ug/mLHRP mark PCT antibody, in addition also containing 0.01% Proclin300；
The kit is made using following preparation method：
（1）PCT ANTIGEN DESIGNThes and preparation, including：PCT antigens amino acid sequence and base sequence design, sequence table reach, plasmid structure Build, plasmid extraction, protein expression, protein purification and detection；
（2）PCT Antibody preparations, including：Immune blood sampling, hybridoma fusion screening；
（3）Acridine derivatives mark PCT antibody；
（4）HRP marks PCT antibody；
（5）Sandwich method detects.
Preferably, the PCT antigens amino acid sequence is such as：SEQIDNO.1, base sequence is such as：SEQIDNO.2.
Preferably, the plasmid construction includes synthetic gene sequence, including including 5 ' end EcoRI, 3 ' end XhoI digestions positions Point, N-terminal addition Kozak (GCCGCCACC) and CD33 signal peptide sequences (ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCT), C-terminal addition HisTag sequences (CATCATCACCATCACCAT) and termination codon (TGATAG), and by this sequence construct to pcDNA4.0TomycHisA carry Body, the gene order of synthesis is such as：SEQIDNO.3.
Preferably, endotoxin-free plasmid is largely extracted with carrier for expression of eukaryon, the endotoxin-free plasmid extracted is carried out Cell transfecting, cell conditioned medium and cell pyrolysis liquid are collected, using Ni post affinity chromatographys, collect sample and carry out SDS- PAGEWesternblot is examined, and Concentration Testing, qualified samples is dialysed to PBS, and by protein concentration extremely>1.0mg/ml.
Preferably, the hybridoma fusion screening comprises the following steps：
（1）Before fusion prepared by feeder cells, including macrophage and thymocyte, prepares and is carried out in gnotobasis；
（2）Before fusion prepared by myeloma cell；
（3）Extract the spleen cell of immune success rate；
（4）Cell fusion；
（5）ELISA is detected；
（6）Subclone screening.
It is using the beneficial effect of technical scheme：
1st, the luminous separation of solid and liquid saved required for out-phase chemiluminescence of homogeneous chemistry and cleaning step, reactions steps are simple, inspection Quick, device simple is surveyed, realizes that chemiluminescence POCT is detected；
2nd, testing result is accurate, continues the advantage of chemiluminescence platform, high sensitivity, the range of linearity is wide, and stability is good；
3rd, animal doctor's infectious disease Testing index is increased, for PCT with respect to CRP and blood routine, diagnostic value is higher, can instruct animal doctor's antibiosis Element uses.
Brief description of the drawings
The present invention is further described with reference to the accompanying drawings and examples.
Fig. 1 is the protein purification result figure of the present invention.
Fig. 2 is the ELISA testing result figures of the present invention.
Fig. 3 is the technical schematic diagram of the present invention.
Fig. 4 is the schematic diagram of Procalcitonin quantitative test in embodiments of the invention one.
Fig. 5 is the schematic diagram of Procalcitonin quantitative test in embodiments of the invention two.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to embodiment The present invention is further detailed explanation.
First, " one embodiment " or " embodiment " referred to herein refers to may be included at least one realization side of the present invention Special characteristic, structure or characteristic in formula." in one embodiment " that different places occur in this manual not refers both to Same embodiment, it is not the single or selective embodiment mutually exclusive with other embodiment yet.
First, PCT ANTIGEN DESIGNThes are with preparing
1.PCT antigens amino acid sequence (SEQIDNO.1)：
APFRSALESSPADPATLSEDEARLLLAALVQNYVQMKASELEQEQEREGSSLDSPRSKRCGNLSTCMLGTYTQ DFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN
Base sequence (SEQIDNO.2)：
GCTCCTTTCCGCAGCGCTCTCGAATCTAGCCCCGCCGACCCAGCTACACTGTCCGAGGACGAGGCTAGACTGC TGCTGGCCGCCCTGGTGCAGAACTATGTACAGATGAAGGCTTCTGAGCTGGAGCAGGAACAAGAGCGGGAGGGGTCG AGCCTCGACTCTCCTAGATCGAAGCGGTGCGGAAACCTGTCTACCTGTATGCTCGGAACATACACCCAGGACTTCAA CAAGTTCCACACATTCCCTCAGACAGCTATTGGCGTGGGCGCTCCCGGCAAGAAGCGCGACATGTCTTCTGACTTAG AAAGAGACCACCGGCCTCACGTTTCCATGCCACAGAACGCCAAC
2. expression system
Expressed using 293F eukaryotic systems.
The recombinant protein of HEK293 cells expression has accurate posttranscriptional modification function, the glycosylation pharmaceutical protein of expression Close to native protein molecule in terms of molecular structure, physicochemical property and biological function.293F can be hanged using serum-free Floating culture, serum free suspension culture are that a kind of cell of animal blood serum is replaced with the albumen or hormone in known people source or animal origin Training method, it can reduce later-period purification work, improve product quality.
3. plasmid construction
Synthetic gene sequence：Including 5 ' end EcoRI, 3 ' end XhoI restriction enzyme sites, N-terminal addition Kozak (GCCGCCACC) and CD33 signal peptide sequences (ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCT), C-terminal addition HisTag sequences (CATCATCACCATCACCAT) and termination codon (TGATAG).And by this sequence construct extremely PcDNA4.0TomycHisA carriers.The gene order of synthesis is following (SEQIDNO.3)：
gaattcGCCGCCACCATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGCTCCTTTCC GCAGCGCTCTCGAATCTAGCCCCGCCGACCCAGCTACACTGTCCGAGGACGAGGCTAGACTGCTGCTGGCCGCCCTG GTGCAGAACTATGTACAGATGAAGGCTTCTGAGCTGGAGCAGGAACAAGAGCGGGAGGGGTCGAGCCTCGACTCTCC TAGATCGAAGCGGTGCGGAAACCTGTCTACCTGTATGCTCGGAACATACACCCAGGACTTCAACAAGTTCCACACAT TCCCTCAGACAGCTATTGGCGTGGGCGCTCCCGGCAAGAAGCGCGACATGTCTTCTGACTTAGAAAGAGACCACCGG CCTCACGTTTCCATGCCACAGAACGCCAACCATCATCACCATCACCATTGATAGctcgag
4. plasmid extraction, protein expression, protein purification and detection
Endotoxin-free plasmid (MaxExtract) is largely extracted with carrier for expression of eukaryon.The endotoxin-free plasmid extracted is carried out Cell transfecting.Cell conditioned medium and cell pyrolysis liquid are collected, using Ni post affinity chromatographys, sample is collected and carries out SDS- PAGEWesternblot is examined, Concentration Testing, by qualified samples (purity>90%) dialyse to PBS (phosphate buffer), and By protein concentration extremely>1.0mg/ml, protein purification result are as shown in Figure 1.
2nd, PCT Antibody preparations
1. immune and blood sampling
1) 3 BALB/c mouses (female, more than 6 week old) are chosen to be immunized.
2) the 0th week：Before immune, the μ l/ of mouse orbit venous blood collection 50 only, after 37 DEG C are placed 1h, are centrifuged with 5000rpm 5min, prepare negative serum, -20 DEG C of preservations.It is immune for the first time to carry out mouse, antigen dose is 100 μ g/, and Freund is helped completely Agent (Sigma-F5881), divides multiple spot to be subcutaneously injected.
3) the 2nd week：Mouse is immune for the second time, and antigen dose is 50 μ g/, Freund's incomplete adjuvant (Sigma-F5506), is injected Mode and the same initial immunity of dosage.
4) the 4th week：Mouse third time is immune, and immunizing dose and immunization method are immune with second.
5) the 5th week：The μ l/ of mouse orbit venous blood collection 50 only, after 37 DEG C are placed 1h, centrifuge 5min with 5000rpm, prepare blood Clearly, ELISA detections are carried out, testing result is as shown in Figure 2.Remaining -20 DEG C of preservations of serum.Titre no longer changes after taking serum turn sun Mouse (the 2nd mouse, 1/4000 dilution when, ELISA values be more than 1) stop be immunized, the next day booster immunization, the μ of antigen dose 50 G/ only, carries out cell fusion after 3 days.
2. hybridoma fusion screens
2.1. feeder cells are prepared (including macrophage and thymocyte, prepare and carried out in gnotobasis) before merging
1) 5 week old mouse are put to death using de- cervical approach, immersed in 75% ethanol.Mouse part skin is torn, is drawn with 5ml syringes 20%IMDMHT5ml, lavation mouse peritoneal (the flat thorn of syringe needle, internal organ can not be injured).Hold two legs and rock mouse up and down, will contain The nutrient solution of macrophage is drawn back, and is placed in 50ml centrifuge tubes, as far as possible all suctions out the liquid in abdominal cavity.
2) two week old mouse, the infiltration of 75% ethanol are put to death.Mouse thoracic cavity is opened in del along breastbone both sides, takes out chest Gland, it is put into the disposable screen cloth of BD companies, grinds grinding with the inner tube of 2ml syringes, cell homogenates is sucked 20% In IMDMHT.
3) mixed-culture medium containing peritoneal macrophage and thymocyte is added in loading slot, adds appropriate carbonic acid and delay Fliud flushing, mix, 96 orifice plates are spread with the density in 1.0 × 104/ holes, 100ul/ holes, 8 pieces.96 porocyte culture plates for completing cell are put Enter 37 DEG C, in 5%CO2 incubator, observation is such as pollution-free, then places standby in incubator.
2.2. before merging prepared by myeloma cell
1) 5-7 days before merging, defrosting myeloma cell, carry out cell maintenance and expand culture, adjust cell state, reach pair In number growth period, Cell viability is more than 95%.
2) myeloma cell of exponential phase is taken out from incubator, outwells cell culture fluid, add 3-5mlIMDM Nutrient solution jog culture dish, outwells nutrient solution into culture dish.Add the fresh IMDM nutrient solutions of 3ml, with pipettor by cell from Blown down on culture dish, collect cell suspension in 50ml centrifuge tubes, 1000rpm centrifugation 5min, discard nutrient solution, it is new to add 20ml Cell is resuspended in fresh IMDM nutrient solutions, 1000rpm centrifugation 5min, abandons supernatant.
3) cell is resuspended with fresh IMDM nutrient solutions 10ml, is placed on standby in super-clean bench.
2.3. the spleen cell of immune success rate is extracted
1) before merging, mouse is plucked into eyeball and takes blood, after 37 DEG C of 1h, it is right as the positive that serum is prepared using 1000rpm5min centrifugations According to -20 DEG C of preservations, the mouse of execution immerses 5-10min in 75% ethanol.
2) mouse is inserted in a sterile petri dish, belly is upward, and sterile working is cut off and tears skin, makes rear abdominal muscle Sarcocyte is fully and skin peeling.Use new scissors instead and cut off abdominal cavity film, take out spleen.
3) clean spleen 2 times with the 10ml IMDM nutrient solutions being preheated in culture dish, reject the fat that spleen surface is adhered Fat and connective tissue, then it is transferred in the sterile petri dish of another nutrient solution containing 10mlIMDM.
4) choose different points after drawing nutrient solution with 1ml syringes to be expelled to inside spleen, heave spleen, beneficial to thin Born of the same parents discharge.Spleen is extruded in culture dish with 1ml syringe handles, cell is separated as far as possible.
5) after the nutrient solution for extruding mixing splenocyte is drawn with disposable sterilized pasteur pipet, by cell screen clothes extremely In 50ml centrifuge tubes, after rinsing screen cloth with fresh IMDM nutrient solutions, 1000rpm centrifugation 10min, supernatant is abandoned.Add fresh Splenocyte is resuspended in IMDM nutrient solutions, 1000rpm centrifugation 10min, abandons supernatant.Splenocyte is gently struck to scattered, addition IMDM nutrient solutions Splenocyte is resuspended, then myeloma cell's suspension is added into splenocyte suspension, is settled to 30ml, mixes, 1000rpm centrifugations 10min, supernatant is abandoned, exhausted the liquid of residual with pipettor, treat that fusion is used in next step.
2.4. cell fusion
1) scattered cell is gently struck, the concentration that 1ml37 DEG C of preheating is slowly added to 2ml pipettes is 50% PEG1500, is softly blown Cell 100s is inhaled, PEG (polyethylene glycol) is fully contacted with cell.
2) the IMDM nutrient solutions of 15ml37 DEG C of preheating are rapidly added, is mixed, is terminated PEG reaction, cell is resuspended, are centrifuged Pipe is placed in 37 DEG C of water-bath 10min.
3) 1000rpm centrifuges 10min, abandons supernatant.
4) take 10mlIMDMHAT nutrient solutions that cell is resuspended, cell liquid is transferred in loading slot, add 80mlIMDMHAT trainings Cell is resuspended in nutrient solution, and cell suspension is well mixed, and is added and has been spread 96 orifice plates of feeder layer, 8 pieces of bed board, 100 μ l/ holes, then Cell plates are placed in 37 DEG C, cultivated in 5%CO2 incubators.
5) see whether to pollute after merging 2-3 days, if pollution-free, nutrient solution in 96 orifice plates is carefully vacantly drawn with pipettor Abandon, exchange fresh 20%IMDMHT nutrient solutions for and cultivated, observation clone growing way, carries out ELISA detections after 5-6 days.
2.5.ELISA detection
1) PCT antigens are dissolved in coating buffer by debita spissitudo；
2) 100 μ l antigens are added in corresponding hole, 4 DEG C overnight；
3) empty liquid and pat dry residual liquid, cleaning solution rinses 5 times；
4) 200 μ l confining liquids are added per hole, 37 DEG C are incubated 1 hour or 4 DEG C overnight；
5) empty liquid and pat dry residual liquid, cleaning solution rinses 3 times；
6) 100 μ l primary antibodies are added per hole, 37 DEG C are incubated 1 hour；
7) empty liquid and pat dry residual liquid, cleaning solution rinses 7 times；
8) 100 μ l secondary antibodies are added per hole, 37 DEG C are incubated 1 hour；
9) empty liquid and pat dry residual liquid, cleaning solution rinses 7 times；
10) residual liquid in hole is patted dry, 100 μ l nitrite ions, 37 DEG C of lucifuges colour developing 10min are added per hole；
11) add 50 μ l2MH2SO4 color development stoppings per hole, and read 450nmOD values immediately.It is positive, cell health to select ELISA Clone goes to 24 orifice plates and expands culture (ELISA positive criteria for selections：ELISAOD values are more than 1.0, or dilute more than negative 1000 times Release value 3 times).
2.6. subclone screening
1) 10-20 polyclonal, Cell viability more than 90%, cell density 60%-80% are selected；
2) each clone carries out cell count.Cell is diluted according to gradient dilution method, 1 piece of 24 hole culture is got out before clone Plate, 900 μ l IMDMHT nutrient solutions are added per hole, clone cell is well mixed in foramen primum, draws 100 μ l cell suspension Cloned accordingly in hole to ready 24 orifice plate, while micro cell suspension is taken to cell counting count board in stoste is cloned Carry out cell count, the good cell density each cloned of record；
3) according to the number (3 cells/wells) of diluting cells and the plate number of clone, a certain amount of cell is taken to be diluted, until The number of cells of needs is diluted to, is added in loading slot, adds 12ml IMDMHT nutrient solutions, is well mixed.A and B rows inhale 200 μ l/ holes are taken, 6mlIMDMHT nutrient solutions are added in loading slot, are well mixed, takes 200 μ l/ holes to add to C, D, E and arranges.Add 6mlIMDMHT liquid is added in sample groove, is well mixed, takes 200 μ l/ holes to add to F, G, H and arranges, and mark is carried out on clone's plate It is put into CO2gas incubator and cultivates after (clone number, plate number, date, operator, clone's number)；
4) growing way of clone is observed after cloning in good time, typically detects ELISA in 7-14 days sample presentations；
5) clone selected is transferred in 24 orifice plates and cultivated, carry out second of subclone screening, the operating method of clone is the same as first Secondary subclone screens, and does ELISA, WesternBlot detection and hypotype identification after clone in good time.
3rd, acridine derivatives mark PCT antibody
1）Poly-D-lysine is dissolved in 1mL150mMPBS buffer solutions (pH7.4), final concentration 10nmol/L, it is molten to add 10 μ L Solution reacts 1h, with the desalting column of 5mL7KD molecular cut offs in the 10mmol/L acridinium esters of DMSO solvents in 25 DEG C (Thermofish companies) crosses post 3 times, removes free acridine using 150mMPBS (pH7.4) buffer solutions as liquid buffer solution is changed Ester and byproduct of reaction, obtain acridine-Poly-L-Lysine Solution.
2）EDC (final concentration of 5mmol/L) and NHS are added into the above-mentioned acridine-Poly-L-Lysine Solution purified (eventually Concentration is 10mmol/L).After 25 DEG C of addition reaction 30 minutes, final concentration of 10mM mercaptoethanol is added, after being activated Acridine-poly-D-lysine.
3）The anti-PCT antibody of 1mg is being added, is being mixed, 25 DEG C of reaction 1h are being placed in, with the desalting column of 5mL7KD molecular cut offs (Thermofish companies) crosses post 3 times using 150mMPBS (pH7.4) buffer solutions as liquid buffer solution is changed, remove free NHS and Byproduct of reaction, obtain conjugate acridinium ester-poly-D-lysine-PCT antibody-solutions.
4th, HRP marks PCT antibody
1）By 5mg peroxidase（HRP）It is dissolved in 1.2mL10mMPBS buffer solutions（PH=7.0）In, add the fresh configurations of 0.3mL 0.1M sodium periodate solutions, 37 DEG C be incubated 20 minutes.
2）In 10mM sodium-acetate buffers（PH=4.0）Middle dialysis, 4 DEG C overnight.
3）PCT antibody is dissolved in 20mM sodium carbonate buffers（PH=9.5）, concentration 10mg/mL, add 0.5mLPCT and resist In body and HRP, 37 DEG C are incubated 2 hours.
4）100 μ L4mg/mL sodium borohydrides are added, are placed 2 hours at 4 DEG C.
5）In 10mMPBS buffer solutions（PH=7.0）Middle dialysis, 4 DEG C overnight.
5th, sandwich method detects
Technical schematic diagram such as Fig. 3.
1. the assembling of kit
Main component is 10mMPBS buffer solutions in testing tube in kit of the present invention（PH=7.0）, it is 170.uL often to test part Volume, wherein containing 20ug/mL acridinium ester label PCT antibody and 20ug/mLHRP mark PCT antibody, also contain 0.01% in addition Proclin300.
2. the quantitative test of Procalcitonin
1) making of standard curve：PCT standard items are diluted with calf serum, it is 0ng/mL to be made into concentration some row concentration, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 300ng/mL each 150 μ L of Procalcitonin standard items, 30 μ L are taken to add this Testing tube in invention, testing tube is placed in supporting mini-plant, places 15min.Each sample difference replication 3 It is secondary.For necessary instrument according to the proportionate relationship between RLU and PCT concentration, automatic Fitting calculates PCT concentration, takes and determines three times averagely Value, draw standard curve.
2) the μ L of sample 200 to be checked are taken, the testing tube being placed in the present invention, per the μ L of sub-sampling 30, replication 3 times, according to 1）The standard curve of middle drafting can obtain the concentration value of Procalcitonin in measuring samples.
3) result shows：Using the method for the invention by further testing, detection sensitivity 0.06ng/mL, Detection range is 0.01ng/mL-200ng/mL, and CV is less than 5% between batch, and CV is less than 1.5% in batch, and does not occur HOOK effects, Without being diluted processing to detection sample.
Case 1
Conventional method
1. the assembling of kit
Main component is 10mMPBS buffer solutions in testing tube in present case kit（PH=7.0）, it is 170.uL often to test part Volume, wherein containing 20ug/mL acridinium ester label PCT antibody and 20ug/mLHRP mark PCT antibody, also contain 0.01% in addition Proclin300, as shown in Figure 4.
2. the quantitative test of Procalcitonin
1) making of standard curve：PCT standard items are diluted with calf serum, it is 0ng/mL to be made into concentration some row concentration, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 300ng/mL each 150 μ L of Procalcitonin standard items, 30 μ L are taken to add this Testing tube in case, testing tube is placed in supporting mini-plant, places 15min.Each sample difference replication 3 It is secondary.For necessary instrument according to the proportionate relationship between RLU and PCT concentration, automatic Fitting calculates PCT concentration, takes and determines three times averagely Value, draw standard curve.
2) the μ L of sample 200 to be checked are taken, the testing tube being placed in present case, per the μ L of sub-sampling 30, replication 3 times, according to 1）The standard curve of middle drafting can obtain the concentration value of Procalcitonin in measuring samples.
3) result shows：Using present case methods described by further testing, detection sensitivity 0.1ng/mL, inspection Survey scope is 0.02ng/mL-200ng/mL, and CV is less than 5% between batch, and CV is less than 1.5% in batch, and does not occur HOOK effects, nothing Processing need to be diluted to detection sample.
Case 2
Using biotin-Avidin amplification system, because the affinity of biotin and Avidin is higher, and an Avidin divides Son can combine 4 biotin molecules, detection sensitivity is improved 4-5 times than common detection methods；As shown in Figure 5.
1. biotin labeling PCT antibody
Weigh HPDP-LC- biotins to be dissolved in DMF, it is 2mg/ml to make concentration.Add in every milliliter of 1mg/mL PCT antibody-solutions Enter 100 μ L HPDP-LC- biotin solutions, react at room temperature 90min.Unreacted HPDP- is removed with SephadexG25 posts LC- biotin solutions.
2.HRP marks Avidin
1）By 5mg peroxidase（HRP）It is dissolved in 1.2mL 10mM PBSs（PH=7.0）In, add that 0.3mL is fresh matches somebody with somebody The 0.1M sodium periodate solutions put, 37 DEG C are incubated 20 minutes.
2）In 10mM sodium-acetate buffers（PH=4.0）Middle dialysis, 4 DEG C overnight.
3）Avidin is dissolved in 20mM sodium carbonate buffers（PH=9.5）, concentration 10mg/mL, add 0.5mL PCT and resist In body and HRP, 37 DEG C are incubated 2 hours.
4）100 μ L 4mg/mL sodium borohydrides are added, are placed 2 hours at 4 DEG C.
5）In 10mM PBSs（PH=7.0）Middle dialysis, 4 DEG C overnight.
3. kit assembles
Main component is 10mM PBSs in testing tube in present case kit（PH=7.0）, it is 170.uL often to test part Volume, wherein containing 20ug/mL acridinium ester label PCT antibody, 20ug/mL biotin labeling PCT antibody and 100ug/mL SA-HRP, in addition also containing 0.01% Proclin 300.
4. the quantitative test of Procalcitonin
1) making of standard curve：PCT standard items are diluted with calf serum, it is 0ng/ to be made into concentration some row concentration ML, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 300ng/mL each 150 μ L of Procalcitonin standard items, take 30 μ L The testing tube added in present case, testing tube is placed in supporting mini-plant, places 15min.Each sample repeats respectively Measure 3 times.For necessary instrument according to the proportionate relationship between RLU and PCT concentration, automatic Fitting calculates PCT concentration, takes Average value is determined three times, draws standard curve.
2) the μ L of sample 200 to be checked are taken, the testing tube being placed in present case, per the μ L of sub-sampling 30, replication 3 times, Standard curve according to drawing in 1) can obtain the concentration value of Procalcitonin in measuring samples.
3) result shows：Using present case methods described by further testing, detection sensitivity 0.06ng/ ML, detection range 0.01ng/mL-200ng/mL, CV is less than 5% between batch, and CV is less than 1.5% in batch, and does not occur HOOK effects, without being diluted processing to detection sample.
It is complete by above-mentioned description, relevant staff using the above-mentioned desirable embodiment according to the present invention as enlightenment Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention Property scope is not limited to the content on specification, it is necessary to determines its technical scope according to right.

Claims (5)

  1. A kind of 1. kit of homogeneous chemistry luminescence method detection animal Procalcitonin, it is characterised in that：
    The composition in testing tube in the kit is 10mMPBS buffer solutions, and PH=7.0, it is 170.uL volumes often to test part, Wherein containing 20ug/mL acridinium ester label PCT antibody and 20ug/mLHRP mark PCT antibody, in addition also containing 0.01% Proclin300；
    The kit is made using following preparation method：
    （1）PCT ANTIGEN DESIGNThes and preparation, including：PCT antigens amino acid sequence and base sequence design, sequence table reach, plasmid structure Build, plasmid extraction, protein expression, protein purification and detection；
    （2）PCT Antibody preparations, including：Immune blood sampling, hybridoma fusion screening；
    （3）Acridine derivatives mark PCT antibody；
    （4）HRP marks PCT antibody；
    （5）Sandwich method detects.
  2. 2. a kind of kit of homogeneous chemistry luminescence method detection animal Procalcitonin according to claim 1, its feature exist In：The PCT antigens amino acid sequence is such as：SEQIDNO.1, base sequence is such as：SEQIDNO.2.
  3. 3. a kind of kit of homogeneous chemistry luminescence method detection animal Procalcitonin according to claim 1, its feature exist In：The plasmid construction includes synthetic gene sequence, including including 5 ' end EcoRI, 3 ' end XhoI restriction enzyme sites, N-terminal addition Kozak (GCCGCCACC) and CD33 signal peptide sequences (ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCT), C-terminal addition HisTag sequences (CATCATCACCATCACCAT) and termination codon (TGATAG), and by this sequence construct to pcDNA4.0TomycHisA carry Body, the gene order of synthesis is such as：SEQIDNO.3.
  4. 4. a kind of kit of homogeneous chemistry luminescence method detection animal Procalcitonin according to claim 1, its feature exist In：Endotoxin-free plasmid is largely extracted with carrier for expression of eukaryon, the endotoxin-free plasmid extracted is subjected to cell transfecting, collected Cell conditioned medium and cell pyrolysis liquid, using Ni post affinity chromatographys, collect sample and carry out SDS-PAGEWesternblot inspections, it is dense Degree detection, qualified samples are dialysed to PBS, and by protein concentration extremely>1.0mg/ml.
  5. 5. a kind of kit of homogeneous chemistry luminescence method detection animal Procalcitonin according to claim 1, its feature exist In the hybridoma fusion screening comprises the following steps：
    （1）Before fusion prepared by feeder cells, including macrophage and thymocyte, prepares and is carried out in gnotobasis；
    （2）Before fusion prepared by myeloma cell；
    （3）Extract the spleen cell of immune success rate；
    （4）Cell fusion；
    （5）ELISA is detected；
    （6）Subclone screening.
CN201710873498.6A 2017-09-25 2017-09-25 A kind of kit of homogeneous luminescent method detection animal Procalcitonin Pending CN107817347A (en)

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CN110736733A (en) * 2018-07-18 2020-01-31 博阳生物科技（上海）有限公司 procalcitonin homogeneous phase chemiluminescence detection kit, detection method and device
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CN117986328A (en) * 2024-04-03 2024-05-07 江西省转化医学研究院 Cyclic peptide luminescent coupling molecule and preparation method and application thereof

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