CN105131122A

CN105131122A - Preparation and applications of monoclonal antibodies of polychlorinated biphenyl homologs

Info

Publication number: CN105131122A
Application number: CN201510504450.9A
Authority: CN
Inventors: 庄惠生; 孙瑞艳; 杨光昕
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2015-08-17
Filing date: 2015-08-17
Publication date: 2015-12-09

Abstract

The invention provides preparation and applications of monoclonal antibodies of polychlorinated biphenyl homologs. According to the preparation, immunogens are based on PCB37 haptens, and animal immunization method and hybridoma antibody technology are adopted so as to prepare anti-polychlorinated biphenyl PCB37 monoclonal antibodies from the immunogens. Compared with the prior art, antibody specificity is high, titer is high, practicality is excellent, stability is excellent, the preparation technology is simple and practicable, no special instrument is need in the whole preparation process, cost is low, and industrialized production can be easily realized. The prepared anti-polychlorinated biphenyl PCB37 monoclonal antibodies can be applied to immunodetection of polychlorinated biphenyl monomers PCB37 in the environment.

Description

Preparation of a kind of monoclonal antibody of polychlorinated biphenyl congener and uses thereof

Technical field

The present invention relates to a kind of preparation method of monoclonal antibody, particularly the preparation method of the monoclonal antibody of the polychlorinated biphenyl congener of the immunodetection of environmental hormone polychlorobiphenyl PCB37 in a kind of water body and Soil sediment.

Background technology

Polychlorobiphenyl (PCBs), as a kind of organic micro-pollutant with persistence harm, obtains global extensive concern.Different countries is different to the mode of the name of PCBs, and except standardized denomination chemically, PCBs is divided into trichloro biphenyl (PCB by biphenyl by the number (no matter its position of substitution) that chlorine replaces by China traditionally ₃), tetrachloro biphenyl (PCB ₄), pentachlorodiphenyl (PCB ₅), chlordene biphenyl (PCB ₆).According to estimates, nearly 1,000,000 t of the polychlorobiphenyl that the whole world has produced and applied, its semi-invariant in all kinds of environment is estimated to reach 250,000-30 ten thousand t.PCBs has lower water-soluble and high Xin Chun – aqueous systems partition ratio, is therefore easy to just enter ecological circulation, by food chain by enrichment, toxic action is produced to the mankind.Simultaneously because its chemical degradative processes and biodegradation process are quite slow, make this type of material cause long-term pollution to environment, so that repeatedly occur secondary pollution.

Polychlorobiphenyl (PCBs) is as a kind of Endocrine Disruptors being subject to countries in the world environmentalist's extensive concern, and the research of its detection method becomes a study hotspot.Current, being representative using euzymelinked immunosorbent assay (ELISA) (ELISA), immunologic detection method is quick as one, and high specificity, the advantage such as highly sensitive, easy, quick, enjoy the concern of people in recent years.American National Environmental Protection Agency (EPA) also just announced out as standard method using these class methods in 2003, was applied to the detection of polychlorobiphenyl in Soil sediment.Vehicles Collected from Market there is the product of polychlorobiphenyl immunity detection reagent.

But the key setting up immune analysis method obtains height of tiring, antibody that selectivity is good.Although there has been the report about the antibody for PCB12, PCB37, PCB77 at present, they are lower to tiring of target molecule, and selectivity is relatively poor, have impact on sensitivity and the selectivity of detection method.

In to environment polychlorobiphenyl PCB37 detect delay in, the monomer detection as PCB37 pollutent stronger to the single representational toxicity of polychlorobiphenyl in sample has very important significance.Although the enzyme linked immunosorbent assay analysis method set up at present, instrument stratographic analysis can PCBs accurately in testing environment sample, but its sensitivity is relatively low, for the sample that PCBs contained by some exists with trace, often need to realize analyzing and testing to it by concentrated, even can not detect, thus reduce the accuracy of method, be not suitable for the analyzing and testing of Environmental Trace PCBs.But, 2003, the ChadMirkin seminar of Northwestern Univ USA proposes a kind of overdelicate bio-barcode technology based on nanoparticle and detects protein molecular, the method mainly through the specific binding of antigen-antibody and Magneto separate realize to the identification of target molecule be separated; Utilization is modified at the DNA on gold nano grain (goldnanoparticles, GNPs) surface as detection signal, and this DNA is called visually barcode DNA (barcodeDNA) or signal dna (signalDNA).Hundreds of barcode DNA can be modified in GNPs surface, and this makes detection signal effectively be amplified, and through the further amplifying signal of gold label silver stain enhancement techniques, carry out analyzing and testing with flat bed scanner, its sensitivity can reach 10 ^-18molL ^-1.The method is without enzyme labelling or catalytic substrate amplifying signal, and the simple scale effect can modifying a large amount of signal probe with nano-material surface, and utilize the means of pure physics to detect, this also makes bio-barcode technology become the current uniquely a kind of method compared favourably with regard to energy and round pcr without enzyme catalysis amplification reported.In addition, the method is using DNA as detection probes, and the variation of existing DNA detection method is also for the development of biological bar codes technique and application provide wide space.At present, the method has become the study hotspot that biomacromolecule, environmental pollutant etc. analyze context of detection.

In addition, in recent years, along with the combination of bio-barcode technology and other analysing and detecting method, the multiple analytical procedure based on bio-barcode technology is defined.Wherein, quantitative fluorescent PCR biobarcode approach is the detecting stage in system, with barcode DNA for template DNA, utilizes real-time fluorescence quantitative PCR to increase to barcode DNA and analyzes quantitative analysis and detect target molecule; Compare with original method, the method has better specificity, effectively can avoid the cross reaction with similar protein, it also avoid false-positive appearance in Standard PCR using real-time fluorescence quantitative PCR as detection means.Current, indirect competition biobarcode approach based on real-time fluorescence quantitative PCR is mainly used in biology and medical research field, be used for detecting some malignant bacterias or protein, in environmental monitoring field, this technology is also just for the detection to some viruses or protein in environment.Also do not see this technology at present to report for the detection of PCBs class especially PCB37 environmental pollutant.

In prior art, number of patent application is CN201310246024.0, name is called " a kind of preparation method of polychlorobiphenyl monoclonal antibody ", disclose a kind of preparation method of polychlorobiphenyl monoclonal antibody, but prepared by the method is mixed type monoclonal antibody, its specificity is not very strong, easily produces cross reaction, and the phenomenon that when finally causing immunization method to detect polychlorobiphenyl, concentration is higher occurs.

Summary of the invention

For defect of the prior art, the object of this invention is to provide a kind of preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody, monoclonal antibody specificity prepared by the method is stronger, tire higher, improve the affinity of antibody to polychlorobiphenyl monomer, for the needs of the immunoassay technology development meeting polychlorinated biphenyl pollutants provide technical support.

The object of the invention is to be achieved through the following technical solutions:

The invention provides a kind of anti-polychlorobiphenyl monomer 3,4, the preparation method of 4 '-trichloro biphenyl PCB37 monoclonal antibody, said method comprising the steps of:

By PCB37 hapten synthesis immunogen;

The immunogen of synthesis is prepared anti-polychlorobiphenyl PCB37 monoclonal antibody by adopting animal immune method and hybridoma antibody technology.

Preferably, describedly specifically to be comprised the following steps by PCB37 hapten synthesis immunogen:

A1, PCB37 haptens is added aprotic organic solvent a make it dissolve, obtain solution a;

A2, by N, N-dicyclohexyl imide and N-hydroxysuccinimide, add aprotic organic solvent b and make it dissolve, obtain solution b;

A3, described solution b is added in described solution a, stirring reaction; Supernatant liquor is got after having reacted;

A4, bovine serum albumin BSA solution joined in described supernatant liquor and reacts, obtain PCB37 haptens-BSA binding substances, i.e. immunogen.

Preferably, in described steps A 1, described PCB37 haptens is trichloro biphenyl haptens, and in gained solution a, the haptenic concentration of PCB37 is 1 ~ 3mmol/mL; In described steps A 2, the weight ratio of N, N-dicyclohexyl imide and N-hydroxysuccinimide is 1:1.6 ~ 1.8, and in gained solution b, the imido concentration of N, N-dicyclohexyl is 80 ~ 83mg/mL.

Preferably, in described steps A 3, the volume ratio of described solution b and solution a is 3 ~ 5 ︰ 4, and the reaction times is 8 ~ 10h; In described steps A 4, BSA solution is that bovine serum albumin BSA is dissolved in carbonate buffer solution gained, and the concentration of BSA solution is 16mg/mL, and the condition of ice bath lower reaction times is 6 ~ 8h.

Preferably, described non-protic organic solvent a and non-protic organic solvent b is independently selected from DMF or methyl-sulphoxide.

Preferably, described PCB37 haptens is trichloro biphenyl haptens fusing point is 162 ~ 164 DEG C.

Preferably, describedly the immunogen of synthesis prepared anti-polychlorobiphenyl PCB37 monoclonal antibody by animal immune method and hybridoma antibody technology comprise the following steps:

S1, with fully emulsified after the immunogen of purifying and Freund's complete adjuvant mixing, then first immunisation injection carried out to animal, then carry out adding strengthening reaction with Freund's incomplete adjuvant, make to produce anti-polychlorobiphenyl PCB37 monoclonal antibody in animal body;

S2, the animal of anti-polychlorobiphenyl PCB37 monoclonal antibody will be produced in body in step S1, get its splenocyte containing anti-polychlorobiphenyl PCB37 monoclonal antibody and myeloma cell carries out cytogamy, obtain the hybridoma containing anti-polychlorobiphenyl PCB37 monoclonal antibody.

Preferably, described method also comprises employing ascites working system and the hybridoma that step S2 obtains is seeded to animal abdominal cavity, collects ascites, centrifugal, purifying, obtains the step of the anti-polychlorobiphenyl PCB37 monoclonal antibody after purifying.

Preferably, described purifying adopts one or more combinations in ammonium sulfate salting-out process, sad salting-out process, sad-ammonium sulfate precipitation method, DEAE cellulose ion-exchange chromatography method, QAE cellulose ion-exchange chromatography method or affinity chromatography.

Preferably, described purifying adopts sad-ammonium sulfate precipitation method and DEAE cellulose ion-exchange chromatography method to combine.The method purification efficiency is high, simple to operate, is relatively applicable to the purifying of monoclonal antibody.

Preferably, described method is also included in first three sky of cytogamy of step S2, once to make a spurt immunity to animal.

Preferably, the step of the hybridoma that step S2 obtains being carried out the hybridoma cell strain of cloning enlarged culturing acquisition containing anti-polychlorobiphenyl PCB37 monoclonal antibody is also comprised.

Preferably, in step S1, immunogen and the Freund's complete adjuvant of described first immunisation injection employing equivalent are fully emulsified, and immunogenic consumption is 40 ~ 100 μ g/.

Preferably, described Freund's complete adjuvant, comprises the mixture (volume ratio 1 ~ 5:1) of whiteruss and lanolin, immunogen and the cell wall constituent (i.e. bacille Calmette-Guerin vaccine) containing mycobacterium tuberculosis; Freund's incomplete adjuvant, comprise the water in oil emulsion of the mixture (volume ratio 1 ~ 5:1) of whiteruss and lanolin, immunogen composition, adjuvanticity comes from immunogenic sustained release in oil droplet, and stimulates local immunity to react.

Preferably, described animal is Mammals or bird, and Mammals comprises rabbit, sheep, horse, cavy, pig or monkey, preferred rabbit or cavy; Bird comprises chicken, duck, goose or crane quail, preferred chicken; Above-mentioned animal is of the right age, healthy and strong, FFI animal, preferred buck, and the age of animal should be between twenty and fifty, the adult guinea pig at 3 months preferred monthly ages.

Preferably, in step S1, described additional strengthening reaction, first time to strengthen selecting after first immunisation 2 ~ 3 weeks, latter 2 ~ 4 weeks of last immunity are selected in later reinforcement, and described additional strengthening reaction immunogenic consumption used is 0.5 ~ 1.5 times of immunogen consumption first.The immune response demands time, must interval 2 ~ 3 weeks process booster immunization again of adapting to animal one after first immunisation, animal is prevented to be not suitable with the phenomenon generation of external stimulus and death, later booster immunization selects the interval of 2 ~ 4 weeks, also be in order to the biological immune response of animal body does not cause too much excessive injury, as death etc. to animal.The selected of immunizing dose of the additional strengthening reaction in later stage should consider antigenicity power, molecular size range and immunization time.Antigen requirement is many, and the timed interval is long, and dosage can suitably strengthen.Only, only, cavy belongs to animalcule to animalcule about 0.1 ~ 0.6mg/ to large animal antigen dose (being as the criterion with proteantigen) about 0.5 ~ 1mg/.Select and very easily cause immunological tolerance (immunosuppression) because dosage strengthens and meet with unsuccessfully.Immunity pertinent literature is existing to be proved, the protein of a few microgram also immunity can go out antiserum(antisera) well.

Preferably, described additional strengthening reaction, first time to strengthen selecting after first immunisation 3 weeks, later reinforcement selection 2 ~ 3 weeks; Described booster immunization reacts immunogenic consumption used and the former consumption equivalent of first immunisation.

Preferably, after second time booster immunization 2 ~ 3 weeks, measure the antibody titer of animal, after tiring and meeting the requirements, carry out Mammals blood sampling or collect the antibody egg of bird, antibody purification, freeze-drying preservation.

Preferably, the measuring method of antibody titer comprises tube agglutination test, double immunodiffusion test, radioimmunoassay, Enzyme Linked Immunoadsorbent Assay or fluoroimmunoassay, preferred double immunodiffusion test, Enzyme Linked Immunoadsorbent Assay or fluoroimmunoassay.

Preferably, described immunization is a place or many places injection in intravenously, intraperitoneal, intramuscular, intracutaneous, subcutaneous, lymphoglandula.

Preferably, described immunization is one or more combinations in neck dorsal sc multi-point injection, intracutaneous multi-point injection.

Present invention also offers a kind of purposes of the anti-polychlorobiphenyl PCB37 monoclonal antibody prepared according to preparation method, can be used for the immunodetection of polychlorobiphenyl monomer PCB37.

Compared with prior art, the present invention has following beneficial effect:

(1) antibody is practical: the monoclonal antibody preparation technology of anti-polychlorobiphenyl monomer PCB37 has important use value and practical significance, the antibody of preparation has higher cross reacting rate to Polychlorinated Biphenyls monomer PCB37, has reasonable specificity to other compounds similar of structural similitude.This antibody and preparation method thereof provides guarantee for the total amount of the polychlorobiphenyl monomer PCB37 that immunization measures in environment, and good basis has been established in the development also for having the polychlorobiphenyl monomer PCB37 immunity detection reagent of independent intellectual property right based on the indirect competition biobarcode approach technological development of real-time fluorescence quantitative PCR.

(2) Antibody stability is good: the monoclonal antibody of the anti-polychlorobiphenyl monomer PCB37 that this legal system is standby has good stability.After freeze-drying ,-20 DEG C of preservations, its stability at least can keep more than 36 months; 4 DEG C of preservations, its stability at least can keep more than 12 months.

(3) monoclonal antibody of single anti-polychlorobiphenyl monomer PCB37 prepared of method of the present invention, lower with the cross reacting rate of other materials, specificity is good, tire up to 1:640000.

Accompanying drawing explanation

By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

Fig. 1 is the uv absorption spectra of polychlorobiphenyl monomer PCB37 haptens haptenPCB37, the artificial holoantigen PCB37-BSA of BSA, polychlorobiphenyl monomer PCB37;

Fig. 2 is the amplification curve that the indirect competition biobarcode approach based on real-time fluorescence quantitative PCR set up based on the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 detects polychlorobiphenyl monomer PCB37;

Fig. 3 is the partial enlarged drawing that the indirect competition biobarcode approach based on real-time fluorescence quantitative PCR set up based on the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 detects the amplification curve of polychlorobiphenyl monomer PCB37;

Fig. 4 is the standard working curve that the indirect competition biobarcode approach based on real-time fluorescence quantitative PCR set up based on the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 detects polychlorobiphenyl monomer PCB37;

Fig. 5 is the typical curve that the indirect competitive enzyme-linked immunosorbent adsorption analysis method (ic-ELISA) based on monoclonal antibody set up based on the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 detects PCB37.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.

the immunogenic UV absorber of embodiment 1 polychlorobiphenyl monomer PCB37

(1) the immunogenic preparation of polychlorobiphenyl monomer PCB37

Take the fine taper bottle that 72.87mgPCB37 haptens is placed in clean dried, add 0.4mLN, dinethylformamide (DMF) makes it dissolve, and obtains solution a, and controlling haptenic concentration is 1mmol/mL; Take 41.2mgN, N-dicyclohexyl imide (DCC) and 23mgN-hydroxysuccinimide (NHS), add 0.5mLDMF and make it dissolve, obtain solution b, make the weight ratio of DCC and NHS be 1:1.6 ~ 1.8, wherein the concentration of DCC is 80 ~ 83mg/mL; Under the condition of magnetic agitation, added by solution b in solution a, stirred at ambient temperature reaction 8 ~ 10h, the volume ratio of solution b and solution a is that 3 ~ 5 ︰ 4,4 DEG C spends the night; The product prepared is placed in the centrifugal 10min of low-temperature and high-speed whizzer 6000r/min, gets supernatant liquor; Taking 82mgBSA, to be dissolved in 5mL concentration be in the CBS damping fluid of 0.05mol/LpH9.60, and the concentration of BSA in CBS damping fluid is 16mg/mL, then dropwise joins in above-mentioned supernatant liquor, then react 6 ~ 8h under condition of ice bath; After reaction terminates, the solution obtained is transferred in dialysis tubing, with the phosphate buffered saline buffer dialysis 3d of 0.01mol/LpH7.40 under 0 ~ 4 DEG C of low temperature, the centrifugal 15min of 6000r/min, isolate supernatant liquor and obtain PCB37 haptens-BSA binding substances, the i.e. immunogen of PCB37, after ultraviolet-visible pectrophotometer scanning qualification, is sub-packed in-20 DEG C of freezen protective in 1.5mL centrifuge tube for subsequent use.

(2) the immunogenic sign of polychlorobiphenyl monomer PCB37

PCB37 haptens-BSA is suitably diluted, take PBS as blank, measure PCB37 haptens with ultraviolet spectrophotometer respectively at 200 ~ 500nm, BAS, and the light absorption value of PCB37 haptens-BSA, draw uv absorption spectra.As shown in Figure 1, the haptenic charateristic avsorption band of PCB37 is at 271nm place, the charateristic avsorption band of BSA is at 279nm place, and the immunogenic charateristic avsorption band of PCB37 is at 245nm place, blue shift is there occurs compared with the UV spectrum of two above, this may be subject to protein to produce in the impact of 230nm place absorption peak, shows PCB37 haptens and carrier proteins BSA coupling success, has namely successfully prepared the immunogen of PCB37.

the preparation of the monoclonal antibody of embodiment 2 anti-polychlorobiphenyl monomer PCB37

(1) immunity of animal

With PCB37 haptens-BSA for immunogen, adopt mode immunity three cavys of neck dorsal sc multi-point injection, immunization protocol is as shown in table 1, during first time immunity, immunogen and complete Freund's adjuvant are carried out equal-volume mixing and emulsifying, from second time immunity, immunogen and Freund's incomplete adjuvant are carried out equal-volume mixing and emulsifying, per injection 100 μ L, immunizing dose is 50 μ g, first three sky of cytogamy, immunity of once making a spurt.From third time immunity, every one week after each immunity, taken a blood sample by mouse tail, measure antiserum titre with indirect ELISA.

Table 1 immunization protocol

Immune time	Immunization interval	Immunity position	Phylactic agent
				1	--	Neck, back	Immunogen: CFA (1:1)
2	Two weeks	Neck, back	Immunogen: IFA (1:1)
				3	Two weeks	Neck, back	Immunogen: IFA (1:1)
4	Two weeks	Neck, back	Immunogen: IFA (1:1)
				Spurt immunity	First 3 days of cytogamy	Neck, back	Immunogen

Note: CFA refers to not formula Freund's complete adjuvant, and IFA refers to Freund's incomplete adjuvant.

(2) cytogamy

The cultivation of myeloma cell SP2/0: the water-bath that frozen myeloma cell SP2/0 is put into rapidly 37 DEG C, jiggles, makes frozen storing liquid dissolve completely in 1min.In super clean bench, be transferred in centrifuge tube by myeloma cell SP2/0 solution, add 5mLRPMI1640 nutrient solution, under 1000r/min, centrifugal 10min, abandons supernatant liquor; Add 5mLRPMI1640 complete culture solution again, be transferred in Tissue Culture Flask, at 37 DEG C, containing 5%CO after mixing gently ₂incubator in continue cultivate.Growth conditions according to cell changes liquid in time, before cytogamy, makes cell remain on logarithmic phase.

Prepare the splenocyte containing anti-polychlorobiphenyl PCB37 monoclonal antibody: first 3 days of cytogamy, carry out spurt immunity to cavy, the same day of fusion puts to death cavy by disconnected neck, and is soaked in 5min in the ethanolic soln of 75%.In super clean bench, cavy abdominal cavity is opened in aseptic technique subsequently, take out spleen and be placed in glass dish, in spleen, inject RPMI1640 nutrient solution with syringe, then puncture spleen film with bending syringe needle multiple spot, draw nutrient solution with syringe subsequently and repeatedly blow and beat to spleen and bleach completely.In glass dish, fill it up with nutrient solution, and be transferred in centrifuge tube by cell suspension, under 1000r/min, centrifugal 5min, abandons supernatant liquor, and with RPMI1640 nutrient solution re-suspended cell, repeated centrifugation washs 3 times, finally uses 10mL basic culture solution re-suspended cell for subsequent use.

PEG merges: the myeloma cell SP2/0 in vegetative period of taking the logarithm, with basic culture solution centrifuge washing once, and resuspended for subsequent use with basic culture solution.By myeloma cell (2 × 10 ⁸) and splenocyte (1 × 10 ⁷) mixing, add basic culture solution centrifuge washing once, knock bottom centrifuge tube gently, cell precipitation is broken up.In the water-bath of 37 DEG C, add the PEG1500 solution that 0.8mL concentration is 50% gently, leave standstill 90s, in 5min, count 10mLRPMI1640 nutrient solution subsequently.Centrifugal 10min under 1000r/min, abandon supernatant liquor, and with HAT nutrient solution (adding the HAT substratum (50 ×) of 2% and the foetal calf serum of 20% in RPMI1640 basic culture solution) re-suspended cell, inoculate 10 piece of 96 porocyte culture plate (the day before yesterday adds feeder cell) with every hole 100 μ L after mixing, Tissue Culture Plate is placed in 37 DEG C, CO ₂concentration is cultivate in the cell culture incubator of 5%.

(3) screening of hybridoma and colonized culture

After cytogamy 4d, observation of cell growing state, HAT nutrient solution and HT nutrient solution (adding the HT substratum (100 ×) of 1% and the foetal calf serum of 20% in RPMI1640 basic culture solution) is used partly to change liquid respectively, indirect ELISA method is adopted to detect cell conditioned medium liquid after 10d, filter out specificity high, positive strong contains anti-polychlorobiphenyl PCB37 monoclonal antibody hybridoma cell.

Limiting dilution assay is adopted to carry out colonized culture to the positive hybridoma cell screened.Hybridoma is diluted to 80/mL, 40/mL, 20/mL, 10/mL, every hole 100 μ L is inoculated in 96 well culture plates, cultivates in cell culture incubator, inoculates 10 piece of 96 porocyte culture plate altogether.After 10d, detect and only have the hole of a hybridoma colonies, if positive, then repeated cloning in the same way.Through statistics, hole, position 851, Growth of Hybridoma Cell hole; Screening with indirect ELISA the positive hole obtained is 7 holes; Adopt limiting dilution assay to carry out colonized culture to 7 positive holes, through 3 time clonings, obtain 1 strain positive rate reach 100% monoclonal cell strain and high specific, tire high containing anti-polychlorobiphenyl PCB37 cell strain of monoclonal antibody.The monoclonal cell strain that positive rate reaches 100% carries out enlarged culturing through 24 well culture plates and little square vase again, frozen for subsequent use.

(4) a large amount of preparations of the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37

Ascites working system is adopted to prepare the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37.Silica gel H is injected, every 5mg in cavy abdominal cavity.At the hybridoma of cavy intraperitoneal inoculation containing anti-polychlorobiphenyl PCB37 monoclonal antibody after one week, 1 × 10 ⁶/ mL/ only.Cavy intraperitoneal inoculation obviously expands containing cavy belly after the hybridoma 10d of anti-polychlorobiphenyl PCB37 monoclonal antibody, collect ascites, centrifugal 20min under 3500r/min, collect the ascites that supernatant liquor obtains the faint yellow monoclonal antibody containing anti-polychlorobiphenyl monomer PCB37 ,-20 DEG C save backup.

(1) immunity of animal

With PCB37 haptens-BSA for immunogen, adopt mode immunity three cavys of neck dorsal sc multi-point injection, immunization protocol is as shown in table 2, during first time immunity, immunogen and complete Freund's adjuvant are carried out equal-volume mixing and emulsifying, from second time immunity, immunogen and Freund's incomplete adjuvant are carried out equal-volume mixing and emulsifying, per injection 100 μ L, immunizing dose is 100 μ g, first three sky of cytogamy, immunity of once making a spurt.From third time immunity, every one week after each immunity, taken a blood sample by mouse tail, measure antiserum titre with indirect ELISA.

Table 2 immunization protocol

Immune time	Immunization interval	Immunity position	Immunizing dose (μ g)	Phylactic agent
					1	--	Neck, back	100	Immunogen: CFA (1:1)
2	Two weeks	Neck, back	100	Immunogen: IFA (1:1)
					3	Two weeks	Neck, back	100	Immunogen: IFA (1:1)
4	Two weeks	Neck, back	100	Immunogen: IFA (1:1)
					Spurt immunity	First 3 days of cytogamy	Neck, back	50	Immunogen

(2) cytogamy

(3) screening of hybridoma and colonized culture

After cytogamy 4d, observation of cell growing state, HAT nutrient solution and HT nutrient solution (adding the HT substratum (100 ×) of 1% and the foetal calf serum of 20% in RPMI1640 basic culture solution) is used partly to change liquid respectively, indirect ELISA method is adopted to detect cell conditioned medium liquid after 10d, filter out specificity high, the positive strong hybridoma containing anti-polychlorobiphenyl PCB37 monoclonal antibody.

Limiting dilution assay is adopted to carry out colonized culture to the positive hybridoma cell screened.Hybridoma is diluted to 80/mL, 40/mL, 20/mL10/mL, every hole 100 μ L is inoculated in 96 well culture plates, cultivates in cell culture incubator, inoculates 10 piece of 96 porocyte culture plate altogether.After 10d, detect and only have the hole of a hybridoma colonies, if positive, then repeated cloning in the same way.Through statistics, hole, position 851, Growth of Hybridoma Cell hole; Screening with indirect ELISA the positive hole obtained is 7 holes; Adopt limiting dilution assay to carry out colonized culture to 7 positive holes, through 3 time clonings, obtain 1 strain positive rate reach 100% monoclonal cell strain and high specific, tire high containing anti-polychlorobiphenyl PCB37 cell strain of monoclonal antibody.The monoclonal cell strain that positive rate reaches 100% carries out enlarged culturing through 24 well culture plates and little square vase again, frozen for subsequent use.

Ascites working system is adopted to prepare the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37.Silica gel H is injected, every 5mg in cavy abdominal cavity.At the hybridoma of cavy intraperitoneal inoculation containing anti-polychlorobiphenyl PCB37 monoclonal antibody after one week, 1 × 10 ⁶/ mL/ only.After cavy intraperitoneal inoculation hybridoma 10d, cavy belly obviously expands, and collects ascites, centrifugal 20min under 3500r/min, and collect supernatant liquor and obtain the faint yellow ascites containing anti-polychlorobiphenyl PCB37 monoclonal antibody ,-20 DEG C save backup.

the purifying of the monoclonal antibody of embodiment 4 anti-polychlorobiphenyl monomer PCB37

Adopt sad-ammonium sulfate precipitation method and DEAE cellulose ion-exchange chromatography method in conjunction with the ascites of purifying cavy containing anti-polychlorobiphenyl PCB37 monoclonal antibody, concrete steps are as follows: get 1mL ascites in small beaker, add 2mL0.06mol/L, pH is the acetate buffer solution of 5.0, then adjusts pH to 4.8 with the hydrochloric acid of 1mol/L; At room temperature dropwise add 11 μ L while stirring sad, vibrate 30min gently, and under 4 DEG C of cold condition, the centrifugal 30min of 5000r/min, abandons precipitation, collects supernatant liquor, then adjusts about pH to 7 with the sodium hydroxide of 1mol/L; Slowly add the saturated ammonium sulphate solution of equivalent subsequently, at 4 DEG C, leave standstill 2h after mixing gently; Repeated centrifugation once again, abandons supernatant liquor, precipitation is dissolved in 1mLPBS, proceeds to dialysis tubing, and be that dialyzate is dialysed 3d with PBS, every day changes liquid 3-4 time, until whole NH ₄ ⁺(NH is had till being removed ₄ ⁺when existing, add Nessler's reagent and then produce yellow mercury oxide).If have a small amount of insoluble precipitation, then centrifugal segregation after dialysis, finally obtain the anti-polychlorobiphenyl PCB37 monoclonal antibody after purifying, packing is stored in-20 DEG C.

the sign of the monoclonal antibody of embodiment 5 anti-polychlorobiphenyl monomer PCB37

(1) indirect ELISA detects antibody titer

Specific experiment step is as follows, and the coating antigen (PCB37 haptens-OVA) of PCBs is diluted to proper concn with CBS respectively, and add in 96 hole enzyme plates, 100 μ L/ holes, wrap at 4 DEG C and spent the night; Wrap by after enzyme plate in add washings to wash away free coating antigen, 200 μ L/ holes, concussion 3min after dry, wash 3 times, then add the gelatin confining liquid (BSA of 1%) of 0.5%, 200 μ L/ holes, at 37 DEG C close 1h; Outwell confining liquid, wash 3 times dryings with washings, add 100 μ L antiserum(antisera)s, add the antiserum(antisera) of blank rabbit (mouse) in negative control hole, blank adds diluent PBS, incubation 1h at 37 DEG C; Enzyme plate adds two anti-goat anti-rabbit igg-HRP (goat anti-mouse igg-HRP) of 1:1000 dilution after washing three times, 100 μ L/ holes, incubation 1h at 37 DEG C; Wash plate 5 times with washings and dry, adding freshly prepared nitrite ion, 100 μ L/ holes, react 15min under room temperature; Add 2molL ^-1sulfuric acid stop buffer, 50 μ L/ holes, then measure the absorbancy (OD value) of every hole at 450nm and 630nm place, with OD=OD by microplate reader ₄₅₀-OD ₆₃₀for final reading; Calculate the P/N value in every hole, P/N=(OD-OD _blank)/(OD _negative-OD _blank), the antiserum(antisera) maximum dilution multiple corresponding to hole being more than or equal to 2.1 with P/N value is tired for this is sero-fast.Tiring as 1:640000 of the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 is measured in actual procedure.

(2) mensuration of antibody protein content

Measure with 752 type ultraviolet spectrophotometers and saltout and the absorption value A280 of gained antibody at 280nm and 260nm place and A260 after desalination, adopt A280nm and A260nm photoabsorption poor method calculating antibody IgG content.Solution after dialysis through lyophilize, packing cryopreservation.Protein content (mg/mL)=(1.45 × A ₂₈₀-0.74 × A ₂₆₀).After measuring purifying in actual procedure, the protein concentration of the monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 is 5.14mgmL ^-1.

(3) Antibody specificity analyses

For investigating the specificity of the monoclonal antibody obtained, use benzene respectively, dichlorobenzene, chlorobenzene, PCB8, PCB12, PCB15, PCB28, PCB29, PCB37, PCB77, Aroclor1242, Aroclor1248, Aroclor1254, Aroclor1260, as the competitor of antibody and coating antigen, measure its cross reacting rate to anti-polychlorobiphenyl PCB37 monoclonal antibody prepared by the present invention respectively with indirect ELISA.Method of calculation are, CR (%)=(IC ₅₀a)/(IC ₅₀b) × 100, wherein IC ₅₀a is the IC of PCBs ₅₀, IC ₅₀b is the IC that other materials are corresponding ₅₀.

The specificity of anti-PCB37 monoclonal antibody is as shown in table 3, antibody is to based on the Aroclor1242 of low chlordiphenyl, Aroclor1248 cross reacting rate is larger, be respectively 5.38%, 4.60%, to the cross reacting rate of other materials all lower than 5%, this shows that this antibody has good specificity, and compare with anti-PCB37 polyclonal antibody, the specificity of this antibody is stronger.

Table 3 anti-PCB37 monoclonal antibody and anti-PCB37 polyclonal antibody are to the comparative analysis of the cross reacting rate of other materials

the application of the monoclonal antibody of embodiment 6 anti-polychlorobiphenyl monomer PCB37

The monoclonal antibody of anti-polychlorobiphenyl monomer PCB37 prepared by the present invention, is mainly used in the immunodetection of polychlorobiphenyl monomer PCB37 in environment.One of its main application is exactly the method for immunity of the indirect competition biobarcode approach mensuration polychlorobiphenyl monomer PCB37 set up on this basis based on real-time fluorescence quantitative PCR.

The concrete steps that indirect competition biobarcode approach based on real-time fluorescence quantitative PCR measures polychlorobiphenyl monomer PCB37 are as follows: in order to strengthen the adsorptivity of PCR pipe, glutaraldehyde with 0.8% carries out pre-treatment to PCR pipe, every hole adds 20 μ L glutaraldehyde solutions, incubation 6h at 37 DEG C, then use milli-Q water 3 times, dry for subsequent use; Be buffered liquid with bag and PCB37 coating antigen is diluted to proper concn, add in PCR pipe and carry out bag quilt, every hole 20 μ L, wraps at 4 DEG C and is spent the night; Outwell coating antigen, every hole adds 200 μ L washingss and washs, and dries, add 200 μ L confining liquids, incubation 1h at 37 DEG C after washing three times after vibration 3min; Wash plate three times, by antibody dilution to proper concn, every hole adds 10 μ L antibody and 10 μ L samples, adds 20 μ LPBS, incubation 1h at 37 DEG C in blank control wells; Wash plate three times and dry, with containing the PBS of 5% skim-milk by GNPs probe dilution to suitable multiple, every hole adds 20 μ L, incubation 1h at 37 DEG C; First wash plate 5 times with PBST, then dry with ultrapure washing plate 5 times, adopt the amplification system of 20 μ L, every hole adds 10 μ LPCR test kit mixture, and upstream and downstream primer respectively adds 0.25 μ L, ultrapure water 9.5 μ L; When adding pcr amplification reagent, according to the amount of the required reagent of experiment, by various pcr amplification reagent mix together, every hole adds 20 μ L mixed solutions, to reduce the personal errors of repeatedly application of sample generation in single hole, in addition, should bubble be avoided to produce when application of sample as far as possible.Pcr amplification program: 95 DEG C of denaturation 5min; 95 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 20s, carry out 35 circulations with this; 72 DEG C extend 3min.The Ct value measured and the amount of determined antigen are inversely proportional to.Antigen and corresponding Ct value according to adding concentration known make typical curve, thus can obtain the concentration of polychlorobiphenyl monomer PCB37 in corresponding testing sample.The typical curve of its amplification curve measured and foundation is as shown in Fig. 2,3 and 4.

In this experiment, PCB37 coating antigen used adopts active ester method to be prepared, concrete grammar is as follows: take 35.1mgPCB37 haptens in fine taper bottle, add 1mLDMF to dissolve, add 30 μ L n-Butyl Amine 99s and 20 μ L isobutyl chlorocarbonates under magnetic agitation respectively, under ice-water bath, react 1h; Under magnetic agitation, above-mentioned reaction solution is dropwise added in the PBS solution (6mL) containing 60mgOVA, 4h is reacted under ice-water bath, after reaction, solution is transferred in dialysis tubing, to dialyse 3d with PBS at 4 DEG C, change 3-4 water every day, centrifuging and taking supernatant liquor, the i.e. coating antigen of PCB37 after dialysis, PCB37 haptens-OVA ,-20 DEG C of freezen protective after packing.

In this experiment, barcode DNA used takes from a section in PUC19 plasmid DNA.According to design of primers principle, use the primer that primerpremier5.0 software design is suitable ', upstream primer 5 '-GAGGCGGTTTGCGTATTG-3 ' and downstream primer 5 '-AGCGAGGAAGCGGAAGAG-3 ', the preparation synthesis of student on commission's work biotechnology (Shanghai) limited-liability company.

The PBS of the standard model of PCB37 containing 5% is diluted to 0pg/L, 5pg/L, 10pg/L, 50pg/L, 100pg/L, 500pg/L, 1000pg/L, 5000pg/L, under optimum experimental condition, sets up the typical curve detecting PCB37 based on monoclonal antibody.As shown in Figure 2, as shown in Figure 3, from left to right the concentration of PCB37 is respectively 0pg/L to the partial enlarged drawing of black box to amplification curve corresponding to each concentration, 5pg/L, 10pg/L, 50pg/L, 100pg/L, 500pg/L, 1000pg/L, 5000pg/L.Due to the competitive relation of PCB37 in sample and coating antigen PCB37-BSA, along with the increase of sample concentration, Ct value increases gradually.As shown in Figure 4, when polychlorobiphenyl monomer PCB37 concentration is at 5pg/L-5000pg/L, linear dependence is better, and linear equation is Ct=0.97lgC+14.80 (R ²=0.99, n=6), LOD is 2.03pg/L.

embodiment 7 detects PCB37 based on the indirect competitive enzyme-linked immunosorbent adsorption analysis method (ic-ELISA) of monoclonal antibody

For contrasting the superiority of the monoclonal antibody that present method is set up further, now establish based on the PCB37 in indirect competitive enzyme-linked immunosorbent adsorption analysis method (ic-ELISA) the testing environment sample of monoclonal antibody, concrete grammar is as follows: the coating antigen bag of PCB37 is buffered liquid (0.05MpH9.60 carbonate buffer solution) and is diluted to proper concn, the 100 every holes of μ L add in enzyme plate, and 4 DEG C of bags are spent the night; Coating buffer is outwelled, washes plate with washings (0.01MpH7.40PBST), the 200 every holes of μ L, dry after concussion 3min, wash three times, then add confining liquid, every hole 200 μ L, incubation 1h at 37 DEG C; With PBS by antibody dilution to suitable multiple, confining liquid is outwelled, wash plate 3 times with washings and dry, add the sample of 50 μ L and the antibody of 50 μ L respectively, the cumulative volume of every hole solution is made to be 100 μ L, blank control wells adds 100 μ LPBS, and negative control hole adds the negative antiserum(antisera) of 100 μ L, incubation 1h at 37 DEG C; Reaction solution is outwelled, washes plate 3 times with washings and dry, with PBS, goat anti-rabbit igg-HRP (goat anti-mouse igg-HRP) is diluted to suitable multiple; Add in enzyme plate, every hole 100 μ L, incubation 1h at 37 DEG C; Reaction solution is outwelled, washes plate 5 times with PBST and dry, add nitrite ion (400 μ L2.5mg/mL3,3 ', 5, the H of 5 '-tetramethyl benzidine substrate solution and 10mL phosphoric acid salt-citric acid substrate buffer solution, 10 μ L30% ₂o ₂be mixed to form nitrite ion) 100 μ L/ holes, react 10min under room temperature; Every hole adds 50 μ L stop buffers (2mol/L sulphuric acid soln) with color development stopping, then measures each hole in 450nm and 630nm place absorbance (OD value), with OD=OD by microplate reader ₄₅₀-OD ₆₃₀as final reading.Experimental result inhibiting rate represents, and is ordinate zou with inhibiting rate, and the logarithmic value of standard model concentration is X-coordinate drawing standard curve, and the standard working curve of foundation as shown in Figure 5.Wherein, inhibiting rate (%)=(1-A/A ₀) × 100, wherein A is the OD value in the hole having sample to exist, A ₀for there is no the OD value in the hole of sample.

Choose the concentration 0.01 μ g/L of PCB37 standard model, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L set up the typical curve that indirect competitive enzyme-linked immunosorbent adsorption analysis method detects PCB37, and equation is y=20.96LogC _pCB37+ 48.10 coefficient R ²=0.98, wherein IC ₅₀represent the sensitivity of detection method, analyte concentration 1.23 μ g/L corresponding when namely inhibiting rate is 50%; IC ₁₅the Monitoring lower-cut of method for expressing, analyte concentration 0.027 μ g/L corresponding when namely inhibiting rate is 15%; IC ₂₀-IC ₈₀represent linearity range, i.e. 0.015 μ g/L ~ 8.965 μ g/L.Compared with traditional instrument analytical procedure, sensitivity improves 100-500 doubly; Compared with the ic-ELISA of documents CN201310246024.0, the sensitivity of the method improves more than 45 times.In addition, between the method hole, the variation coefficient is 3.85% ~ 5.46%, and between plate, the variation coefficient is 4.25% ~ 6.45%, illustrates that the stability of the method is better.

Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims

1. a preparation method for anti-polychlorobiphenyl PCB37 monoclonal antibody, is characterized in that, said method comprising the steps of:

By PCB37 hapten synthesis immunogen;

2. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 1, is characterized in that, described is specifically comprised the following steps by PCB37 hapten synthesis immunogen:

A3, described solution b is added in described solution a, stirring reaction; Supernatant liquor is got after reaction terminates;

A4, bovine serum albumin BSA solution is joined linked reaction in described supernatant liquor, obtain PCB37 haptens-BSA binding substances, i.e. described immunogen.

3. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 2, is characterized in that, in described steps A 2, the weight ratio of N, N-dicyclohexyl imide and N-hydroxysuccinimide is 1:1.6 ~ 1.8; In described steps A 3, the volume ratio of described solution b and solution a is 3 ~ 5 ︰ 4, and the reaction times is 8 ~ 10h; In described steps A 4, BSA solution is that bovine serum albumin BSA is dissolved in carbonate buffer solution gained, and the reaction times is 6 ~ 8h.

4. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 1, it is characterized in that, described the immunogen of synthesis is prepared anti-polychlorobiphenyl PCB37 monoclonal antibody by animal immune method and hybridoma antibody technology comprise the following steps:

S1, with fully emulsified after the immunogen of purifying and the mixing of Fu Shi Freund's complete adjuvant, then first immunisation injection is carried out to animal, then carry out adding strengthening reaction with not formula Freund's incomplete adjuvant, make to produce anti-polychlorobiphenyl PCB37 monoclonal antibody in animal body;

5. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 4, it is characterized in that, also comprise and adopt ascites working system that the hybridoma that step S2 obtains is seeded to animal abdominal cavity, collect ascites, centrifugal, purifying, obtains the step of the anti-polychlorobiphenyl PCB37 monoclonal antibody after purifying.

6. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 5, is characterized in that, described method is also included in first three sky of cytogamy of step S2, once to make a spurt immunity to animal.

7. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 5, it is characterized in that, also comprise and the hybridoma that step S2 obtains is carried out the step that cloning enlarged culturing obtains the hybridoma cell strain containing anti-polychlorobiphenyl PCB37 monoclonal antibody.

8. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 5, is characterized in that, in step S1, immunogen and the Freund's complete adjuvant of described first immunisation injection employing equivalent are fully emulsified, and immunogenic consumption is 40 ~ 100 μ g/.

9. the preparation method of anti-polychlorobiphenyl PCB37 monoclonal antibody according to claim 5, it is characterized in that, in step S1, described additional strengthening reaction, first time to strengthen selecting after first immunisation 2 ~ 3 weeks, latter 2 ~ 4 weeks of last immunity are selected in later reinforcement, and described additional strengthening reaction immunogenic consumption used is 0.5 ~ 1.5 times of immunogen consumption first.

10. the purposes of anti-polychlorobiphenyl PCB37 monoclonal antibody that obtains of preparation method according to claim 1, is characterized in that, for the immunodetection of polychlorobiphenyl monomer PCB37.