CN1598586A - Immune antibody for detecting pesticide organic phosphorus residus and its application - Google Patents
Immune antibody for detecting pesticide organic phosphorus residus and its application Download PDFInfo
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Abstract
The invention discloses a rudimental immune antibody which is used to detect organic phosphorus pesticide residue and its application, the character of the invention is using diethyl phosphonic acid acetic acid as hapten, produce artificial immunogen through coupling with bovine serum albumin(BSA), and produce several clone antibody through immune Zelanian giant blanc or produce mono clone antibody through chmice immune, cell amalgamation, filtration, clone planting and other steps, the several and single antibody can be used to indirect emulative ELISA, direct ELISA, or immune test paper to detect the rudimental phosphorus pesticide. The excellences of the invention including: the selected hapten has several organic phosphorus pesticides general structure, the structure reforming is not necessary, it can immune animal after coupling with protein, it can also be used to detect organic phosphorus pesticide in food and water source, this show the rapid and convenient of the immune detecting tehchnology.
Description
Technical field:
The present invention relates to a kind of immune antiboidy and application thereof of residues of pesticides immune detection, especially a kind of immune antiboidy and application thereof that is used to detect organophosphorus pesticide.
Background technology:
Agricultural chemicals is that current agricultural production is used for anti-curing the disease, worm, weeds be to the indispensable material of murrain, to promoting that agricultural produce has epochmaking effect, but bring serious environmental problem simultaneously: disruption of ecological balance, quality of agricultural product descends, and human body health is compromised.At present, organophosphorus, pyrethroid pesticide have insecticidal effect efficiently, are the main forces of chemical protection of plant.Along with the cry of environment in recent years protection is more and more higher, organophosphorus insecticide uses the environmental problem paid more and more attention of bringing.
But with regard to present situation, still hunger stares at sb. in the face for the mankind, poor puzzlement, and the use of completely forbidding agricultural chemicals is unpractical, in order to reach highly efficient and productive target, the status of agricultural chemicals can't replace in a short time.Given this, be to ensure people health, rationally the using and its residual quantity is monitored of agricultural chemicals during effectively control is produced developed a kind of fast, reliable, sensitive, and the trace analysis method that is suitable for the residues of pesticides on-site supervision has important practical significance.The residue analysis method of organophosphorus insecticide mainly is by after extracting, purifying at present, be equipped with efficient HPLC or GC again, this detection method expense is relatively costly, sample pretreatment process is complicated, instrument and equipment is had relatively high expectations and needed consumes a large amount of organic solvents, thereby causes second environmental pollution.
Immunoassay is a kind of quick, sensitive, simple to operate, a kind of detection method that expense is low.Agricultural chemicals mostly is micromolecular compound, need just can be used for immune animal after connecting with high molecular weight protein class material lotus root, and the stimulation animal immune system produces the antibody at the micromolecule agricultural chemicals.Because most of agricultural chemicals itself do not possess the site that directly connects with protide macromolecular substances lotus root, therefore, need transform agricultural chemicals usually, research and development in early stage expense is higher.And, usually the specificity to predetermined substance is stronger owing to antibody, many of the immune analysis methods of setting up are applicable to the check and analysis of single persticide residue, have limited the application of immune analysis method, can not give full play to quick, simple to operate, the advantage that testing cost is low of immunoassay.
Summary of the invention:
The objective of the invention is to: the specificity to predetermined substance is stronger usually at the antibody that exists in the present residues of pesticides immune detection, many of the immune analysis methods of setting up are applicable to the check and analysis of single persticide residue, limited the practical problems that immune analysis method is used, a kind of immune antiboidy and application thereof that is used to detect organophosphorus pesticide is provided.
The object of the present invention is achieved like this: a kind of immune antiboidy that is used to detect organophosphorus pesticide, it is characterized in that, with diethyl phosphonic acids acetate is haptens, by being prepared into artificial immunogen with bovine serum albumin (BSA) coupling, and be prepared into polyclonal antibody or make monoclonal antibody by processes such as mouse immune, Fusion of Cells, screening and cloning cultivations by immune new zealand white rabbit, described artificial immunogen can adopt active ester method or the preparation of water-soluble carbodiimide method.
Active ester method prepares immunogene and is achieved in that diethyl phosphonic acids acetate and the N-hydroxy-succinamide (NHS) of getting equimolar amounts, ring dihexyl carbimide (DCC), with dioxane potpourri is dissolved, the lucifuge reaction is spent the night under the room temperature, centrifugal removal post precipitation, get the supernatant drying, residue is suspended in 3 milliliters of borate buffer solutions that are dissolved with 20mg bovine serum albumin (BSA), potpourri is magnetic agitation 1 hour at room temperature, 4 ℃ of phosphate buffers to 1 liter of pH7.4 (PBS) dialysis is prepared into artificial immunogen; The water-soluble carbodiimide legal system is equipped with artificial immunogen and is achieved in that taking by weighing diethyl phosphonic acids acetate earlier is dissolved in the dimethyl formamide (DMF), and low temperature stirs, and water intaking dissolubility carbodiimide (EDC) is dissolved among the 1mlDMF, slowly joins in the above-mentioned solution.Other gets bovine serum albumin (BSA) 20mg and is dissolved among the 2mlDMF, and 4 ℃ of stirrings slowly join in the above-mentioned mixed liquor.Continue stirring reaction 8h, spend the night in 14 ℃ of reactions, the centrifugal 10min of 2000g next day gets precipitation, and dialysis is prepared into immunogene.
The Polyclonal Antibody Preparation method is: with the immunogene of synthetic, the immunity new zealand white rabbit, physiological saline dilution auricular vein injection with doubled amount antigen, when reaching the best when tiring, the heart blood sampling, earlier with behind the ammonium sulfate step-by-step precipitation method preliminary purification, again through Sephadex G-200 be further purified high-quality broad spectrum activity antibody.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method is: with the immunogene of synthetic, and immune balb/c mice, every mouse is got 100 μ g immunogenes, and is even with equal-volume Fu Shi Freund's complete adjuvant mixing and emulsifying, injects in the abdominal cavity film along groin.After 4 week, booster immunization, dosage is constant, and adjuvant changes the Fu Shi Freund into.Behind the booster immunization three times, blood sampling is surveyed and is tired, when treating that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10: 1 ratio is mixed in the 50ml centrifuge tube and merges, cell after the fusion treats that after cultivating the hybrid cell quantity in the hole reaches 300 when above, get cell culture supernatant and be used for enzyme linked immunosorbent detection, the each detection is the detection of multiple hole, second day duplicate detection after each the detection is with confirmed results.Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the cell in all positive hole is the hybridoma cell strain of secrete monoclonal antibody.Hybridoma cell strain is used for preparing ascites through after the enlarged culture.Carry the last week with 0.5ml paraffin oil injection mouse peritoneal, 106 hybridomas are suspended in the 1ml serum free medium, inject in the mouse peritoneal.After 10 days, mouse web portion obviously expands, and collects ascites.Come monoclonal antibody purification with caprylic acid-ammonium sulfate precipitation method.
The application of above-mentioned antibody in detection is characterized in that, described polyclonal antibody or monoclonal antibody can be used for indirect competition ELISA, direct ELISA or immune test paper detects the residual of organophosphorus insecticide.
The invention has the advantages that: on the basis of the multiple organophosphorus insecticide structure of comparative analysis, according to its common structure characteristics design with diethyl phosphonic acids acetate as haptens, selected haptens has the universal architecture of multiple organophosphorus insecticide, and do not need to carry out structure of modification, directly can carry out immune animal after the coupling with protein, prepared antibody is to chlopyrifos, omethoate, basudin, ethyl parathion, Profenofos, multiple organophosphorus insecticide such as phoxim all has specificity, can be used for food, the screening of multiple organophosphorus insecticide detects in the water source, has fully shown the quick of immunoassay technology, easy characteristics.
Description of drawings
Fig. 1 is immunogene and the coating antigen that adopts the active ester method preparation;
Fig. 2 is immunogene and the coating antigen that adopts the preparation of water-soluble carbodiimide method.
Embodiment
The invention will be further described below in conjunction with accompanying drawing.
Embodiment one, active ester method prepare immunogene and envelope antigen
Get the diethyl phosphonic acids acetate (0.0196g) and the NHS (0.0109g) of equimolar amounts (0.1mmol), 0.11mmol DCC (0.0226g), be added in the glass tube, with dioxane potpourri is dissolved, the lucifuge reaction is spent the night under the room temperature, the centrifugal 10min of 15000g next day, remove precipitation, get supernatant and drain, residue is suspended in the sodium borate buffer liquid that 3ml is dissolved with 20mg bovine serum albumin (BSA) (being used for synthetic artificial immunogen) or ovalbumin (OVA) (being used for synthetic envelope antigen) in 35 ℃ of vacuum.Potpourri is magnetic agitation 1 hour at room temperature, and 4 ℃ to 1LPBS dialysed overnight (changing liquid 4 times).Envelope antigen is the conjugate of haptens and ovalbumin, and the preparation method is identical with immunogene.Dialyzed sample is carried out full wavelength scanner with BACKMAN DU640 ultraviolet scanner and is identified the coupling result.
Fig. 1 a is the haptenic conjugate of BSA-(immunogene) that utilizes the active ester method preparation, and Fig. 1 b is the haptenic conjugate of OVA-(coating antigen) that utilizes the active ester method preparation.Three kinds of materials are respectively carrier protein BSA, carrier protein BSA and hapten conjugation thing, haptenic ultraviolet absorpting spectrum from top to bottom among Fig. 1 a.At 254nm place haptens one trough is arranged as can be seen, carrier-antigen conjugates also has trough to occur, and the stack of absorption peak takes place herein for the two.At 210nm, 238nm place tangible absorption superposition phenomenon is arranged also in addition.Illustrate reaction has taken place between the two, can infer haptens and carrier protein BSA success coupling.Three kinds of materials are respectively carrier protein OVA, carrier protein OVA and hapten conjugation thing, haptenic ultraviolet absorpting spectrum from top to bottom among Fig. 1 b.At 254nm place haptens one trough is arranged as can be seen, carrier-antigen conjugates also has trough to occur, and the stack of absorption peak takes place herein for the two.At 210nm, 238nm place tangible absorption superposition phenomenon is arranged also in addition.Illustrate reaction has also taken place between the two, can infer haptens and carrier protein OVA success coupling.
Embodiment two, water-soluble carbodiimide legal system are equipped with immunogene and envelope antigen
Take by weighing diethyl phosphonic acids acetate 0.0392 and be dissolved in the 2ml dimethyl formamide (DMF), low temperature stirs.Take by weighing water-soluble carbodiimide (EDC) 8.0mg again and be dissolved among the 1ml DMF, slowly join in the DMF solution of above-mentioned diethyl phosphonic acids acetate.Other gets BSA (being used for synthetic artificial immunogen) or OVA (being used for synthetic artificial envelope antigen) 20mg is dissolved in 2ml DMF, and 4 ℃ of stirrings slowly join in the above-mentioned mixed liquor.4 ℃ are continued stirring reaction 8h, spend the night in 14 ℃ of reactions again, and the centrifugal 10min of 2000g next day gets precipitation, and the sample after the PBS solution of PH7.4 was dialysed 3 days carries out full wavelength scanner with BACKMAN DU640 ultraviolet scanner and identifies the coupling result.
Fig. 2 a is the haptenic conjugate of OVA-(coating antigen) that utilizes the preparation of water-soluble carbodiimide method, and Fig. 2 b is the haptenic conjugate of BSA-(immunogene) that utilizes the preparation of water-soluble carbodiimide method.Three kinds of materials are respectively carrier protein OVA, carrier protein OVA and hapten conjugation thing, haptenic ultraviolet absorpting spectrum from top to bottom among Fig. 2 a.At 254nm place haptens one trough is arranged as can be seen, carrier-antigen conjugates also has trough to occur, and the stack of absorption peak takes place herein for the two.At 210nm, 238nm place tangible absorption superposition phenomenon is arranged also in addition.Illustrate reaction has taken place between the two, can infer haptens and carrier protein OVA success coupling.Three kinds of materials are respectively carrier protein BSA, carrier protein BSA and hapten conjugation thing, haptenic ultraviolet absorpting spectrum from top to bottom among Fig. 2 b.At 254nm place haptens one trough is arranged as can be seen, carrier-antigen conjugates also has trough to occur, and the stack of absorption peak takes place herein for the two.At 210nm, 238nm place tangible absorption superposition phenomenon is arranged also in addition.Illustrate reaction has also taken place between the two, can infer haptens and carrier protein BSA success coupling.
Embodiment three, polyclonal antibody preparation.
According to 3 new zealand white rabbits of each immunity of two kinds of immunogenes of embodiment 1 or the preparation of embodiment 2 methods, concrete immunization protocol such as table 1.
Table 1 preparation organophosphorus polyclone antiantibody animal immune scheme
| Immune time | The time interval | The immunity position | Immunizing dose | Serum is gathered |
| 1 | 1 week (apart from gathering negative serum) | The back is subcutaneous | 0.1mg/ only immunogene adds equal-volume Fu Shi Freund's complete adjuvant | — |
| 2 | 4 weeks | Subcutaneous, vola, back | 0.1mg/ only immunogene adds the equal-volume freund 's incomplete adjuvant | — |
| 3 | 3 weeks | Subcutaneous, vola, back | The same | The ear vein blood sampling is surveyed and is tired |
| 4 | 3 weeks | The back is subcutaneous, vola, ear vein | The same | The ear vein blood sampling is surveyed and is tired |
| 5 | 1 week | Ear vein | 0.2mg/ only immunogene adds equivalent physiological saline | The ear vein blood sampling is surveyed and is tired |
| - | 10 days | - | - | Heart blood sampling preparation antiserum |
Finish in the table 1 behind the immune programme for children, utilize the square formation titrimetry that two kinds of antiserums are detected with two kinds of envelope antigens respectively, determine best Ag-Ab combination and best effort concentration thereof, the results are shown in Table 2.And utilize the antigen-antibody best operating condition of setting up to analyze the inhibiting effect of different types of organophosphorus pesticide to this Ag-Ab association reaction.With diethyl phosphonic acids acetate is object, sets up regression equation, calculates its I
10And I
50Choose 13 agricultural chemicals and comprise ethyl parathion, parathion-methyl, Rogor, omethoate, acephatemet, phoxim, chlopyrifos, basudin, Profenofos, Hostathion, malathion, phenthoate dimephenthoate cidial, isocarbophos, the medicament inhibiting rate is carried out regretional analysis to drug concentration, draw regression equation, and calculate the I of each medicament antagonist reaction
50Value the results are shown in Table 3.
As can be seen from Table 2, compare relatively poor with active ester method with the effect of the synthetic immunogen immune animal of EDC method.The antibody that active ester type antigen produces has faint recognition capability to the antigen of EDC method preparation; The antibody that EDC type antigen produces to the coating antigen of active ester method preparation without any effect.In the antibody that is obtained by active ester method antigen, it is the highest to tire with No. 4310 rabbit secretory antibodies, is 1: 25600, is research object with this antibody in the research of this paper back.Utilize the antibody of No. 4310 rabbit generations to carry out the square formation titration, the gained result is that antigen, antibody best effort concentration are 1: 2000.
Table 2 antibody titer measurement result
| Coating antigen | Active ester method OP-BSA antibody titer | EDC method OP-BSA antibody titer | ||||
| 4324 | 4310 | 4316 | 4320 | 4343 | 4358 | |
| Active ester legal system OP-OVA | 12800 | 25600 | 6400 | - | - | - |
| The EDC legal system is equipped with OP-OVA | 200 | 400 | 200 | 6400 | 3200 | 3200 |
With diethyl phosphonic acids acetate is research object, and the regression equation of foundation is I=0.6614+0.229lgC, r=0.9699, I
50=0.1820 μ g/ml, I
10=0.003536 μ g/ml.The I of multiple organophosphorus medicament antagonist
50Be shown in table 3 as calculated.
The inhibition ability of the different agricultural chemicals antagonists of table 3
| Pesticide variety | ????I 50(μg/ml) |
| Diethyl phosphonic acids acetate | ????0.18 |
| Chlopyrifos | ????0.12 |
| Basudin | ????0.15 |
| Omethoate | ????0.21 |
| Ethyl parathion | ????0.88 |
| Profenofos | ????0.97 |
| Phoxim | ????2.5 |
| DDVP | ????32 |
| Acephatemet | ????89 |
| The malathion | ????200 |
| Parathion-methyl | ????215 |
| Isocarbophos | ????- |
| Phenthoate dimephenthoate cidial | ????- |
As can be seen from Table 3, gained antibody has produced recognition reaction to Multiple Pesticides.Antibody resists former guide's thing diethyl phosphonic acids acetate stronger affinity, I
50Be 0.18 μ g/ml.The selective difference of this method between Rogor and omethoate is bigger, and the two structural difference is that P-O is different with the P-S key, and all the other groups are all identical.Antibody has stronger compatibility to omethoate, but very weak to the affinity of Rogor, this shows that in the process of antigenic activation immuning system generating antibody, the P=O key has played conclusive effect.But antibody is to the I of chlopyrifos, basudin, ethyl parathion
50All less than 1 μ g/ml, these 3 kinds of agricultural chemicals all contain a public structure (C2H50-), illustrate that C2H50-also exists as an antigenic determinant, plays important effect.Profenofos is because contain a C
2H
5O--and a P=O, and make antibody stronger recognition reaction be arranged to it, I
50Be 0.97 μ g/ml.It is relatively poor that isocarbophos, malathion, parathion-methyl etc. suppress ability to antigen-antibody reaction, illustrates that antibody does not have or have only faint recognition capability to these medicaments.From above result as can be seen, at diethyl phosphonic acids acetate c-terminus coupling carrier albumen, can make the C on the phosphonyl group
2H
5O--and a P=O fully expose as antigenic determinant, stimulate animal to produce antibody.
Embodiment four, Monoclonal Antibody
Get 4 of Balb/C mouse, every mouse 100 μ g immunogenes, even with equal-volume Fu Shi Freund's complete adjuvant mixing and emulsifying, inject in the abdominal cavity film along groin.After 4 week, booster immunization, dosage is constant, and adjuvant changes the Fu Shi Freund into.Behind the booster immunization 5 times, docking blood sampling has in 4 mouse that antibody titer reaches 1: 25600 in 3 serum, has 1 to reach 1: 51200 and no longer rise, and choose No. 3 the highest mouse extracting spleen cells of tiring and do fusion this moment.
Under aseptic technique, with splenocyte and murine myeloma cell in 5-10: 1 ratio is mixed in the 50ml centrifuge tube, adds 30ml serum-free IPMI1640 nutrient culture media, and 1200r/min is centrifugal, and 10min abandons supernatant, with the cell mass pine that shakes gently, place 37 ℃ of water-baths.1ml 50%PEG 4000 is slowly added in the cell, in 1min, drip off, stir bottom settlings gently simultaneously, leave standstill 1min.Slowly add serum-free medium along tube wall and stop fusion process.Slowly at the uniform velocity added 1ml in preceding 30 seconds, added 2ml in back 30 seconds, add the 27ml serum-free medium then fast, the centrifugal 10min of 1200r/min abandons supernatant.With 30mlHAT nutrient culture media suspension cell, suspending liquid immigration cultivation is in advance had in the 96 porocyte culture plates of feeder cells, be placed on 5%CO
2, cultivate in 37.3 ℃ of cell culture incubators.And simultaneously the myeloma cell who does not add fusion agent and splenocyte Mixed culture with as negative control.Merge after 3 days, it is clear that background begins to become, and non-fused cell is broken dead, and the cell of fusion forms little population of cells.Merge after 7 days, cell clone is highly purified.Show that with ELISA method Preliminary detection result the hole of positive reaction accounts for 87%, light absorption value accounts for 6.7% greater than 1 strong positive hole.Through 4 time clonings, the hybridoma that obtains 1 strain stably excreting monoclonal antibody is 3C3 with the cell in the strong positive hole.With this strain cell enlarged culture, implant and injected in the mouse body of paraffin oil, after 7 days, there are 4 mouse web portions to expand, moving difficulty, 1 death, 1 belly does not produce and expands.
Cell after the fusion screens in the HAT selective medium earlier, changes the HT nutrient solution after 5 days into, treats that the hybrid cell quantity hole in reaches 300 when above, carries out the detection of multiple hole with indirect ELISA pair cell culture supernatant, and next day, duplicate detection was with confirmed results.Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the hole inner cell that all is positive is the hybridoma of secrete monoclonal antibody.Through enlarged culture, a part of cell cryopreservation is in liquid nitrogen container with hybridoma, and a part of cell is used for preparing ascites.
Choose 6 multiparity Balb/C mouse, carry and be expelled to mouse peritoneal with the 0.5ml paraffin oil the last week, about 106 hybridomas are suspended in the 1ml serum-free IPMI1640 nutrient culture media, inject in the mouse peritoneal.After 10 days, treat to collect when mouse web portion obviously expands ascites.Come purifying ascites with caprylic acid-ammonium sulfate precipitation method.Measure the A278nm/A251nm value of IgG with the nucleic acid-protein ultraviolet scanner, and measure the value of A278nm, tentatively judge whether globulin into IgG according to the gained data.
After ascites antibody was purified, the value that records the A278nm/A251nm of antibody on the ultraviolet protein analyzer was about 2.5.A278nm/A251nm according to mammal IgG is about 2.5-3.0, and this ratio of other protein is the characteristics of 1-1.5, and the albumen that obtains after can the preliminary judgement purifying is IgG albumen.
According to the principle of square formation titration, 2000 times of antigen diluents, concentration is 25 μ gml
-1, during about 4000 times of antibody dilution, light absorption value is 1, can determine that antibody best effort concentration is 4000 times.Embodiment five, solid phase indirect competition ELISA detect step
1, bag quilt: every hole adds 100ul coating buffer (envelope antigen dissolves with carbonate buffer solution) in 96 microwell plates, and 4 ℃ are spent the night or 37 ℃ of incubations 2 hours;
2, sealing: take out 96 microwell plates, discard coating buffer,, dry with PBST (phosphate buffer that contains 0.05% Tween-20, down with) washing three times, and in every hole adding 200ul confining liquid (1%OVA), 37 ℃ of incubations 1 hour;
3, application of sample: take out ELISA Plate, with PBST washing three times, dry, sample is removed suspended impurity after, mix with equal-volume antibody mixed liquor, press and inject in 96 microwell plates shown in the table one, every hole 100ul, simultaneously, ELISA Plate G1-G6 hole adds the antibody of 50ul and the PBS of 50ul, capable each hole of H adds the PBS of 100ul, 37 ℃ of incubations 2 hours;
4, enzyme-added mark SPA (staphylococcal protein A): take out ELISA Plate,, dry, in every hole, add 100ul enzyme mark SPA dilution, 37 ℃ of incubations 45 minutes with the PBST washing.
5, add substrate solution: take out ELISA Plate,, dry with PBST washing three times.In every hole, add the substrate solution now join, 37 ℃ of incubations 15 minutes.
6, cessation reaction: add 50ul 2M sulfuric acid solution cessation reaction in every hole.
7, reading adds and blots the enzyme mark with thieving paper immediately behind the sulfuric acid and pull the bottom, and ELISA Plate is placed on the microplate reader, and is capable of serving as blank the zeroing with H under the 450nm wavelength, measures the OD value in each hole.
8, the mean value of the OD value of setting control wells G1-G6 is BO, and the OD value mean value of the repeating hole of same sample is Byp (samples that the yp representative is different).Every group of data all are as the criterion with the mean value of three repetitions.The inhibiting rate of each sample determination correspondence is I=100%[(B
0--Byp)/Bo].
9, the result judges, the OD value is calculated inhibiting rate per sample, if inhibiting rate＞20% shows possibility organophosphorus pesticide residue in the sample, need further carry out instrument and confirm.
During enforcement, described antibody can adopt monoclonal antibody also can adopt polyclonal antibody, and described sample can adopt vegetables and fruits class or liquid matter.In the present embodiment, described antibody is the polyclone clonal antibody, and described sample is a liquid matter.
Case study on implementation six: solid phase directly detects step
1, bag quilt: every hole adds 100ul envelope antigen dilution in 96 microwell plates, and 4 degrees centigrade are spent the night or 37 degrees centigrade of incubations 2 hours;
2, sealing: take out 96 microwell plates, discard coating buffer, with PBST washing three times, dry, and in every hole, add the 200ul confining liquid, 37 degrees centigrade of incubations 1 hour;
3, application of sample: take out ELISA Plate, with PBST washing three times, dry, sample is extracted with 5: 95 mixed liquors with methyl alcohol and water, the extract thickening-purification technology is handled the back and is mixed with equal-volume horseradish peroxidase (HRP) labelled antibody mixed liquor, inject in 96 microwell plates by shown in the table 4-4, every hole 100ul, simultaneously, capable each hole of ELISA Plate H adds the PBS of 100ul, the G1-G6 hole adds the SPA labelled antibody of 50ul and the PBS of 50ul, 37 facility degree incubations 2 hours;
4, add substrate solution: take out ELISA Plate,, dry with PBST washing three times.In every hole, add the substrate solution now join, 37 degrees centigrade of incubations 15 minutes.
5, cessation reaction: add 50 μ l 2M sulfuric acid solution cessation reactions in every hole.
6, reading blots enzyme base number of a tender portion with thieving paper after adding sulfuric acid immediately, ELISA Plate placed on the microplate reader, and under the 450nm wavelength, serve as blank zeroing with capable each hole of H, measure the OD value in each hole.
7, the mean value of the OD value of setting control wells G1-G6 is BO, and the OD value mean value of the repeating hole of same sample is Byp (samples that the yp representative is different).Every group of data all are as the criterion with the mean value of three repetitions.The inhibiting rate of each sample determination correspondence is I=100%[(BO----Byp)/Bo].
8, the result judges, the OD value is calculated inhibiting rate per sample, if inhibiting rate＞20% shows possibility organophosphorus pesticide residue in the sample, need further carry out instrument and confirm.
During enforcement, described antibody can adopt monoclonal antibody also can adopt polyclonal antibody, and described sample can adopt vegetables and fruits class or liquid matter.In the present embodiment, described antibody is monoclonal antibody, and described sample is the vegetables and fruits class.
Application of sample empirical model in table 4 ELISA Plate
| ??1 | ??2 | ??3 | ??4 | ??5 | ??6 | ??7 | ??8 | ??9 | ??10 | ??11 | ??12 | |
| ?A | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 |
| ?B | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 |
| ?C | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 |
| ?D | Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Sample 24 |
| ?E | Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Sample 24 |
| ?F | Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Sample 24 |
| ?G | Contrast | Contrast | Contrast | Contrast | Contrast | Contrast | ||||||
| ?H | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank |
Embodiment seven, immune detection trial-production method detect
Immunity test strip comprises: thieving paper A, the plain film S of glass fibre, nitrocellulose filter T and nitrocellulose filter C end, the concrete structure of immunity test strip is: with the mutual juxtaposition of joint between thieving paper A, the plain film S of glass fibre, nitrocellulose filter T and the nitrocellulose filter C, be pasted on the plastic sheet successively; Adsorbing the labelled antibody that multiple organophosphorus is had specific recognition on the plain film S of glass fibre, nitrocellulose filter T bag is by the excessive antigen of linear, and nitrocellulose filter C is fixing goat anti-rabbit antibody IgG.In immunity test strip, be respectively the carrier of antigen and goat anti-rabbit igg with nitrocellulose filter T and nitrocellulose filter C, the plain film S of glass fibre is the carrier of labelled antibody, with thieving paper A leaching sample liquid, quantitative mark antibody point on the plain film S of glass fibre, is coated on excessive coating antigen by on the nitrocellulose filter T that soaks the sample end, as the test section again, goat anti-rabbit igg point is in nitrocellulose filter C end, district in contrast.The excessive coating antigen bag of nitrocellulose filter T is achieved in that nitrocellulose filter T is put as sealing 30min in 1% bovine serum albumin (BSA), uses phosphate buffer (PBS) washing 3 times then, 37 ℃ of dry 3h.
During detection, thieving paper A one end is put in the sample liquid, sample liquid moves to the other end under effect capillaceous, when liquid arrives the plain film S of glass fibre place, labelled antibody dissolved and with sample in antigen-reactive and form compound, liquid stream continues to move forward to nitrocellulose filter T place, when if sample is negative, labelled antibody has surplus, can be again combine with antigen on the film, adding substrate has chromogenic reaction, and unnecessary labelled antibody continues to move forward to nitrocellulose filter C end, chromogenic reaction can take place compare.On the contrary, when sample was positive, test zone T antigen can not the incorporation of markings antibody complex adds substrate and does not have chromogenic reaction, and compound continues reach and is fixed on check plot (C) goat anti-rabbit igg combination, added substrate and formed the colour developing control line.If do not develop the color in the check plot, illustrate that then test strips lost efficacy.
To detect the dish sample is example, take by weighing 20g dish sample before the detection earlier, add acetonitrile 50ml homogenate 1min, add 5-7g sodium chloride behind the suction filtration and saltout, its supernatant is a sample extracting solution, keeps flat taking out behind the test strips thieving paper A end immersion sample liquid 10s, the 10min observations, have only the check plot colour developing, the expression sample is negative, and measuring in district's colour developing expression dish sample has residues of pesticides.
Claims (9)
1, a kind of immune antiboidy that is used to detect organophosphorus pesticide, it is characterized in that, with diethyl phosphonic acids acetate is haptens, by preparing immunogene, and be prepared into polyclonal antibody or prepare monoclonal antibody by processes such as mouse immune, Fusion of Cells, screening and cloning cultivations by immune new zealand white rabbit with bovine serum albumin (BSA) coupling.
2, the immune antiboidy that is used to detect organophosphorus pesticide according to claim 1 is characterized in that: described artificial immunogen adopts active ester method or the preparation of water-soluble carbodiimide method.
3, the immune antiboidy that is used to detect organophosphorus pesticide according to claim 2, it is characterized in that: described employing active ester method prepares artificial immunogen and is achieved in that diethyl phosphonic acids acetate and the N-hydroxy-succinamide (NHS) of getting equimolar amounts, ring dihexyl carbimide (DCC), with dioxane potpourri is dissolved, the lucifuge reaction is spent the night under the room temperature, remove post precipitation and get the supernatant drying, residue is suspended in 3 milliliters of borate buffer solutions that are dissolved with 20mg bovine serum albumin (BSA), potpourri is magnetic agitation 1 hour at room temperature, 4 ℃ of phosphate buffers to 1 liter of pH7.4 (PBS) dialysis is prepared into immunogene.
4, the immune antiboidy that is used to detect organophosphorus pesticide according to claim 2, it is characterized in that: described employing water-soluble carbodiimide legal system is equipped with artificial immunogen and is achieved in that taking by weighing diethyl phosphonic acids acetate earlier is dissolved in the dimethyl formamide (DMF), low temperature stirs, water intaking dissolubility carbodiimide (EDC) is dissolved among the 1mlDMF, slowly joins in the above-mentioned solution.Other gets bovine serum albumin (BSA) 20mg and is dissolved among the 2mlDMF, and 4 ℃ of stirrings slowly join in the above-mentioned mixed liquor.4 ℃ of stirring reaction 8h spend the night in 14 ℃ of reactions again, and the centrifugal 10min of 2000g next day gets precipitation, and dialysis is prepared into immunogene.
According to the application of the described immune antiboidy of claim 1 in detection, it is characterized in that 5, immune antiboidy can be used for the residual of organophosphorus insecticide detected.
According to the application of the described immune antiboidy of claim 5 in detection, it is characterized in that 6, described organophosphorus insecticide comprises chlopyrifos, omethoate, basudin, ethyl parathion, Profenofos, phoxim, diethyl phosphonic acids acetate.
7, according to claim 5 or 6 application of described immune antiboidy in detection, it is characterized in that: the monoclonal antibody of acquisition or polyclonal antibody can be used for conventional indirect competition ELISA and detect.
8, according to claim 5 or 6 application of described immune antiboidy in detection, it is characterized in that: the monoclonal antibody of acquisition or polyclonal antibody are used for direct ELISA after by gold, enzyme, fluorescent-labeled antibody and detect.
9, according to claim 5 or 6 application of described immune antiboidy in detection, it is characterized in that: after the monoclonal antibody of acquisition or polyclonal antibody are made immunity test strip, the detection of organophosphorus pesticide in the fruits and vegetables.
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