Preparation and the Specificity of Monoclonal Antibody against Saxitoxin
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Saxitoxin(STX)was conjugated to bovine serum albumin(BSA) as immune antigen.Inoculated mouse with STX-BSA,gained one hybridoma cell strains secreting monoclonal antibodies against STX stably.The subclass of the McAb was IgM.The titer of the McAb was 1∶102400 in ascites according to the indirect ELISA determination.The affinity constant was 3.71×106 L/mol.Keywords:
Saxitoxin
Subclass
Bovine serum albumin
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Nine colchicine specific monoclonal antibodies have been developed by immunizing BALB/c mice with a colchicine-keyhole limpet hemocyanin (Col-KLH) conjugate prepared using a bishydroxysuccinimide coupling reagent. Of four immunization procedures examined, intraperitoneal injection of the antigen attached to acid treated E. coli resulted in the maximum antigen specific antibody titers. A colchicine bovine serum albumin (Col-BSA) conjugate, prepared using a water soluble carbodiimide coupling technique, formed the basis of an enzyme linked immunosorbent assay used for screening hybridomas for colchicine specific antibody secretion and for determining the relative affinity and specificity profile of the monoclonal antibodies. All antibodies demonstrated high affinity, saturable binding to colchicine and low cross-reactivity with a panel of compounds structurally related to colchicine. The IC50 for the highest affinity antibody, C44, was 3.6±0.84 nM colchicine in the competitive enzyme immunoassay. The affinity of this antibody determined from Scatchard analysis of antibody binding to tritiated colchicine was 0.66±0.11 nM. Antibody C44 has the level of specificity and affinity suitable for a sensitive and selective immunoassay of colchicine for monitoring therapeutic drug levels. In addition, this antibody provides a specific pharmacologic antagonist for studies of colchicine's therapeutic mechanism and has the potential to reverse colchicine toxicity.
Colchicine
Keyhole limpet hemocyanin
Conjugate
Bovine serum albumin
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Paraoxon (E600) was conjugated to bovine serum albumin(BSA) or tachypleus tridentatus hemocyanin (TTH) by diazotization. Two hybridoma cell lines secreting monoclonal antibodies(McAb) against paraoxon have been established by fusing mouse myeloma cells and splenocytes from Balb/c mice immunized with E600-BSA. The chromosomes of the hybridoma cell 2B10 were analyzed. The immunoglobulin of the McAb was classified. The affinity and specificity of this antibody were determined. The hybridoma cells have fairly reserved the antibody-producing capacity after continuously growing or stored in liquid nitrogen for 10 weeks.
Paraoxon
Splenocyte
Bovine serum albumin
Fetal bovine serum
Hemocyanin
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Brevetoxin(BTX) was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by mixed anhydride coupling method.BTX-BSA and BTX-OVA were used as immunizing antigen and coating antigen,respectively.BTX-BSA was vaccinated into Balb/c mice for monoclonal antibody(McAb) production.A hybridoma(named 3B4) which secrected McAb against brevetoxin was obtained and some characteristics of the McAb were studied.The antibody titer was 10-5.And the McAb has no cross reactions with other marine toxins.The McAb lays a foundation for the establishment of an immunological detection method on brevetoxin.
Bovine serum albumin
Antibody titer
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Neomycin was conjugated to carrier protein BSA and OVA by the method of EDC to form the immunizing antigen BSA-NEO and the coating antigen OVA-NEO.IR and SDS-PAGE were used to identify the NEO artificial antigen.Balb/c mice were immunized with BSA-NEO.The titre of polyclonal antibody was detected by indirect ELISA and blocking ELISA.The hybridoma lines that secrete NEO mAb were established with monoclonal antibody hybridoma technology.The immunological traits such as titer,affinity,sensitivity and specificity of the mAb were characterized.The results showed that NEO artificial antigen was synthesized successfully.Three hybridoma cell lines of 1E9、4E8 and 1G1 were screened for specificity to NEO.The indirect ELISA titer of the mAb were 1∶2.56×103,1∶1.28×103 and 1∶5.12×103 in supernatant,and 1∶5.12×105,1∶2.56×105 and 1∶1.02×106 in ascites.The affinity constant(Ka)of 1G1 was about 3.75×1010(L/mol).The mAb of 1G1 showed good sensitivity with an IC50 of 2.57 ng/mL to NEO.No cross-reactivity to other compounds was detected,snch as Gentamicin,Streptomycin,Oxytetracycline,Ciprofloxacin,Difloxacin,etc.The NEOmAb with high-titer,sensitivity and specificity had been generated,and could be used to establish the immunoassay of NEO residues in feed and animal food.
Polyclonal antibodies
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Objective:To determine the relative affinity of three monoclonal antibodies (4A9,4C3,9F12) against type O foot-and-mouth disease.Methods:A method was developed in which serial dilution of purified antigen of type O FMDV was coated.The monoclonal antibodies (4A9,4C3,9F12) were purified from the ascites of mice inoculated with the hybridonas.Relative affinity of the purified monoclonal antibodies was determined with the indirect ELISA method.Results:The optimal dilution of the antigen and enzyme-labeled antibodies were 1∶800 and 1∶10 000,respectively.The relative affinity of the monoclonal antibodies (4A9,4C3,9F12) were 5.242 μg/ml(1∶600),0.763 μg/ml(1∶5 000) and 0.127 μg/ml(1∶40 000),respectively,with the relative affinity of 9F124C34A9.Conclusion:Relative affinity of the monoclonal antibodies (4A9,4C3,9F12) can be determined by ELISA,and it provides a theoretical foundation for the monoclonal antibody which is suitable for used in further studies of FMDV.
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For development of monoclonal antibodies against Microcystin,a hybridoma cell line steadily secreting monoclonal antibodies against Microcystin,coded G5,was obtained by fusing murine Sp2/0 cells with spleen cells from mice immunized with MC-KLH conjugate and subcloning for 3 to 4 cycles.The ascites containing monoclonal antibodies against MC was gained via inoculation of the hybridoma cells into the abdominal cavity of BalB/c mice.The antibody subclass is IgG2a,affinity constant is 2.9×10-11 mol/L,molecular weight is 150 kD,recovery is 81.5%～125.0%,cross-reaction 1%。The monoclonal antibodies thus prepared are highly sensitive and specific against microcystin.
Subcloning
Subclass
Conjugate
Cell fusion
Microcystin-LR
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The monoclonal antibodies against BSA were generated by fusion spleen cells from immunized BALB/c mouse and SP2/0 mouse myeloma cells,which can be used in determination of BSA contamination in biological preparations. The culture of hybridoma lines were first screened by sandwich-ELISA in detection of mouse IgG,then mouse ascites were prepared.Four hybridoma lines which secreted monoclonal antibodies against BSA were determined and developed,named as 2A5,3A3,3G6,4A8 and isotyping as IgG2a/κ,IgG1/κ,IgG1/κ,IgG1/κ respectively.These McAbs with titers over 10-5 to BSA by indirect ELISA,and purity of purified ascites attained more than 90%.Forthermore,the MCAbs showed a high specificity to BSA with a MW 68000 Da by Western blotting and had a low reactivity to serum albumin from human and other animals,which can recognize two different epitopes on BSA.Both of their relative affinity and sensitivity are in an order 3A32A53G64A8.These hybridoma lines could be cultured for continuous three months and kept in a stable condition.
Bovine serum albumin
Subclass
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Hybridoma (4C4) secreting monoclone anti-idiotype antibody (McAb2) against anti-phenolic glycolipid-I (PGL-I) antibody (Ab1) was produced by fusion of SP2/0 myeloma cells and spleen cells of syngeneic mice which had been previously tolerant to human IgM 4C4 monoclone anti-idiotype antibody was identified with a series of experiments including competitive and neutralizing inhibition ELISA. It was found that the binding of McAb2 with rabbit anti-PGL-I antibody could be competitively inhibited by NT-O-BSA (synthetic analog of PGL-I) and neutralized by polyclonal anti-PGL-I antibody derived from various origins (human or rabbit); McAb2 could block the binding of purified human Ab1 with NT-O-BSA. The assay of McAb2 as mimic antigen demonstrated that McAb2 could substitute for NT-O-BSA in leprosy serodiagnosis. These results show that anti-idiotype antibody produced by 4C4 is a monoclone anti-idiotype antibody bearing internal image of PGL-I and possibly can be used in leprosy serodiagnosis.
Polyclonal antibodies
Mycobacterium leprae
Idiotopes
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