Screening And Analysis Of Monoclonal Antibody With Broad-spectrum Against A Variety Of Fish Serum Ig

There were a wide variety of commcial fishes and quite different in genetic relationship.None detection of fish immunoglobulin reagents commonly used.The use of 20 monoclonal antibodies against of Siniperca chuatsi,Epinephelus awoara Pseudosciaena crocea,Anguilla anguilla to study 26 fish serum Ig in order to filter out the broad recognition spectrum of monoclonal antibodies,or monoclonal antibody combinations.1, Fish serum was collected and total serum protein was analysed.Twenty-six commercial fish serum was collected. Twenty-six commercial fish included Pseudosciaena crocea,Epinephelus awoara,Hapalogenys nitens Richardson,Sciaenops ocellatus,Sebastiscus marmoratus,Micropterus salmoides and so on.The total serum protein concentration of twenty six commercial fish species was about 9.00-50.0mg.ml-1.The standard deviation was above 1.90 in different individuals of the same fish species.There was significant differernce. The total serum protein concentration was also significant differernce in different fish species.2,Immunoglobulin was extracted, then the purity and activity were analysed. Purification of serum immunoglobulin was carried out by means of euglobulin as well as ammonium sulfate precipitation.Applications SDS-PAGE,ELISA and Western-blot analysed of extracts by means of the purity and activity.Experimental results showed that euglobulin extraction method was clearly superior to ammonium sulfate precipitation.Purified by euglobulin precipitation, the resultant material analyzed by SDS-PAGE showed two distinct protein bands, indicating a higher purity of the A. anguilla serum immunoglobulin than that obtained by ammonium sulfate precipitation. Furthermore, The ELISA and Western-blot analysis showed that the serum immunoglobulin purifid by euglobulin precipitation had a higher antibody activity than that purified by ammonium sulfate precipitation. The results suggest that euglobulin method could be used as a fast,simple and efficient method of extracting fish serum Ig.3,The monoclonal antibody cross-reactivity spectrum was constructed.Twenty monoclonal antibody against Siniperca chuatsi,Epinephelus awoara,Pseudosciaena crocea,Anguilla anguilla reacted with 26 commercial fish spieces serum Ig. The monoclonal antibody cross-reactivity spectrum against twenty six fish spieces was constructed.Monoclonal antibody 2H5F4 could react with Siniperca chuatsi,Micropterus salmoides,Trachinotus ovatus,Tilapia nilotica,Lateolabarax japonicus,Tilapia sp.,Epinephelus awoara,Pseudosciaena crocea,Sebastiscus marmoratus,Sciaenops ocellatus,Sparus macrocephalus,Scophthatmus maximus,Cyprinus carpio,Spinibaebus caldwelli,Erythroculter ilishaeformis,Megalobrama terminalis,Carassius auraatus cuvieri,Sebastiscus marmoratus 18 fish serum Ig.Monoclonal antibody 7E2 could react with Anguilla anguilla,Siniperca chuatsi,Cyprinus carpio,Erythroculter ilishaeformis,Megalobrama terminalis,Carassius auraatus cuvieri,Muraenesox cinereus,Ictalurus punctatus,Pseudobagrus fulvidraco,Clarias lazera,Silurus asotus Linnaeus serum Ig.The monoclonal antibody that 2H5F4 mixed proportional with 7E2 could react with 25 fish species serum Ig. These two monoclonal antibodies could be used as excellent alternative antibody detection kit.4,Common epitope of different fish immunoglobulin was analysed.The common epitope of different fish spieces serum Ig was studied by monoclonal antibody of 7F12F6 and 2H5F4. Monoclonal antibodies 7F12F6 could bind to Siniperca chuatsi,Micropterus salmoides,Lateolabarax japonicus,Pseudosciaena crocea,Sciaenops ocellatus,Sparus macrocephalus serum Ig L chain. It showed that the six fish serum Ig shared the same epitope in the light chain.The monoclonal antibody 2H5F4 could bind to some fish serum Ig H chain. Western-blot in reduced condition showed that monoclonal antibody could bind to Pseudosciaena crocea,Epinephelus awoara,Hapalogenys nitens Richardson,Sciaenops ocellatus,Sebastiscus marmoratus serum Ig H chain. Western-blot in non-reduced condition showed that monoclonal antibody could react with Siniperca chuatsi,Micropterus salmoides,Trachinotus ovatus,Tilapia nilotica,Lateolabarax japonicus,Tilapia sp.,Sparus macrocephalus,Scophthatmus maximus,Cyprinus carpio,Spinibaebus caldwelli,Erythroculter ilishaeformis,Megalobrama terminalis,Carassius auraatus cuvieri besides the five fish serum Ig in reduced conditions.The result showed that there was common epitope in different fish spieces serum Ig.The monoclonal antibody 2H5F4 to Pseudosciaena crocea,Epinephelus awoara,Hapalogenys nitens Richardson,Sciaenops ocellatus,Sebastiscus marmoratus serum Ig was sequential epitope, and it didn't effect by denaturant.However, the monoclonal antibody 2H5F4 to the other 13 fish serum Ig was conformation dependent.and it effected by denaturant.5,Reaction specificity of Monoclonal antibody verified.Application of monoclonal antibody 2H5F4 to determine of immune and non-immune Nile tilapia and Common carp serum specific anibody levels.The immune group had higher specific antibody levels than non-immune groups. The anibody could be applied to Nile tiapia,Common carp immunoassay.Screening and analysis of monoclonal antibody with broad-spectrum against a variety of fish serum Ig had played an important role in the development of fish immunology.