Determination of dilution factor of three proteins.

(A) Mean OD values of animals’ sera/plasma with corresponding protein dilutions. The broken red lines indicate a similar mean OD value (corresponding to the dilution factor) between proteins A and AG and between proteins G and AG. Protein dilutions of 1:128,000 for protein A, 1:32,000 for protein G, and 1:16,000 for protein AG were used in ELISA for the same enzyme activity. The dilution factor of 1:128,000, 1:32,000, or 1:16,000 for proteins A, G, or chimeric protein AG whose mean OD values are slightly above 2.0. However, the dilution factor 1:8000 for proteins for proteins A, G or chimeric protein AG was not considered in the study. (B) Confirmation of appropriate dilutions of proteins. Using the determined dilution factor for each protein, no significant differences were observed in the mean OD values of the animal sera (p > 0.05). The mean OD values of protein A and protein AG for pigs at dilution factors 1:128,000 and 1:16,000, respectively, were 2.06 and 2.09, while for dogs at the same dilution factors, the values were 2.03 and 2.11; similarly, the mean OD values of protein G and protein AG for cows at dilution factors 1:32,000 and 1:16,000, respectively, were 2.01 and 2.01, while for goats at the same dilution factors, the values were 1.97 and 2.05. Abbreviations: OD, optical density; ELISA, enzyme-linked immunosorbent assay.</p

OD values of the individual experiment.

In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds’ species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.</div

General information on the pinniped species used in the study.

General information on the pinniped species used in the study.</p

OD values of the individual experiment.

In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds’ species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.</div

General information on the cetacean species used in the study.

General information on the cetacean species used in the study.</p

Marine mammal species used in this study and immunoglobulin-protein binding properties.

Marine mammal species used in this study and immunoglobulin-protein binding properties.</p

Development of Sheath-Flow Probe Electrospray Ionization Mass Spectrometry and Its Application to Real Time Pesticide Analysis

For the real time and direct analysis of chemical constituents from living beings and dry sample, sheath-flow probe electrospray ionization mass spectrometry (SF-PESI-MS) has been newly developed. The components from dry or semidry biological tissues can be extracted using the solvent and picked up by the needle for electrospray. This technique was applied to real-time pesticide analysis of living plants. The results have been validated with that of a well-known system, liquid extraction surface analysis mass spectrometry (LESA-MS). It is demonstrated that SF-PESI-MS can produce reasonable ionization efficiency, which is confirmed by LESA-MS