Specificity and sensitivity of an indirect fluorescent antibody test to detect antibodies against bovine viral diarrhea virus.

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab')2 fragment anti-bovine IgG [F(ab')2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab')2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab')2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated (r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype).

Both IRBIT and long-IRBIT bind to and coordinately regulate Cl-/HCO3- exchanger AE2 activity through modulating the lysosomal degradation of AE2.

Anion exchanger 2 (AE2) plays crucial roles in regulating cell volume homeostasis and cell migration. We found that both IRBIT and Long-IRBIT (L-IRBIT) interact with anion exchanger 2 (AE2). The interaction occurred between the conserved AHCY-homologous domain of IRBIT/L-IRBIT and the N-terminal cytoplasmic region of AE2. Interestingly, AE2 activity was reduced in L-IRBIT KO cells, but not in IRBIT KO cells. Moreover, AE2 activity was slightly increased in IRBIT/L-IRBIT double KO cells. These changes in AE2 activity resulted from changes in the AE2 expression level of each mutant cell, and affected the regulatory volume increase and cell migration. The activity and expression level of AE2 in IRBIT/L-IRBIT double KO cells were downregulated if IRBIT, but not L-IRBIT, was expressed again in the cells, and the downregulation was cancelled by the co-expression of L-IRBIT. The mRNA levels of AE2 in each KO cell did not change, and the downregulation of AE2 in L-IRBIT KO cells was inhibited by bafilomycin A1. These results indicate that IRBIT binding facilitates the lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, suggesting a novel regulatory mode of AE2 activity through the binding of two homologous proteins with opposing functions.

Synthesis of 5-tert-butyl-1-(3-tert-butyldimethylsiloxy)phenyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptanes and their fluoride-induced chemiluminescent decomposition: effect of a phenolic electron donor on the CIEEL decay rate in aprotic polar solvent.

Four bicyclic dioxetanes bearing a phenolic substituent, 3-tert-butyldimethylsiloxy-4-chlorophenyl (3a), 5-tert-butyldimethylsiloxy-4-chloro-2-ethylphenyl (3b), 5-tert-butyldimethylsiloxy-2-ethylphenyl (3c), and 3-tert-butyldimethylsiloxy-4-ethylphenyl (3d), were synthesized. All dioxetanes 3a-3d gave intense blue light on treatment with tetrabutylammonium fluoride (TBAF) in DMSO or acetonitrile. Kinetic study on the fluoride-induced CIEEL decay of these dioxetanes 3a-3d and the parent dioxetane 2b revealed that the para-substitution with chlorine on the phenolic moiety of dioxetane increases free energy of activation (DeltaG++), while the para-substitution with ethyl on the aryl decreases DeltaG++. On the other hand, substitution with an ethyl at the ortho-position instead of the para-position was found to increase DeltaG++ and to suppress the CIEEL decay. This fact is attributed to the steric factor of the ortho-ethyl group which would prevent the aromatic ring from rotating freely around the axis joined to the peroxide ring, and supports the suggestion for a CIEEL-active dioxetane bearing a phenolic moiety that an intramolecular electron transfer occurs preferentially from the phenolic donor to O-O of the dioxetane ring, when the aromatic ring lies in a certain conformation(s).