An immunohistochemical assay on human tissue using a human primary antibody.

Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10(Biotin)) and Cy3-conjugated streptavidin (Strep(Cy3)). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low Strep(Cy3) background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.

Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C.

AIM: To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C. METHODS: The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 µL reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysates. Reactions were terminated after 10, 30 or 60 min of incubation with Doles medium. RESULTS: Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl beta-D-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni(2+)-affinity chromatography. Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5, as well as recombinant rat ACSL1 (sensitive control), but not recombinant rat ACSL5 (insensitive control). The IC50 for human ACSL5 was about 10 µmol/L. The inhibitory triacsin C effect was similar for different incubation times (10, 30 and 60 min) and was not modified by the N- or C-terminal location of the 6xHis-tag. In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment, stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed. In both models, ACSL5 peak activity was found at pH 7.5 and pH 9.5, corresponding to the properties of recombinant human ACSL5 protein. In the presence of triacsin C (25 µmol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa. CONCLUSION: The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.