Antigen-specific messenger RNA for immunological research: purification and characterization of RNA from intestinal epithelial cells using specific antibody.
A murine organ-specific antigen system (ECAC B1,B2) has been described that is reactive with sera and lymphocytes from patients with chronic inflammatory bowel disease. However, the amino acid sequence of macromolecules comprising ECAC is unknown, making difficult synthesis of a genetic (DNA) probe for molecular biologic studies. As an alternative to obtain a specific cDNA clone, we developed a methodology to immuno-purify and characterize ECAC-specific messenger RNA from murine and human gut mucosal epithelium. Advantages of this new approach include that it is rapid, non-radioactive, applicable to heterologous or monoclonal immunoglobulin, and of low cost. 100 X 10(6) intestinal epithelial cells were lysed with 0.35% V/V NP-40, and the supernatant layered on a 65% W/V sucrose solution for isolation of polyribosomes by ultracentrifugation. ECAC B1/B2-specific mRNA was isolated from this polyribosome fraction using protein A column-purified specific immunoglobulin from heterologous hyperimmune serum or from monoclonal ascites, followed by adsorption of antibody-bound polyribosomes on a solid phase composed of Sepharose-protein A. After messenger RNA elution, the poly-adenine-containing fraction was further purified by oligo-dT chromatography and characterized. The yield of highly purified messenger RNA (A260/A280 greater than or equal to 2) was 10-20 micrograms/10(8) epithelial cells, and formaldehyde agarose gel electrophoresis demonstrated a limited number of RNA bands had been isolated (150-1400 base-pairs in size). The function of murine and human (normal and disease-involved) messenger RNA isolates was determined in a cell-free in vitro translation system, demonstrating synthesis of single components of ECAC identified by immunoblotting, enzyme-linked immuno-sorbent assay, and analytical polyacrylamide gel electrophoresis of lysate fractions after immuno-affinity chromatography. We conclude that heterologous or monoclonal antibody can be used to isolate antigen-specific messenger RNA from a species other than that from which the antigen was originally isolated, that the functional status for translation of newly purified mRNA can be easily determined by one of several non-radioactive means, and that the technique can be applied even to cells with a high enzymatic degradative capacity, such as those in gut epithelium.
Paul
J W
JW
Roche
J K
JK
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000032:analysis; D006801:Humans; D007158:Immunologic Techniques; D007413:Intestinal Mucosa / Q000032:analysis*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D011917:Rats, Inbred Lew
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger