A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 2. The individual immune molecule.
In the accompanying original article, the universal theoretical and experimental framework was developed for quantifying one and the same single (selfsame), individual fluorescent-tagged biological molecule without immobilization, hydrodynamic flow or photon burst analysis of fluorescence intensity traces. In the present original article, we describe an application to the detection and identification of circulating anti-glomerular basement membrane antibodies (BMAs) in Goodpasture syndrome. The same single, individual two-color molecule complex was observed among many other molecules. The molecule consisted of the green-tagged antigen, sandwiched autoantibody and red-tagged secondary (detecting) antibody. A 200-fold increase in sensitivity was obtained as compared to the conventional ELISAs on solid phase. This novel concept has several advantages, namely (i) the sensitivity to detect an individual molecule in solution; (ii) the association of the signal with the reaction event, independent of any immobilization procedure and the artifacts thereof; (iii) the assessment of the broad field of natural antibodies. The theoretical and experimental results obtained bring advanced ultrasensitive analytics to the direct investigation of one and the same single, individual immune molecules as exemplified by the experiments performed with Goodpasture antibody. The novel universal theoretical and experimental framework for continuous measuring the same single, individual immune molecule can be readily transferred to other applications.
Földes-Papp
Zeno
Z
Clinical Immunology and Jean Dausset Laboratory, Graz University Medical School and Hospital, Auenbruggerplatz 8, A-8036 Graz, LKH, Austria. Zeno.Foldes-Papp@uni-graz.at
Demel
Ulrike
U
Tilz
Gernot P
GP
D019867:Anti-Glomerular Basement Membrane Disease / Q000175:diagnosis / Q000276:immunology*; D000906:Antibodies / Q000032:analysis* / Q000097:blood / Q000276:immunology; D001323:Autoantibodies / Q000032:analysis* / Q000097:blood / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D024141:Collagen Type IV / Q000276:immunology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000906:Antibodies; D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D005456:Fluorescent Dyes; D012235:Rhodamines; C051951:antiglomerular basement membrane antibody; C057112:type IV collagen alpha3 chain