Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection

Coupling laser capture microdissection (LCM) with sensitive quantitative ch emiluminescent immunoassays has broad applicability in the field of proteom ics applied to normal, diseased, or genetically modified tissue. Quantitati on of the number of prostate-specific antigen (PSA) molecules/cell was cond ucted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 mu m in diameter captured specific cells from the heterogeneous tissue sectio n onto a polymer transfer surface. The cellular macromolecules from the cap tured cells were solubilized in a microvolume of extraction buffer and dire ctly assayed using an automated (1.5 hour) sandwich chemiluminescent immuno assay, Calibration of the chemiluminescent assay was conducted by developin g a standard curve using known concentrations of PSA, After the sensitivity , precision, and linearity of the chemiluminescent assay was verified for k nown numbers of solubilized microdissected tissue cells, it was then possib le to calculate the number of PSA molecules per microdissected tissue cell for case samples, In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximate ly 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6.3 x 10( 6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and inva sive carcinoma, Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochem ical staining intensity paralleled the LCM quantitative immunoassay for eac h tissue subpopulation and verified the heterogeneity of PSA content betwee n tissue subpopulations in the same case. Extraction buffers were successfu lly adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure p opulations of tissue cells.