title: Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.
abstract: Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible 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 approximately 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 invasive 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 immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.
authors.affiliation: Laboratory of Pathology, National Cancer Institute, and the Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: N L
authors.initials: NL
authors.lastname: Simone
authors.forename: A T
authors.initials: AT
authors.lastname: Remaley
authors.forename: L
authors.initials: L
authors.lastname: Charboneau
authors.forename: E F
authors.initials: EF
authors.lastname: Petricoin
authors.forename: J W
authors.initials: JW
authors.lastname: Glickman
authors.forename: M R
authors.initials: MR
authors.lastname: Emmert-Buck
authors.forename: T A
authors.initials: TA
authors.lastname: Fleisher
authors.forename: L A
authors.initials: LA
authors.lastname: Liotta
mesh_terms: D002138:Calibration; D002277:Carcinoma / Q000737:chemistry / Q000473:pathology; D004210:Dissection / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007150:Immunohistochemistry; D053685:Laser Therapy; D008297:Male; D008866:Microsurgery; D009361:Neoplasm Invasiveness; D009375:Neoplasms, Glandular and Epithelial / Q000737:chemistry; D011467:Prostate / Q000737:chemistry; D017430:Prostate-Specific Antigen / Q000032:analysis; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000473:pathology; D011506:Proteins / Q000032:analysis*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011506:Proteins; D017430:Prostate-Specific Antigen