A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. I. A comparison of cell fixation and storage techniques.
A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks' balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.
Walker
K W
KW
Transplantation Laboratory, University of California, Irvine 92717.
Llull
R
R
Balkian
G K
GK
Ko
H S
HS
Flores
K M
KM
Ramsamooj
R
R
Black
K S
KS
Hewitt
C W
CW
Martin
D C
DC
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000954:Antigens, Surface / Q000276:immunology*; D002021:Buffers; D015925:Cryopreservation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005557:Formaldehyde; D007552:Isotonic Solutions; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D000432:Methanol; D051381:Rats; D011919:Rats, Inbred Strains; D016707:Tissue Fixation / Q000379:methods*
D000906:Antibodies; D000954:Antigens, Surface; D002021:Buffers; C072670:Hanks Balanced Salt Solution; D007552:Isotonic Solutions; D005557:Formaldehyde; D000432:Methanol