Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells

Abstract

A quantitative flow cytometry method for the analysis of membrane‐associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double‐antibody reaction; a flow cytometer with a 2‐W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty‐two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 103 molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG‐3 choriocarcinoma cells revealed the expression of membrane‐associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.

hCG, the wonder of today's science

“…In the years that followed Acevedo and Krichevsky [126] and Regelson [127] used cancer membrane flow cytometry methods to show that all advanced cancers produced an hCG free ß-subunit variant. An investigation into hCG degradation at that time showed that hCG free ß-subunit (circulating 1/2-life 0.72 h) is removed from the circulation much more rapidly than hCG (circulating 1/2-life 36 h) [128].…”

Human chorionic gonadotropin (hCG), the hormone of life and death: a review

“…Three years later, using quantitative analytical flow cytometry and a battery of monoclonal antibodies directed to different epitopes of the hCG molecule, Acevedo and his associates demonstrated the expression of membrane‐associated hCG, its subunits and fragments by live cancer cells of every type and origin (10–12). Using the same methodology, and the nude mouse model, Acevedo and Hartsock pointed out that there was a direct in vivo correlation between human cancer cells that metastasize spontaneously in nude mice, and the expression of membrane‐associated complete hCGβ.…”

“…Since all the normal cells from our immune system, macrophages, NK cells and B cells, express in their membranes a “normal” negative charge, the high negative charge of hCG and its subunits demonstrated to be present in the cell membranes of embryonic and fetal cells, in sperm cells in every stage of development (Figures 3,4) (60–62), and in all cancer cells irrespective of type or origin as membrane‐associated hCG (10–13), make all these cells immunologically inert . The cells from the immune system cannot approach, cannot touch, since negative charges repel.…”

“…The thorough mapping of the hormone and its isoforms, the availability of a large group of monoclonal antibodies (MoAb) directed to specific, well characterized, antigenic determinants of the hCG molecule ( Figure 5) and the development of a quantitative method using analytical flow cytometry, permitted us to demonstrate with live cancer and fetal cells, that hCG in all of its forms is an integral part of the cell membrane in all of these cells, irrespective of their type and origin. The membrane‐associated material can only be isolated by rupturing the cells and the use of detergents such as Triton X (10–13).…”

“…Anti‐cancer vaccines It was logical that the demonstration of membrane‐associated hCG in all cancer cells , irrespective of type or origin (10–13), indicated the possibility of using the anticonceptional vaccine as a vaccine against cancer. The first experimental studies using the Population Council vaccine were done in 1982 by Kellen, Kolin and Acevedo (86,87) at the Departments of Clinical Biochemistry and Pathology at Sunnybrook Medical Centre, Toronto, Canada, and the Department of Pathology, Allegheny General Hospital, Pittsburgh, PA, USA.…”