The expression of human chorionic gonadotropin (hCG), its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double‐antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its α sub‐unit (hCGα), and its β subunit (hCGβ); and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane‐associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, its subunits, and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG‐holo), hCGβ hCGβ‐free, the carboxy terminal peptide (CTP) of hCGβ, and an epitope of hCGα. The expression of the membrane‐associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCGβ, free and as part of hCG‐holo, recognized by the MoAb against hCGβ‐CTP, was expressed by a high percentage of cells of most cell lines. There was great variability in the expression of hCG‐holo, recognized by MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCGα and/or hCGβ than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding; the expression of small amounts of hCG‐holo was caused by a low degree of hCGα synthesis.

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.

Using the indirect fluorescein-labeled and indirect peroxidase-antiperoxidaselabeled immunohistochemical techniques, and utilizing both antiserum specific for the f8-subunit of choriogonadotropin and antiserum for the total hormone, we have demonstrated the presence of a choriogonadotropin-like immunoreactive material in a strain of Eubacterium lentum that was originally isolated from a rectal tumor. In contrast, both immunohistochemical reactions were negative when applied to a strain of Corynebacterium parvum and to pathogenic and nonpathogenic strains of Agrobacterium tumefaciens. Our results demonstrate for the first time the expression of the choriogonadotropin-like antigen in an obligate anaerobe and support our previous findings that the choriogonadotropinlike material appears to be expressed only in "cancer-associated bacteria" but that not all bacteria associated with the malignant neoplasms have the capacity to express the antigen, at least in amounts detectable by immunohistochemistry.

Immunocytochemical studies using antisera to whole human choriogonadotropin (hCG), to its alpha- and beta-subunits and to the COOH-terminal peptide of hCG beta, and two monoclonal antibodies to hCG beta, demonstrated expression of hCG-like material, its individual subunits and/or fragments in nine bacterial strains. Seven of these were isolated from patients with cancer and were definitely identified as Streptococcus faecalis (three strains), Staphylococcus haemolyticus (two strains) and Staphylococcus epidermidis and Escherichia coli (single strains). The other two strains were cell-wall-deficient (CWD) variants, one identified as Streptococcus bovis, isolated from the blood of a patient with a fever of unknown origin and a possible brain abscess. The other was a Gram-negative diphtheroid isolated from the urine of a pregnant woman, which during the period of study reverted to a Gram-positive Corynebacterium identified as a 'C. ulcerans' strain and expressed the hCG-like factor only during its phase as Gram-negative diphtheroid. Electron microscopy of these nine strains (including negative controls of strains of the same species subjected to the same immunocytochemical analyses and under identical cultural conditions) revealed morphological alterations in the bacterial cell walls and cytoplasmic material and/or bizarre forms of reproduction in six of the nine strains expressing hCG-like material including the two CWD variants. Collectively, these results provided evidence that (1) hCG-producing bacteria isolated from patients with overt cancer are not a new and unique species as claimed by others, and (2) there is a close resemblance between the bacterial protein and the human trophoblastic hormone, based on immunochemical recognition of different parts of the hCG molecule.(ABSTRACT TRUNCATED AT 250 WORDS)