SECRETION IN NONPREGNANT HUMANS AND

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1 HUMAN CHORIONIC GONADOTROPIN-LIKE PROTEINS: SECRETION IN NONPREGNANT HUMANS AND PRODUCTION BY BACTERIA* WILLIAM D. ODELLt, and (by invitation) JEANINE GRIFFIN, SANJEEV GROVER and DOUGLAS T. CARRELL SALT LAKE CITY Human Chorionic Gonadotropin Secretion by Normal Humans: Human chorionic gonadotropin (hcg) and the other human glycoprotein hormones (luteinizing hormone [hlh], follicle stimulating hormone [hfsh], and thyroid stimulating hormone [htsh]) are biochemically very similar and are composed of two subunits, alpha and beta, which are not covalently bound. The amino acid sequence of the alpha subunit is identical for all four glycoprotein hormones and this protein is encoded by a single gene (1, 2). The beta subunit of hlh is also encoded by a single gene, whereas the beta subunit for hcg is encoded by up to eight genes or pseudogenes (3). The beta subunit of hlh differs from that of hcg in two ways: a) hcg has a carboxyl-terminal extension or prolongation of 37 amino acids, not present on hlh, b) the remainder of the hcg beta has 82% homology to that of hlh (4, 5). In mammalian systems, LH and hcg bind to the same receptor and possess similar biological functions. Human CG has usually been considered a hormone of pregnancy, acting to prolong corpus luteum function or of neoplasms derived from trophoblastic tissue (6). However, in the years , several case reports appeared, describing patients with various types of nontrophoblastic neoplasms that produced hcg (7-9). The first radioimmunoassay for hcg, reported from our laboratory in 1967 (6, 1), reacted equally with hlh and hcg. This assay, and also the bioassays then used to quantify hcg, therefore depended predominantly on quantitative differences in the amount of hcg present to distinguish it from hlh; in normal pregnancy, for example, we estimate the serum concentrations of hcg to be 1-8 M, whereas hlh circulates in nonpregnant humans at 1-1 M. In 1972, Vaitukaitis et al. (11), using polyclonal antisera developed against the beta subunit of hcg, developed an assay with increased specificity for hcg. Human LH still reacted in this assay, * Department of Internal Medicine, University of Utah Medical Center, Salt Lake City, Utah. t Address correspondence and reprint requests to: William D. Odell, M.D., Ph.D., Department of Internal Medicine, University of Utah School of Medicine, 5 North Medical Drive, Salt Lake City, UT

2 CHORIONIC GONADOTROPIN 239 but the dose response line for hlh differed from that of hcg, and significant cross-reaction was seen only when hcg and hlh were present in about equal molar concentrations-a phenomenon that was not then known to occur physiologically. However, employing this improved hcg assay, surprising findings were reported. Braunstein et al. (12) reported that 7% of sera from 828 nonpregnant patients with a variety of carcinomas, including stomach, colon, lung, and pancreas, contained an hcglike material. Furthermore, since testicular teratocarcinomas produce hcg, these workers studied extracts of normal human testes and reported that human testes contained an hcg-like material (13). Braunstein et al. (13) employed a concanavalin A (Con-A) purification technique, and using this technique, they reported that extracts of other tissues did not contain hcg. Con-A is a plant lectin that binds carbohydrate-rich placental hcg avidly. In 1977, however, a series of studies from our laboratory, employing a glacial acetic acid extraction technique that does not depend on carbohydrate presence on hcg, revealed, surprisingly, that extracts of many normal human tissues (e.g., liver, kidney, lung) contained an hcg-like material (14, 15). Further studies showed that this "normal tissue hcg" showed very little binding to Con-A and that on gel chromatography, it had a molecular volume compatible with carbohydrate-free hcg (16-18). In addition, hcg could be extracted from all carcinomas studied, irregardless of histological type (14, 17). The hcg from carcinomas showed highly variable binding to Con-A-ranging from 4% to 86% (Table 1). Braunstein et al. (19) confirmed that extracts of most of all normal tissues contained an hcg-like substance. These studies of hcg, and additional similar studies of ACTH-like materials, vasopressin and the free alpha subunit of hcg, led us to the hypothesis that so-called "ectopic hormone syndromes" were caused by increased production by a cancer, of protein hormone-like substances, usually produced in smaller amounts by normal tissues (14, 16); that is, so-called ectopic hormone production is not ectopic. TABLE 1 hcg Binding to Concanavalin A % Bound ± SEM Range Normal Tissue (1) 6.1 ± 1.6 (.-14.6) Cancer Tissue (9) 31.2 ± 9.1 (4.-86.) Placenta (4) 92.5 ±.9 ( ) Pregnant Serum (3) 1. Cancer Serum (8) 54.7 ± 11.9 ( ) Reproduced from Odell WD and Saito E: Protein hormone-like materials from normal and cancer cells-"ectopic" hormone production. In: Mirand EA, Hutchinson WB, Mihich E, eds. Cancer Management, The 13th International Cancer Congress, Part E. New York: Alan R. Liss, Inc.; 1983:247.

3 24 WILLIAM D. ODELL In 1978, Matsuura et al. (2) developed an entirely specific assay for hcg. This assay used polyclonal antibodies against the unique carboxyltail portion of the beta subunit of hcg. This assay, while very specific, lacked sensitivity because of the low affinity of these antisera. Nevertheless, the great specificity of this assay permitted Matsuura et al. (21) and Chen et al. (22) to show that urine and pituitary from nonpregnant humans contained an hcg-like material. Borkowski et al. (23, 24) subsequently extracted large volumes of plasma from nonpregnant subjects and demonstrated the presence of an hcg-like material. These findings of production of hcg and its circulation in blood of normal humans depended on three generations of progressively more specific assay systems. The fourth generation employed immunoradiometric (IRMA) or so-called sandwich assays. These assays employed monoclonal antibodies and were capable of greatly increased specificity as well as specificity. Employing such an assay, Armstrong et al. (25) confirmed that hcg was detectable in urine of nonpregnant humans. Further studies from our laboratory were directed to determining the tissue source of the hcg present in blood and urine of nonpregnant humans. To assist in answering this question we developed an extremely sensitive (.4 miu/ml) and entirely specific assay for the intact hcg molecule (holo-hcg) (26). A second assay based on similar principles was developed for hlh (27). The hcg assay showed no reaction with the free alpha or beta subunits of hcg and no reaction with htsh or hfsh. Immunochemical grade hlh showed.15% reaction in the assay, which interestingly, we were later able to show, was caused by contamination of this pituitary hlh with small amounts of hcg from pituitary (28). When we further purified this hlh, we also showed hlh had no cross-reaction in the hcg assay (Fig. 1). Using these assay systems, we found that if repeated serum samples were obtained, small amounts of hcg were detectable in every normal eugonadal or postmenopausal subject (29, 3). Furthermore, we were able to show that this hcg was secreted in a pulsatile fashion, in parallel with hcg in postmenopausal women (29), and in women during the normal menstrual cycle (29) (Figs. 2-4). These studies from our laboratory, as well as quite independent studies by Stenman et al. (31), also showed that hcg was stimulated by gonadotropin releasing hormone (GnRH) and suppressed by GnRH agonists and estrogens (29, 32) (Fig. 5). Human CG concentrations in sera from hypopituitary patients or from men with prostate cancer, treated with GnRH agonists, were very low or undetectable. All these findings suggested that the major source of hcg in blood of normal humans was the pituitary gland, and not other peripheral tissues. In further studies (reported in 199) employing human fetal pituitaries

4 5,- CHORIONIC GONADOTROPIN 241 4, hcg z 3,- m ~ X 2,- 1,- A/ ~~~~u~~7-%~ eta hcga 1hFSH htsh HORMONE CONC. (pg) a z M. U hcg hlh hlh affinity purified B HORMONE (pg/tube) FIG. 1A. Immunoradiometric assay for hcg. The free alpha subunit and beta subunit, as well as purified hfsh and htsh showed no reaction (<.1%) in the assay. Purified hlh showed.15% reaction. (From Odell and Griffin [29]. Reprinted by permission of The New England Journal of Medicine, 1987; 317: 1688.) FIG. 1B. Reaction of hlh, showing larger doses than in Figure 1A. This reaction of hlh in the hcg assay was abolished when the hlh was further purified (affinity purified). (Reproduced from Odell et al. [33]; copyright by The Endocrine Society.) in tissue culture, we directly demonstrated pituitary secretion of hcg in vitro (33) (Fig. 6). To further evaluate the source within the pituitary gland, we employed a monoclonal antibody specific for the beta subunit of hcg, as well as the polyclonal antibody against the carboxyl-tail region of hcg to immunostain pituitary glands. Hammond et al. (34) were able

5 242 WILLIAM D. ODELL 'i,a U E - A TIME (min) E cl E D EX-J U B TIME (min) FIG. 2. Serum hlh and hcg concentrations measured at 1-minute intervals for six hours in two postmenopausal women. Significant peaks (P <.25) are indicated by triangles for hlh and by asterisks for hcg. (Reproduced from Odell and Griffin [29]. Reprinted by permission of The New England Journal of Medicine, 1987; 317: 1688.) to identify a previously unknown human pituitary cell that immunostained for hcg, but not for hlh or other anterior pituitary hormones. This cell had unique morphological characteristics which also could distinguish it from the hlh cell (Fig. 7). This report, initially presented at the Endocrine Society meeting in 1988, was met with considerable skepticism and had a very delayed acceptance for complete publication,

6 F - FOLLICULAR PHASE Progesterone =.2 ng/ml H i D HE -78 U -52 A -1 S I I Con '4uU TIME IN MINUTES 41 F- FOLLICULAR PHASE Progesterone=.4 ng/ml 2 -J H I -j H 4U I B Ij H LI ISO C TIME IN MINUTES FIG. 3. Concentrations of hcg and human LH (hlh) in serum determined each 1 minutes in three women during the follicular phase of the menstrual cycle. *, Significant peaks (P <.25) for hcg; A, significant peaks for LH. 39F, 39-year-old female; 41F, 41- year-old female; 46F, 46-year-old female. (Reproduced from Odell and Griffin [3]; copyright by The Endocrine Society.) 243

7 244 WILLIAM D. ODELL 4-28F- LUTEAL PHASE Progesterone =3.6 ng/ml z -j D H 2- _ H E IX _ -I- 1- U -13 = A O- U TIME IN MINUTES u ,..,..,%... P_ D H X -i J E H E U B 3 - l l l l t [ l [ [ l l l 5 1 IS 2 25 TIME IN MINUTES 41F-LUTEAL PHASE Progesterone = 9.9 ng/ml J 6 H II H E 3 5D C I I I I 5 1 IS TIME IN MINUTES FIG. 4. See Figure 3 for symbol explanation. Concentrations of hcg and human LH (hlh) determined each 1 minutes in three women during the luteal phase of the menstrual cycle. (Reproduced from Odell and Griffin [3]; copyright by The Endocrine Society.) 4

8 CHORIONIC GONADOTROPIN 245 LUJ z LUI (I) LL. LU1 z ai TIME (min) FIG. 5. Changes in serum concentrations of hcg and hlh caused by intravenous injection of gonadotropin-releasing hormone (2 Mg) in 1 men. Hormone responses are given as the percentage changes from base line. Each point represents a mean ± SEM. (Reproduced from Odell and Griffin [29]. Reprinted by permission of The New England Journal of Medicine, 1987; 317: 1688.) IV a. C, 2 I CIz -J DAY OF CULTURE FIG. 6. Secretion of CG (upper panel) and LH (lower panel) by the cultured cells of three female human fetal pituitary glands for 11 days in culture. DCells from each pituitary gland were dispersed into one to three wells, depending on the number of cells available. The medium was completely changed at 1, 4, 7, and 11 days, and medium from each well at each change was assayed for LH and CG. Each point represents the mean of the values from one to three wells at each time from each cultured pituitary. (Reproduced from Odell et al. [33]; copyright by The Endocrine Society.)

9 246 WILLIAM D. ODELL *.tn FIG. 7. Electron micrographs of cells corresponding to those immunostaining with hcg. A, A low power electron micrograph showing an acinus containing hcg-type cells. Original print magnification, x 5,. B, The cellular detail of one hcg-type cell. Original print magnification, x 12,5. Sections were counterstained with uranyl acetate and lead citrate. (Reproduced from Hammond et al. [34]); copyright by The Endocrine Society.) finally appearing in 1991 (34). Editors were finally convinced when we employed a double-labelling technique-fluorescein-labelled anti-hlh and rhodamine-labelled anti-hcg. The rhodamine dye and fluorescein dyes are visible through different filters. Tissue sections were stained with both labelled antibodies simultaneously. Since each dye is visible through a different filter, examination of identical microscope fields revealed populations of cells immunostaining for hcg, but not LH, and a different population of cells staining for hlh and not hcg. These findings may be summarized: 1) Human CG is secreted into blood by the normal human pituitary, in parallel with hlh. 2) Human CG is extractable from all normal human tissues, but these tissues do not appear to contribute to blood hcg. 3) The function of the hcg secreted by the pituitary is not known. 4) The function of the hcg present in normal non-pituitary tissues is also unknown.

10 CHORIONIC GONADOTROPIN 247 Production of hcg-like Proteins by Bacteria: In 1974, Livingston and Livingston (35) reported that an hcg-like material was produced by a bacteria they called Progenitor cryptocides. These investigators employed an equilibrium-type radioimmunoassay to identify hcg. Acevedo et al. (36) later identified this bacteria as Staphylococcus epidermis. Subsequently, Slifkin et al. (37), Backus and Affronti (38), and Dominique et al. (39) employed immunocytochemical methods to demonstrate that a number of bacteria obtained from cancer patients possess immunoaccessible hcg-like material. Acevedo et al. (36, 4-44) published a series of papers, again using immunocytochemical techniques, to show that epitopes reacting with hcg polyclonal antibodies were present on several strains of bacteria including: Staphylococcus (ATCC 19433, 27848), Enterococci coli (ATCC 25922), and Pseudomonas maltophilia (ATCC 13637). In 1977, Richert and Ryan (45) reported that Pseudomonas maltophilia possessed an hcg/hlh binding site. Recently, studies from our laboratory (46) led to purification of an hcg-like protein from Pseudomonas maltophilia. We employed an immunoglobulin-saturation technique to block immunoglobulin Fc binding sites on the surface of Pseudomonas (these sites interfere with use of monoclonal antibodies to study the hcg-like protein) and a Zwitterion detergent, membrane solubilization technique to isolate and characterize this hcg-like protein. This protein reacted in a polyclonal rabbit hcg immunoassay, in two monoclonal anti-hcg immunoassays, and in the carboxyl-tail hcg assay (Fig. 8). The protein showed no reaction in immunoassay designed to quantify htsh, hlh, and the free alpha subunit of hcg (Fig. 9), demonstrating that assay reaction was not a methodological artifact. The protein was purified by sequential chromatography on phenyl sepharose and then affinity chromatography. The purified protein exhibited a single band on sodium dodecyl-polyacrylamide gel electrophoresis with MW of 48.5 kda (46). The purified protein did not bind to concanavalin A, which avidly binds human placental hcg, and also did not bind to mammalian hcg/lh receptors on rat Leydig cells. This hcg-like protein has been partially sequenced, and the portions available show approximately 4% amino acid homology with hcg. In further studies (47), we characterized the hcg-binding site previously reported by Richert and Ryan (45) and showed that hcg binds to this site with two affinities, a high affinity 3. x 1-1o Kd and a lower affinity 1. x 1- Kd (Fig. 1). Richert and Ryan (45) did not detect the higher affinity site in their studies because the techniques used, saturated this site with 1251-hCG. Strikingly, this high-affinity site is very specific for hcg, and shows no reaction with hlh, hfsh, or htsh (Fig. 11). This is the first binding site to be described in nature which distinguishes hcg from hlh. The

11 248 WILLIAM D. ODELL Rb. anti CG VOLUME (ul) I a n.1 -' a \. 1.b.1 t DOSE OF CG (ng) o hcg CR-121 * Ps. maoo. MOUSE ANTI CG(#4) mco 1zu B VOLUME (ul) o hcg CR-121 * Ps. maoo. 2 In u v v 1 s DOSE OF hcg (ug) FIG. 8. Dose-response curves for purified pregnancy CG (CR-121), used as a reference standard, and for the immune-saturated solubilized membrane preparation of Pseudomonas maltophilia used for four immunoassays. Various antibodies were used. For comparison, different volumes of the crude preparation have been plotted for all of the antibodies used. The protein concentration would be approximately 471 Mg/1 Ml solubilized preparation. A, Polyclonal equilibrium-type RIA for CG using rabbit anti-cg antiserum. B, Monoclonal equilibrium-type RIA using mouse anti-cg 4. C, Monoclonal equilibrium-type RIA using mouse anti-cg 9. D, Equilibrium-type RIA using the polyclonal rabbit anti-o-cooh tail peptide of CG. B/Bo, Bound/free ratio. (Reproduced from Grover et al. [46]); copyright by The Endocrine Society.)

12 A O m m m m '..,,,,,,..,,, n,,, * s, * - - n * n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CHORIONIC GONADOTROPIN MOUSE ANTI CG (#9) VOLUME (ul) Q- -o - 8 o hcg CR-121 * Ps. malta i,,,.,, 1- T I - I DOSE OF CG (ug) ANTI B-COOH Ab. VOLUME (ul) D 1. o Q \ o hcg CR * Ps. malta \ n DOSE OF CG (ug)

13 U mt vv vw w w...,. w-s ws wwwww www-s w w w wwv-to w w wss v w w w wwess w w w-w w w w sw w w-sw w w wwses w w w wn Rb. ANTI hlh VOLUME (ul) A o so o hlh * Ps. malo I I I 1 1 DOSE OF hlh (ng) MOUSE ANTI htsm(#21) VOLUME (ul) B o, s htsh * 6 Ps. matto. 4 2 n DOSE OF htsh (ng) MOUSE ANTI ALPHA CG VOLUME (ul) % *o 3o thj 1 so b% hcg CR * Ps. molo e\\ 2 ",1 9 1 o DOSE OF CG (ug) FIG. 9. Solubilized Pseudomonas membrane preparation reaction in three additional RIAs. A, Polyclonal equilibrium-type RIA using rabbit anti-lh# antibody. B, Monoclonal equilibrium-type RIA using mouse anti-htsh antibody. C, Monoclonal equilibrium-type RIA using mouse anti-cga antibody. The concentration of protein of the crude extract was 471 Mg/1 Al. B/Bo, Bound/free ratio. (Reproduced from Grover et al. [46]); copyright by The Endocrine Society.) 25

14 CHORIONIC GONADOTROPIN 251 UL B (pmole) FIG. 1. Scatchard plot of hcg binding to the Pseudomonas binding site. Binding studies were performed using 2-4 pg '25I-hCG. The mean Kd of the high-affinity binding site, collected from 6 assays, was 1.3 x 1-1 (±.4 = SEM). The mean Kd of the lower affinity site was 2.3 x 1- (±.9 = SEM). (Reproduced from Carrell and Odell [47].) 1 c -j z C) a* 9C 8C 7a 6C 5C 4C u9 FIG. 11. Dose-response curves for the native Pseudomonas hcg-like protein (PmCG) and for hcg, measured by displacement of 125I-hCG binding to the high-affinity Pseudomonas binding site. The hormone hcg and the Pseudomonas hcg-like protein compete equally well for '25I-hCG binding. The other hormones, hlh, htsh, and hfsh, showed little or no ability to displace 1251-hCG. (Reproduced from Carrell and Odell [47].)

15 252 WILLIAM D. ODELL purified Pseudomonas hcg-like protein also binds to this Pseudomonas binding site and with affinity identical to hcg (Fig. 11). We presume the hcg-like protein is the native ligand for this binding site. It is striking that a bacterium produces a protein with homology to hcg, and also possesses a high-affinity binding site for both hcg per se and the hcg-like protein. Humans and bacteria are separated in evolutionary terms by large distances. Furthermore, hcg has not been convincingly demonstrated in any non-primate vertebrates, nor in amphibians, reptiles, or fish. These studies of hcg lead to further questions of the functions of the hcg-like protein in bacteria and of any relations to the functions of hcg in nonpregnant normal humans. REFERENCES 1. Fiddes JC, Goodman HM. The gene encoding the common alpha subunit of the four human glycoprotein hormones. J Mol Appl Genet 1981; 1: Boothby R, Ruddon RW, Anderson C, et al. A single gonadotropin a-subunit gene in normal tissue and tumor-derived cell lines. J Biol Chem 1981; 256: Boorstein WR, Vamvakopoulos NC, Fiddes JC. Human chorionic gonadotropin fisubunit is encoded by at least eight genes arranged in tandem and inverted pairs. Nature 1982; 3: Keutmann HT, Williams RM. Human chorionic gonadotropin. Amino acid sequence of the hormone-specific COOH terminal region. J Biol Chem 1977; 252: Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981; 5: Odell WD, Hertz R, Lipsett MB, et al. Endocrine aspects of trophoblastic neoplasms. Clin Obstet Gynecol 1967; 1: Fusco FC, Rosen SL. Gonadotropin-producing anaplastic large-cell carcinomas of the lung. N Engl J Med 1966; 275: Beck JS, Porteous IB, Uilyot JL. Gonadotropin-secretingbronchial carcinoma: aberrant endocrine activity of trophoblastic differentiation? J Pathol 197; 11: Floyd WS, Cohn SL. Gonadotropin producing hepatoma. Obstet Gynecol 1973; 41: Paul WE, Odell WD. Radiation inactivation of the immunological and biological activities of human chorionic gonadotropin. Nature 1964; 23: Vaitukaitis JL, Braunstein GD, Ross GT. Radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 1972; 113: Braunstein GD, Vaitukaitis JL, Carbone P, et al. Ectopic production of human chorionic gonadotropin by neoplasms. Ann Intern Med 1973; 78: Braunstein GD, Rasor J, Wade ME. Presence in normal human testes of a chorionicgonadotropin-like substance distinct from human luteinizing hormone. N Engl J Med 1975; 293: Odell WD, Wolfsen AR, Yoshimoto Y, et al. Ectopic peptide synthesis: a universal concomitant of neoplasia. Trans Assoc Am Physicians 1977; 9: Yoshimoto Y, Wolfsen AR, Odell WD. Human chorionic gonadotropin-like material in nonendocrine tissues of normal subjects. Science 1977; 197: Odell WD, Saito E. Protein hormone-like materials from normal and cancer cells: "ectopic" hormone production. In: Mirand EA, Hutchinson WB, Mihich E, eds. 13th

16 CHORIONIC GONADOTROPIN 253 International Cancer Congress. Part E. Cancer Management. New York: Alan R. Liss; 1983: Yoshimoto Y, Wolfsen AR, Hirose F, et al. Human chorionic gonadotropin-like material: presence in normal human tissues. Am J Obstet Gynecol 1979; 143: Yoshimoto Y, Wolfsen AR, Odell WD. Glycosylation, a variable in the production of hcg by cancers. Am J Med 1979; 67: Braunstein GD, Kamdor V, Rasor J, et al. Widespread distribution of a chorionic gonadotropin-like substance in normal human tissues. J Clin Endocrinol Metab 1979; 49: Matsuura S, Chen H-C, Hodgen GD. Antibodies to the carboxyl-terminal fragments of human chorionic gonadotropin 13-subunit: characterization of antibody recognition sites using synthetic peptide analogues. Biochemistry 1978; 17: Matsuura S, Ohashi M, Chen H-C, et al. Physicochemical and immunological characterization of an hcg-like substance from human pituitary glands. Nature 198; 286: Chen H-C, Hodgen GD, Matsuura S, et al. Evidence for a gonadotropin from nonpregnant subjects that has physical, immunological and biological similarities to human chorionic gonadotropin. Proc Natl Acad Sci USA 1976; 73: Borkowski A, Muquardt C. Human chorionic gonadotropin in the plasma of normal, nonpregnant subjects. N Engl J Med 1979; 31: Borkowski A, Puttaert V, Gyling M, et al. Human chorionic gonadotropin-like substance in plasma of normal nonpregnant subjects and women with breast cancer. J Endocrinol Metab 1984; 68: Armstrong EG, Ehrlich PH, Birken S, et al. Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal nonpregnant and pregnant individuals. J Clin Endocrinol Metab 1984; 59: Griffin J, Odell WD. Ultrasensitive immunoradiometric assay for chorionic gonadotropin which does not cross-react with luteinizing hormone nor free beta chain of hcg and which detects hcg in blood of nonpregnant humans. J Immunol Methods 1987; 13: Odell WD, Griffin J. Two-monoclonal-antibody "sandwich"-type assay of human lutropin, with no cross-reaction with choriogonadotropin. Clin Chem 1987b; 33: Sawitzke AL, Griffin J, Odell WD. Purified preparations of human luteinizing hormone are contaminated with small amounts of a chorionic gonadotropin-like material. J Clin Endocrinol Metab 1991; 72: Odell WD, Griffin J. Pulsatile secretion of human chorionic gonadotropin in normal adults. N Engl J Med 1987; 317: Odell WD, Griffin J. Pulsatile secretion of chorionic gonadotropin during the normal menstrual cycle. J Clin Endocrinol Metab 1989; 69: Stenman U-H, Alfthan H, Ranta T, et al. Serum levels of human chorionic gonadotropin in nonpregnant women and men are modulated by gonadotrophin releasing hormone and sex steroids. J Clin Endocrinol Metab 1987; 64: Kyle CV, Griffin J, Jarrett A, et al. Inability to demonstrate an ultrashort loop feedback mechanism for LH. J Clin Endocrinol Metab 1989; 69: Odell WD, Griffin J, Bashey HM, et al. Secretion of chorionic gonadotropin by cultured human pituitary cells. J Clin Endocrinol Metab 199; 71: Hammond E, Griffin J, Odell WD. A chorionic gonadotropin secreting human pituitary cell. J Clin Endocrinol Metab 1991; 72: Livingston VW-C, Livingston AM. Some cultural, immunological and biochemical properties of Progenitor cryptocides. Proc NY Acad Sci 1974; 36: Acevedo HF, Slifkin M, Pouchet PR, et al. Immunohistochemical localization of

17 254 WILLIAM D. ODELL choriogonadotropin-like protein in bacteria isolated from cancer patients. Cancer 1978; 41: Slifkin M, Pardo M, Pouchet-Melwin GR, et al. Immunoelectron microscopic localization of a choriogonadotropin-like antigen in cancer-associated bacteria. Oncology 1979; 36: Backus BT, Affronti LF. Tumor-associated bacteria capable of producing a human choriogonadotropin-like substance. Infect Immunol 1981; 32: Dominque GJ, Acevedo HF, Powell JE, et al. Antibodies to bacterial vaccines demonstrating specificity for human choriogonadotropin (hcg) and immunochemical detection of hcg-like factor in subcellular bacterial fractions. Infect Immunol 1986; 53: Acevedo HF, Slifkin M, Pouchet-Melvin GR, et al. Choriogonadotropin-like antigen in an anaerobic bacterium, eubacterium lentum, isolated from a rectal tumor. Infect Immunol 1979; 24: Acevedo HF, Campbell-Acevedo EA, Slifkin M. Immunodetection of choriogonadotropin-like antigen in bacteria isolated from cancer patients. In: Segal SJ, ed. Chorionic Gonadotropin. New York: Plenum Press; 1981: Acevedo HF, Acevedo EAC, Kloos WE. Expression of human choriogonadotropin-like material in coagulase-negative Staphylococcus species. Infect Immunol 1985; 5: Acevedo HF, Acevedo EAC, Koide SS, et al. Choriogonadotropin-like antigen in a strain of Streptococcus and a strain of Staphylococcus simulans: detection, identification and characterization. Infect Immunol 1981; 31: Acevedo HF, Pardo M, Acevedo EC, et al. Human choriogonadotropin-like material in bacteria of different species: electron microscopy and immunocytochemical studies with monoclonal and polyclonal antibodies. J Gen Microbiol 1987; 133: Richert ND, Ryan RJ. Specific gonadotropin binding to Pseudomonas maltophilia. Proc Natl Acad Sci USA 1977; 74: Grover S, McGee ZA, Odell WD. Isolation of a 48.5-kDa membrane protein from Pseudomonas maltophilia which exhibits immunologic cross-reaction to the,b-subunit of human chorionic gonadotropin. Endocrinology 1991; 128: Carrell DT, Odell WD. A bacterial binding site which binds human chorionic gonadotropin but not human luteinizing hormone. Endocrine Res To be published, April 1992.