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S Birken

Publications and source records attributed to S Birken.

At least 19 recordsLinked to original sources
PubMed2003

Analysis of human choriogonadotropin core 2 o-glycan isoforms.

Measurements of human choriogonadotropin (hCG) isoforms containing core 2 o-glycans may be useful for diagnosis of Down Syndrome pregnancies and trophoblastic disease. As shown here, this isoform is also present in pituitary extracts, early pregnancy urine, and urine of postmenopausal women. Although, measurements of hCG isoforms may be useful in several clinical settings, this remains to be determined due to the lack of suitable standards and the difficulties of comparing data obtained in different laboratories. Here, we report that monoclonal antibodies B152 and CTP104 recognize the third (Ser132) and fourth (Ser138) o-glycans, respectively, in the carboxyterminal portion of the hCG beta-subunit. The proximity of these sites prevents B152 and CTP104 from binding simultaneously to isoforms containing core 2 o-glycans. Unlike B152, which binds only the core 2 isoform, CTP104 recognizes both glycan moieties. By measuring hCG with CTP104 in the presence and absence of B152, one can quantify both isoforms using the same readily available standard.

Antibodies, Monoclonal↗
PubMed2002

Differential expression of human chorionic gonadotropin (hCG) glycosylation isoforms in failing and continuing pregnancies: preliminary characterization of the hyperglycosylated hCG epitope.

Human chorionic gonadotropin (hCG) glycoforms change as pregnancy progresses. We have developed an antibody (B152) which can measure a hyperglycosylated early pregnancy isoform of hCG. This putative hyperglycosylated form of hCG arises very early in pregnancies and is rapidly replaced by an isoform that predominates for the remainder of the pregnancy. The profiles of these hCG glycoforms are measured as a ratio of values of two immunometric assays. The profiles of these ratios differ between pregnancies which persist and those which will experience early failure. In this report, daily urine hCG isoform ratios from donor eggs (no exogenous hCG pretreatment), in vitro fertilization pregnancies were profiled and analyzed from the first day following embryo transfer (ET). Significant differences were found between continuing pregnancy and pregnancy loss throughout days 5-20 post-ET. When hCG isoform ratios were analyzed from the first day of detectable hCG, pregnancy loss could be predicted in the case of a single fetus both during the 5- to 10-day time segment (P=0.018) and the 10- to 15-day time segment (P=0.045). When single and multiple fetus pregnancies were analyzed together significance was approached in the 10- to 15-day time period (P=0.058). In a second population of pregnant women who conceived naturally, in whom urine samples were collected at approximately weekly intervals to either term birth or clinical spontaneous abortion, the ratio could discriminate between miscarriages and normal term pregnancies (P=0.043). In later pregnancy, the ratio of hCG isoforms declined more rapidly in miscarriages than in term pregnancy. Antibody B152 was produced using a choriocarcinoma-derived hCG (C5), which was hyperglycosylated at both N- and O-linked sites and was 100% nicked at position beta(47-48). Western blot analyses supported the assay results showing that early pregnancy urine does not contain nicked C5-like hCG. Also, the early pregnancy hCG appeared to be the same size as later pregnancy hCG as judged by SDS gel electrophoresis. A series of Western blot analyses and immunoassays conducted with the samples either non-reduced or reduced showed that B152 is directed to a linear epitope located in the COOH-terminal peptide region of the beta subunit. This indicated that only the O-glycan groups and not the N-linked glycans are part of the antibody epitope.

Abortion, Spontaneous↗
PubMed2001

Metabolism of gonadotropins: comparisons of the primary structures of the human pituitary and urinary LH beta cores and the chimpanzee CG beta core demonstrate universality of core production.

The gonadotropins are a family of closely related heterodimeric glycoprotein hormones homologous in structure to disulfide-knot growth factors. Metabolic proteolytic processing in vivo of this disulfide cross-linked region results in urinary excretion of a residual highly stable core structure. The primary structure of the pituitary form of the hLH beta core was reported earlier, but it has proved difficult to isolate the urinary core, although antibodies to the pituitary core demonstrated its presence. By conventional and immunoaffinity methods, the urinary core has been isolated and its structure determined by both chemical and mass spectrometric methods. The urinary hLH beta core is the same as the pituitary-extracted hLH beta core, beta 6-40 disulfide bridged to beta 55-93, except that the pituitary core is more heterogeneous containing also beta 49-93. These findings imply a dual origin of urinary cores, both directly from a secreting tissue and by kidney processing of circulating hormone. We also found that pregnant chimpanzees excrete a CG beta core with a primary structure identical to that of the human CG beta core of pregnancy. In conclusion, gonadotropin core generation and urinary excretion of nearly identical gonadotropin metabolites is common among primates. Although possible biological functions of these core fragments remain unproven, they have diagnostic utility because of their stability and abundance.

Amino Acid Sequence↗
PubMed2000

Preparation and analysis of the common urinary forms of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) is the hormone of pregnancy and forms the basis of all pregnancy tests as well as diagnostic assays for a variety of pathological states including certain types of cancers and some diseases of pregnancy and genetic abnormalities. In recent years, the discovery of the diagnostic utility of measurement of the free subunits and fragments of the hormone, especially in urine, has proven of special use for diagnosis of very early pregnancy loss, an important phenomenon related to infertility, as well as part of screening programs for Down Syndrome and gynecological cancers. This article summarizes existing and new methods for the preparation of hCG, its subunits, and the beta core fragment from urinary sources. The methods for proper analyses of these materials are also described to enable investigators to prepare and analyze these materials in various quantities in their own laboratories.

Chorionic Gonadotropin↗
PubMed1999

Differences in urinary excretion patterns of the hLH beta core fragment in premenopausal, perimenopausal, and postmenopausal women.

OBJECTIVE: The heterodimeric luteinizing hormone beta core fragment (hLH beta cf) is a highly stable urinary analyte reflective of circulating hLH. It is measured easily because of its high molar content and has none of the multiple isoforms and subunit dissociation problems of LH urinary measurements. As part of a long-term effort to develop new biochemical assays to stage women during the perimenopausal transition, we have examined the patterns of urinary excretion of this metabolite of hLH in premenopausal, perimenopausal, and postmenopausal women. DESIGN: We measured the concentration of the hLH beta cf in 10 consecutive first morning void urine specimens from premenopausal, perimenopausal, and postmenopausal women. Day 1 of collection was the first day of menses in the cycling women. RESULTS: Postmenopausal women exhibited a widely fluctuating pattern of LH beta core fragment excretion, which is not correlated with hLH measured by immunofluorometric assay or with follicle-stimulating hormone measured by immunofluorometric assay. The postmenopausal group was easily distinguished from premenopausal women on the basis of an area-under-the-curve concentration function. Perimenopausal women displayed intermediate hLH beta cf concentrations; some clearly were in postmenopausal ranges, and others were in the premenopausal ranges. CONCLUSIONS: The pattern of excretion and concentrations of the hLH beta cf is significantly different between premenopausal and postmenopausal women. Perimenopausal women exhibited intermediate changes. The capability to measure this type of stable urinary metabolite as a reflection of changes in dynamics of its parent circulating hormone offers new possibilities in the development and application of large-scale testing that does not require blood sampling.

Adult↗
PubMed1999

Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG.

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its beta-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its beta COOH-terminal region. Two-site immunometric assays have been developed using these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the beta-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

Animals↗
PubMed1999

Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG.

Human chorionic gonadotropin (hCG) exhibits molecular heterogeneity in both its protein and carbohydrate moieties. This communication describes changes in hCG isoforms detected directly in clinical samples. These isoforms, quantified in blood or urine specimens, show a progression of change throughout normal pregnancy. Early pregnancy produces a type of hCG that resembles, in terms of immunoreactivity, a major form of hCG excreted in choriocarcinoma. The isoforms predominate for the first 5-6 weeks of gestation and then diminish, being replaced with the hCG isoforms which predominate throughout the remainder of pregnancy. The alteration in hCG isoform content occurs in both blood and urine. The progression of isoforms is best delineated by calculating the change in the ratio of the two forms, as many hCG assays either do not detect or fail to discriminate among these isoforms. An analogous pattern of hCG isoforms was observed in patients with in vitro fertilization pregnancies. hCG isolated from the pituitary displayed binding characteristics similar to those of the hCG derived from normal pregnancy urine. The early pregnancy hCG isoforms appear to have a differential expression in normal pregnancy as opposed to pregnancies which will not carry to term, suggesting that a determination of the relative balance of hCG isoforms may have diagnostic application in predicting pregnancy outcome.

Biomarkers↗
PubMed1999

Evaluation of nicked human chorionic gonadotropin content in clinical specimens by a specific immunometric assay.

We report the development and characterization of an IRMA for the direct measurement of nicked human chorionic gonadotropin (hCGn) in blood and urine. hCGn derived from a reference preparation of hCG used as an immunogen elicits monoclonal antibodies (mAbs) with enhanced recognition of human luteinizing hormone epitopes. The most specific assay for pregnancy hCGn is an IRMA composed of one mAb to choriocarcinoma-derived hCGn (C5) and a second mAb developed from immunization with normal-pregnancy hCGn. This assay was used to evaluate hCGn profiles in normal, in vitro fertilization, Down syndrome, and ectopic pregnancies. In all pregnancies, hCGn was usually present in much lower concentrations than the non-nicked hCG isoform. Our results suggest that some form of physical separation from the overwhelming quantities of non-nicked hCG present in clinical specimens will be required before accurate immunochemical estimations of hCGn can be made.

Abortion, Spontaneous↗
PubMed1998

Effects of a urinary factor from women in early pregnancy on HIV-1, SIV and associated disease.

The effects of clinical grade crude preparations of human chorionic gonadotropin (hCG) on Kaposi's sarcoma, HIV, SIV and hematopoiesis were examined in vitro and in vivo. In contrast to previous studies, we report that the antiviral activity of hCG associated factors is not due to the native hCG heterodimer, including its purified subunits or its major degradation product, the beta-core. Using gel permeation chromatography of the clinical grade hCG and urine concentrates from pregnant women, we demonstrate that an as yet unidentified hCG associated factor (HAF) with anti-HIV, anti-SIV, anti-KS and pro-hematopoietic activities elutes as two peaks corresponding to 15-30 kDa and 2-4 kDa.

Animals↗
PubMed1998

The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay.

Human gonadotrophins undergo metabolic transformations which result in the presence of several smaller, structurally and immunologically related forms of gonadotrophins in the urine. For luteinizing hormone (LH), a beta core fragment (LHbeta cf) has been isolated from the pituitary and characterized. The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physicochemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of LHbeta cf have a different structure, probably in the carbohydrate moieties. This communication characterizes the expression of LHbeta cf in the urine of both reproductive and post-reproductive age women and in men, employing assays highly specific for the pituitary form of the fragment. It was found that LHbeta cf is the predominant LH associated molecular form in the urine during peri-ovulatory period, peaking 1-3 days later than intact LH and reaching a concentration of approximately 600 fmol/mg creatinine, 7-fold higher than either LH or LH free beta subunit. Corresponding concentrations of human chorionic gonadotrophin (HCG) beta cf were <1% that of LHbeta cf. LHbeta cf cross-reaction with some LH or LHbeta monoclonal antibodies may well interfere with the accurate estimation of the day of the LH surge when urinary tests are utilized.

Adolescent↗
PubMed1996

Metabolism of hCG and hLH to multiple urinary forms.

Human chorionic gonadotropin (hCG) is synthesized primarily in the placenta while human luteinizing hormone (hLH) is produced in the pituitary. Both hormones are highly homologous in structure and both appear to be altered to analogous molecular forms as the hormones are proteolytically processed, or metabolized, from tissue of origin, through the circulation, and finally to the urine. Placental hCG is excreted into urine as heterodimeric hormone, heterodimeric nicked hCG, free subunits (some nicked), and predominantly as the hCG beta core fragment. A pituitary form of heterodimeric hCG, which is partly sulfated as is pituitary hLH, was recently isolated and is likely the form of hCG observed in the urine of healthy postmenopausal women and nonpregnant premenopausal women as well. A pituitary form of the hLH beta core fragment, highly analogous in structure to that of urinary hCG beta core fragment, has been used to develop specific monoclonal antibody assays to measure urinary hLH beta core fragment which is excreted at significantly higher molar concentrations than is hLH in the urine of ovulating women 1 or 2 days after the LH surge. This fragment of LH appears in the urine of postmenopausal women as well. The development of the capability to distinguish the hCG beta core fragment from the hLH beta core fragment in urine may have useful applications in tumor marker assays, pregnancy tests, and menopause. While hCG urinary assays have been widely employed, urinary assays for hCG and hLH metabolites are much less used since the urinary molecular forms are only partly known. Our studies of hCG and hLH urinary metabolites are directed towards improvement of the utility of urinary measurements of molecules derived from these hormones. Since many of the molecular forms of these two hormones in urine differ from their forms in blood, it may be necessary to produce new immunoassays as well as novel urinary reference preparations to accurately measure these molecules within their urinary matrix.

Chorionic Gonadotropin↗
PubMed1996

Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures.

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein backbone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C- and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Animals↗
PubMed1996

Isolation and characterization of human pituitary chorionic gonadotropin.

Human CG (hCG) is the hormone associated with the maintenance of pregnancy. Although three related glycoprotein hormones, LH, hFSH, and hTSH, are secreted by the pituitary, HCG is the only one of this family of glycoprotein hormones that is produced by the placenta in primates to maintain the steroid hormone secretions of the corpus luteum. Although hCG is not considered to be a pituitary hormone, hCG-like immunoreactive materials have been reported in pituitary tissue, blood, and urine from healthy nonpregnant individuals for 2 decades, but it was never isolated. We now report the purification and characterization of pituitary hCG from acetone-preserved human pituitary glands. After gell permeation chromatography, the fractions containing hCG molecules were pooled and purified by immunoaffinity chromatography using antibodies to the COOH-terminal region of hCGbeta. Amino acid analyses, amino-terminal sequence analyses, as well as mass spectrometric studies gave similar results for both pituitary hCG and hCG purified from the urine of pregnant women. Analyses for sulfate and sialic acid contents demonstrated that pituitary hCG contained both sulfate and sialic acid. In vitro biological activity of pituitary hCG indicated that it was 50% as active as hCG purified from the urine of pregnant women in cAMP assays.

Animals↗
PubMed1995

The expression, characterization, and crystallization of wild-type and selenomethionyl human chorionic gonadotropin.

Although the glycoprotein hormone hCG was crystallized over 4 yr ago, it is only now that three-dimensional structural information is available. This manuscript reports the method for successful production of modified expressed hormone, the characteristics of the crystallized protein, and unexpected observations during the crystallization process. Two different routes of solution to the structure of hCG were followed. The first was based on the traditional method of heavy atom isomorphous replacement, and the second was the more novel method of expressing the protein with selenomethionine substituting for methionine and applying multiwavelength anomalous diffraction analysis. Selenomethionyl hCG was employed to successfully grow the crystals used for the solution of the structure of hCG after partial deglycosylation by hydrogen fluoride (HF) treatment. The selenomethionyl hCG proved to be more hydrophobic than the expressed form of native hCG. Furthermore, expressed forms of hCG that were deglycosylated by HF proved to be more intact and less susceptible to peptide bond cleavages during the crystallization process than the urinary form of HF-treated hCG studied previously. It was found that addition of reducing agent during the crystallization period was necessary for the growth of crystals of HF-treated selenomethionyl hCG suitable for diffraction studies. Growth of crystals of HF-treated expressed hCG were accelerated by the addition of dithiothreitol, but would successfully grow without reductant. HPLC analysis of the HF-treated hormones before and during the crystallization process was used to identify alterations in the molecules, including oxidation and aggregation, both of which may affect the growth of crystals.

Animals↗
PubMed1994

Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.

Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.

Animals↗