ELISA for the measurement of serum and urinary chorionic gonadotropin concentrations in the laboratory macaque

A rapid, sensitive, enzyme-linked immunosorbant assay (ELISA) for the measurement of chorionic gonadotropin (CG) in serum and urine samples of laboratory macaques is reported. The ligand (CG) is captured by a readily available, widely used, and well-characterized monoclonal anti-body (Mab, 518B7) generated against the β subunit of bovine luteinizing hormone (LH). This Mab, while specific for LH, shows very little species specificity, and has been shown to detect LH and CG by radioimmunoassay (RIA) in both human and non-human primates. A polyclonal antiserum raised in rabbits against human chorionic gonadotropin (hCG) is conjugated to horseradish peroxidase, and is used as the second anti-body signal. This anti-hCG antiserum cross reacts with CG secreted by both the human (hCG) and macaque (mCG). The ELISA utilizes hCG as the standard, and results are based on the relative concentrations of mCG in serum and urine. Total assay time is less than 5 hours. Range of the standard curve is 0.002 to 0.5 ng hCG/well, and the least detectable concentration of hCG is 0.0023 ± 0.0007 ng/well. Pregnancy was detected in early pregnant macaques (M. fascicularis) on 9 (N = 1/16), 10 (N = 1/16), 11 (N = 1/16), 12 (N = 6/16), 13 (N = 1/16), 14 (N = 4/16), and 15 (N = 2/16) days following the pre-ovulatory urinary estrone conjugate peak. The detection of pregnancy by urinary mCG occurred approximately 24 to 72 hours after its detection in serum. Am. J. Primatol. 41:307–322, 1997. © 1997 Wiley-Liss, Inc.     

Upconverting phosphor reporters in immunochromatographic assays

Immunochromatographic assays have become popular diagnostic tools in a variety of settings because they are sensitive, fast, and easy to use. Here, we describe the use of a novel reporter, upconverting phosphors (UCP), in this assay format. UCP are submicron-sized, inorganic crystals that are excited with infrared light and that emit photons in the visible range depending on the ion composition of the crystal. Using human chorionic gonadotropin (hCG) as a model analyte to describe the properties of phosphors in immunochromatographic assays, a detection limit of 10 pg hCG in a 100-microl sample has been achieved on a regular basis, with occasional detection of 1 pg hCG. This represents at least a 10-fold improvement over conventional reporter systems such as colloidal gold or colored latex beads. Quantitation of analytes is possible over at least 3 orders of magnitude. Furthermore, an example is given of how UCP can be used for analyte multiplexing using a two-plexed wick for the detection of mouse IgG and ovalbumin. Thus, UCP lateral flow assays can be used for applications that are currently limited by assay sensitivity, and they can increase the probability of a diagnosis by verifying the presence of several analytes in the same sample.     

Noninstrumented enzyme-linked immunosorbant assay for detection of early pregnancy in macaques

A practical, noninstrumented enzyme-linked immunosorbant assay (NELISA) for the measurement of urinary monkey chorionic gonadotropin (mCG) has been developed for the detection of early pregnancy in macaque monkeys for use in both the laboratory and the field. Five rhesus monkeys (Macaca mulatta) and six crab-eating monkeys (Macaca fascicularis) were tested for the presence of mCG in urine on gestational days (GDs) 12 to 35. The mCG NELISA detected pregnancy as early as GD 14, with an average earliest detection at GD 16.5 +/- 1.4 (n = 11). Out of 90 tests, 27 false-negative and zero false-positive tests were obtained, for an accuracy of 70.0%. Without the aid of a spectrophotometer, the presence of mCG in pregnant monkey samples was indicated by a dark green color change. Nonpregnant monkey urine samples, on the other hand, exhibited no color change. These findings suggest that the simple, economical, and reliable urinary mCG NELISA may be useful for diagnosing early pregnancy in these and related species. Because capture and restraint are unnecessary for collecting urine samples, the mCG NELISA has widespread potential for confined and free-ranging animals.     

Le point sur les dosages de l’hormone gonadotrophine chorionique : lequel prescrire pour quelle prise en charge ?

The point on chorionic gonadotropin assays: which tests prescribe for the best take care? hCG assays are very often prescribed for biological investigations or before medical investigations. Physicians and biologists have to discuss about these different available hCG assays for an optimal patient's follow-up. Patients must be advised to have blood sample in the same laboratory to avoid analytical variations, possibly prejudiciable in case of cinetic observations.     

Antibodies and immunoassays

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a single molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation variants, such as its free beta (hCGbeta) and alpha (hCGalpha) subunits and clipped, cleaved, terminally differently sialylated, and overglycosylated forms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is highly dependent on the selection of target molecules and the natural variability of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immunoassays are still the state-of-the-art technique for both clinical and research applications but a major problem is the different extents of recognition of hCG variants by mAbs used in different immunoassays. On the whole, construction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, cross-reactivities, epitope locations and compatibilities, overlap and overhang in specificities (pairs of mAbs), and, finally, overspecificity. Consequences of overhang and overlap in antigen recognition of coating and detection mAb specificities are nondesirable assay cross-reactions and competitive interference by antigenic variants. The general agreement on the most favorable assay design is contrasted by the variety of isotopic and nonisotopic detection systems in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity. By measuring substrate absorption values off the absorption maximum, the measuring range of any IEMA can be extended significantly, as shown for 3,3',5, 5'-tetramethylbenzidine (TMB), without jeopardizing assay characteristics. Sensitivity of the IEMA can be enhanced by modifying the horseradish peroxidase (HRPO) labeling technique by using highly purified mAb preparations and higher-input HRPO/mAb ratios. We have also compared the assay characteristics of time-resolved fluoroimmunoassay (IFMA), IEMA, immunoradiometric assay (IRMA), and competitive radioimmunoassay (RIA) based on identical mAbs. Reasons for the observed superiority of the IFMA lie in its concept of signal detection and the high specific labeling of the detection mAb which on a molar basis can be up to 7-fold and 15-fold higher compared with (125)I and HRPO, respectively.     

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.     

Preliminary studies on the indium slide immunoassay for estimation of human chorionic gonadotropin and antihuman chorionic gonadotropin antibody

Human chorionic gonadotropin (hCG) is synthesized and secreted as early as 170 hr after fertilization and has been used as an index for pregnancy. Neutralization of hCG with a beta-subunit hCG vaccine(s) has been proposed as a contraceptive technique. To monitor the duration of effectiveness of the vaccine, it will be necessary to monitor the anti-hCG antibodies, especially those responsible for inhibiting the hCG bioactivity. We report a simple, rapid technique using an indium slide immunoassay for the qualitative estimation of hCG and to monitor a bioeffective anti-hCG antibody. The sensitivity of the indium slide assay to measure hCG ranged from 1 microgram/ml to 1 ng/ml, depending on the format of the assay. The indium slide assay also detected anti-hCG antibodies generated against a specific determinant on hCG recognized by a neutralizing monoclonal antibody (P3W80) in women immunized with a contraceptive vaccine.     

Screening of monoclonal antibodies for hCG for affinity and specificity

Using human chorionic gonadotropin (hCG) as a model polypeptide, we have developed a strategy that allows the direct screening of supernatant fluids from hybridomas for the presence of monoclonal antibodies of high affinity and predefined specificities. The assay evaluates the competition between 125I-labeled and unlabeled homologous or heterologous antigens in a solid-phase two-site immunoradiometric assay. This assay is fast and accurate, and is of general use provided the antigen of interest can be purified in nanomolar quantities. This strategy led to the isolation of nine new monoclonal antibodies for hCG, two of which could be used for elaborating a sensitive two-site immunoradiometric assay for this hormone.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit

The complete amino acid sequence of the beta-subunit of equine chorionic gonadotropin (eCG beta) has been established by both automated Edman and manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradations. Specific fragments were produced by cleavage with Staphylococcus aureus V8 protease, trypsin, or dilute HCl. For the sequence analyses of the heavily glycosylated COOH-terminal portion, a chemical deglycosylation procedure with trifluoromethanesulfonic acid was employed. The peptide chain of eCG beta consists of 149 amino acid residues. Five or more oligosaccharide chains are attached to the protein, 1 unit linked by an N-glycosidic bond to asparagine at residue 13 and four or more units linked by O-glycosidic bonds to serine or threonine at residues in the COOH-terminal portion. The carbohydrate-bearing hydroxy amino acids have not yet been rigorously established. As compared to the beta-subunits of the pituitary gonadotropin hormones, lutropin, follitropin, and thyrotropin, eCG beta possesses a glycosylated COOH-terminal extension of about 30 amino acid residues, as does the human chorionic gonadotropin beta-subunit (hCG beta). When the comparison is restricted inside the disulfide bond-containing core (residues 1-110), the beta-subunit of eCG is highly homologous to hCG beta (66%). On the other hand, although the overall structural features closely resemble each other, much less homology exists in the COOH-terminal extensions of eCG beta and hCG beta.     

Porcine pregnancy-associated glycoprotein family (pPAGs) - as in vitro-produced chorionic ligands for luteal and uterine gonadotropin receptors

This paper compares proteomic interaction-types and binding-effectiveness of secretory chorionic ligands (including pPAGs) with other proteins, i.e. gonadotropin membrane receptors (Rc) isolated from luteal-phase corpora lutea, uterine myometrium and endometrium of cyclic (cCLRc, cMYORc and cENDRc) or pregnant (pCLRc, pMYORc and pENDRc) pigs. Binding-effectiveness of miscellaneous in vitro-produced chorionic ligands (+pPAGs) was compared by radioreceptor assay (RRA) with endometrial (END) proteins of cyclic, pseudopregnant and pregnant gilts - as negative control ligands and porcine LH and hCG - as positive control ligands. The binding-comparison suggests that the pPAGs may play an important role as potential antiluteolytic or luteoprotective chorionic-origin signals during pregnancy, according to the binding-effectiveness of secretory chorionic ligands (+pPAGs) that was relatively comparable to LH/hCG - as classical ligands competing for luteal and uterine gonadotropin receptors of cyclic and pregnant pigs.     

Serum hormone levels in pregnant cynomolgus monkeys

Serum levels of estradiol (E2), progesterone (P), prolactin (Prl), monkey chorionic gonadotropin (mCG), dehydroepiandrosterone sulfate (DHEAS), and cortisol (C) in pregnant cynomolgus monkeys are described. mCG, E2, and Prl patterns resembled those of pregnant rhesus monkeys. P patterns differed from data from the literature. DHEAS patterns did not follow E2 patterns. C levels did not change during pregnancy.     

Immunoaffinity trapping of urinary human chorionic gonadotropin and its high-performance liquid chromatographic-mass spectrometric confirmation

A method has been developed for confirmation of the glycopeptide human chorionic gonadotropin (hCG) in urine. A solid-phase immunoaffinity trapping technique utilizing a monoclonal antibody recognizing both intact hCG and free hCG β-subunits was developed for the extraction of hCG from urine. Recovery of hCG from a urine matrix was essentially quantitative. The hCG was quantitatively eluted with 6 M guanidine hydrochloride, reductively alkylated with vinylpyridine, and subjected to tryptic digestion. The tryptic digest was analysed by HPLC-MS. Ions from three tryptic fragments were monitored with selected ion monitoring to provide specific detection of hCG. The signal observed for a concentration of 25 mIU/ml of hCG could be clearly distinguished from background with a signal-to-noise ratio of 12:1.     

Adsorptive stripping voltammetry of human chorionic gonadotropin and two of its specific antibodies

The electrochemical behaviour of human chorionic gonadotropin (hCG), as well as rat anti-βhCG and rabbit anti-βhCG antibodies, has been studied using cyclic voltammetry and adsorptive stripping voltammetry. Conditions have been optimised for the determination of these species by differential pulse adsorptive stripping voltammetry with respect to accumulation potential, accumulation time, scan rate, pulse amplitude and drop size. This technique has also been used to investigate the interaction of hCG with each of its specific antibodies in solution.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

A label-free amperometric immunosensor for fast and sensitive assay of human serum chorionic gonadotropin (hCG) is presented. hCG was immobilized on nanoporous gold (NPG) foils and using hydroquinone (HQ) redox species as indicator. The variation of amperometric response to the concentration of hCG, the target antigen, was evaluated by cyclic voltammetry in phosphate-buffered solution. Taking advantage of dual-amplification effects of the NPG foils and graphene sheets (GSs), the immunosensor exhibited a specific response to hCG in the range of 0.5–40.00 ng ml⁻¹ with a detection limit of 0.034 ng ml⁻¹ under optimal conditions. It was demonstrated that our proposed method possesses good accuracy, acceptable precision, and reproducibility. The NPG showed a better sensitizing effect and stability as immobilization matrices.     

Highly sensitive optical chip immunoassays in human serum

Over the past decade the ability of refractometric optical sensors to quantitatively measure a wide range of biomolecules has been demonstrated. These include proteins, nucleic acids, microorganisms, and in competitive formats small molecules such as drugs and pesticides. Furthermore, by using high refractive index nanoparticles to amplify the biomolecular binding signal, sensitivities approaching those of well established diagnostic assays have been achieved. However, to date it has not been possible to show rapid detection of analytes in complex bodily fluids such as serum, in a one-step procedure, due to the interference resulting from non-specific binding (NSB) to the sensor surface. We have carried out preliminary work on the control of interference due to NSB using an optical chip based on the Hartman interferometer. This interferometer configuration employs a reference sensing region that can be functionalized separately from the specific sensing region. Optical chips were stored dry after surface functionalization, and rehydrated in serum. The observed level of background drift in serum was reduced by an order of magnitude when an exposed reference was used, compared to a reference which was blind to the sample. An additional 70% reduction in signal drift in serum was achieved by controlling the surface chemistry of the optical chip using a biotin-poly(ethylene glycol) (PEG) blocking agent. This functionalization procedure was combined with a sandwich assay using gold nanoparticles to develop a one-step assay for human chorionic gonadotropin (hCG) in human serum with a detection limit of 0.1 ng/ml for a 35 min assay.     

The estrogen 2-hydroxylase activity of the gonadotropin-stimulated hypophysectomized immature rat ovary

Using high-performance liquid chromatography and a combination of electrochemical and radiometric flow detection for 2-[14C]hydroxyestradiol, changes in estrogen 2-hydroxylase activity in the microsomal fraction of rat ovarian homogenates were followed. Injection of human chorionic gonadotropin (hCG) at 12-hr intervals to hypophysectomized immature rats stimulated hypertrophy of the theca-interstitial tissue and produced a profound increase in enzyme activity. With the last injection of hCG at 96 hr the peak serum concentration of hCG was reached 12 hr later and then decreased exponentially with a half-time of 13 hr. However, enzyme activity remained elevated for at least 60 hr before beginning to fall. Pregnant mare's serum gonadotropin (PMSG) also produced an increase in activity, which was apparently limited to the thecal-interstitial tissue because freshly removed granulosa cells from the mature follicles had undetectable activity levels. Administration of anti-PMSG antiserum after enzyme activity had been increased resulted in a prompt fall in activity, as did injection of hCG to mimic an ovulatory surge of LH. The results indicate that the thecal-interstitial tissue of the rat ovary has estrogen 2-hydroxylase activity that is dependent upon gonadotropic stimulation for expression.     

Modification of sialyl residues of gonadotropic hormones by reductive amination. Influence on biological activity and circulating half-life

The sialic acid residues of human chorionic gonadotropin, human lutropin and human follitropin were quantitatively modified by introduction of an amino compound. In radioreceptor assays, the modified chorionic gonadotropin, lutropin and follitropin saturated the receptors. However, in the low nanogram range, the gonadotropic binding was higher for the control compared to the modified sample.The hormonal activity of the chorionic gonadotropin was testedin vitro. The modified preparations were four- to thirteen-fold less stimulatory compared to the control but elicited the same maximal response. The biological activity of follitropin was determinedin vivo. In this case, the modified preparations were four- to five-fold less stimulatory than the control. Both the modified chorionic gonadotropin and follitropin preparations were found to act as agonists. Modification of the gonadotropin hormones did not significantly alter the immune recognition of these glycoproteins.The apparent circulating half-life in rats of the modified chorionic gonadotropin and follitropin was increased six- to nine-fold compared to that of native hormones; this might be a consequence of resistance of the modified sialyl residues to sialidases and the resultant slower exposure of terminal galactosyl residues; the plasma half-life of modified lutropin remained the same as that of the native hormone.Abbreviations hCG human chorionic gonadotropin - hLH human lutropin or luteinizing hormone - hFSF human follitropin or follicle stimulating hormone - mala methyl ester of alanine - hCG(ala, mala, etc.) human chorionic gonadotropin modified on sialicacid by reductive amination with alanine, methyl ester of alanine, etc. - IRP-HMG intact rat prostrate-human menopausal gonadotropin     

The use of monoclonal antibodies against human chorionic gonadotropin for immunoperoxidase staining of normal placenta, pituitary gland, and pituitary adenomas

A monoclonal mouse antibody to human chorionic gonadotropin (hCG) was used in a modified unlabeled antibody enzyme-bridge staining method to demonstrate the localization of hCG in normal human placenta, pituitary gland, and six pituitary chromophobe adenomas. Mouse ascitic fluid containing monoclonal antibody could be diluted up to 1:500,000 for detection of hCG in the syncytiotrophoblast, whereas no staining was observed in the pituitary or adenomas even with high antibody concentrations (dilutions from 1:500 upward). Nonspecific background staining was negligible. These results demonstrate that monoclonal antibodies are suitable for immunohistochemical localization of antigens in tissues.     

Clinical utility of hyperglycosylated hCG in serum taken before hydatidiform mole evacuation to predict persistent trophoblastic disease

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Studies on hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen, ITA) in PTD are limited. In serum samples taken before evacuation of molar pregnancies we measured the concentrations of free hCG beta-subunit (free hCGbeta), "total" hCG (hCG+hCGbeta) and ITA, and determined whether ITA, the two other hCG analytes, or the calculated ratios of hCGbeta/hCG+hCGbeta, hCGbeta/ITA and hCG+hCGbeta/ITA could predict the later development of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Central Registry for Hydatidiform Moles. The study group comprised 97 patients with hydatidiform moles who did not develop PTD after mole evacuation and 33 patients who did develop PTD. Methods: Serum samples from 130 patients with hydatidiform mole with or without PTD were assayed using specific (radio)immunoassays for free hCGbeta, total hCG, and ITA. From these analytes we also calculated the ratios hCGbeta/hCG+hCGbeta, hCGbeta/ITA, and hCG+hCGbeta/ITA. To predict the development of PTD from these analytes and parameters we performed receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curve (AUCs) that represented the diagnostic accuracy which was rated in a range from excellent (AUC >0.9 or <0.1) to poor (AUC 0.4-0.6). Results: The diagnostic accuracy of ITA was moderate (0.618) and not different from that of free hCGbeta (0.610) and hCG+hCGbeta (0.622). CONCLUSIONS: ITA as well as the other analytes and parameters in serum taken prior to evacuation from patients with molar pregnancies cannot be used to predict the subsequent development of persistent trophoblastic disease.