A silicon sensor-based filtration immunoassay using biotin-mediated capture

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.     

Enhancement in antigen binding by a combination of synergy and antibody capture

The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.     

孕血清中早孕因子的纯化

本文用DEAE纤维素离子交换层析、ConA—Sepharose亲和层析、Sephadex G-100凝胶过滤等技术,分离纯化妊娠3～6周正常早孕妇女静脉血混合血清中早孕因子(Early Pregnancy Factor,EPF).以花结抑制实验检测各阶段层析物中EPF活性,以放射免疫法测定hCG含量.结果表明,经凝胶过滤后,提取物中hCG均为阴性(<3.1ng/ml),高EPF活性部分经SDS-聚丙烯酰胺凝胶电泳显示,分子量分别为24KD.和41KD_α。     

A gold nanoparticles/sol-gel composite architecture for encapsulation of immunoconjugate for reagentless electrochemical immunoassay

A highly hydrophilic, non-toxic and conductive colloidal gold nanoparticle/titania sol-gel composite membrane with a low contact angle was prepared on a glassy carbon electrode via a vapor deposition method. With human chorionic gonadotrophin (hCG) as a model antigen and encapsulation of horseradish peroxidase-labeled hCG antibody (HRP-anti-hCG) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. It displayed a porous and homogeneous composite architecture without the aggregation of the immobilized protein molecules. The presence of gold nanoparticles provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of immunoconjugate by a simple one-step immunoreaction between hCG in sample solution and the immobilized HRP-anti-hCG introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface. Under optimal conditions, the hCG analyte could be determined in two linear ranges from 0.5 to 5.0 mIU/mL and 5.0 to 30 mIU/mL with a relatively low detection limit of 0.3 mIU/mL at 3sigma. The hCG immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications.     

Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine

Human chorionic gonadotrophin (hCG) and its β‐subunit (hCGβ) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C‐terminal hCGβ peptide (hCGβCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGβ mutant (hCGβR68E, designed to eliminate cross‐reactivity with luteinizing hormone) or hCGβCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross‐linkers [1‐ethyl‐3(3‐dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGβR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGβCTP peptide irrespective of the carrier used, how it was cross‐linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self‐protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGβR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGβCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGβ‐positive cancer patients.     

Interleukin-2 production in whole blood cell cultures of women undergoing controlled ovarian hyperstimulation for assisted reproduction technology cycles

PROBLEM: To determine whether human chorionic gonadotropin (hCG) modulates the in vitro release of interleukin (IL-2) from human peripheral lymphocytes and monocytes derived from patients undergoing controlled ovarian hyperstimulation (COH). METHOD OF STUDY: A large university-based IVF unit was used for the study. Blood was drawn thrice from 12 women undergoing our routine IVF long gonadotropin-releasing-hormone-analog protocol during the COH cycle: (1) day on which adequate suppression was obtained (Day-S); (2) day of or prior to hCG administration (Day-hCG); and (3) day of ovum pick-up (Day-OPU). At each point of time, blood was tested for sex-steroid levels and then cultured for 72 hr either without (control-culture) or with hCG (hCG-culture) or with mitogenic stimulation by phytohemagglutinin (PHA-culture). The culture-medium supernatants were tested for IL-2 levels with a commercial sandwich enzyme-linked immunoassay. RESULTS: Whole blood culture IL-2 levels increased significantly during COH until peak E2, and then decreased significantly after hCG administration. IL-2 levels were decreased in the control- and PHA-culture media on Day-OPU compared with Day-hCG. There were no significant correlations between IL-2 levels in the culture media and serum estradiol, progesterone or human chorionic gonadotropin levels. CONCLUSION: Apparently, hCG attenuates IL-2 production by mononuclear cells with and without mitogenic stimulation, irrespective of the estradiol level. This suggests that hCG may indirectly modulate the inflammatory response, resulting in the ovarian hyperstimulation syndrome.     

Clinical comparison of immunoradiometric assays for intact versus beta-specific human chorionic gonadotropin.

Monoclonal immunoradiometric assays (IRMA) for human chorionic gonadotropin (hCG) are available for either intact hCG (IhCG) or beta subunit hCG (beta hCG). The authors evaluated the clinical applications of both methods. Serum samples (N = 180) were divided into the following five clinical groups: Group 1: elevated luteinizing hormone (LH), follicular stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), which share alpha subunits with hCG; Group 2: pregnancy; Group 3: trophoblastic tumors; Group 4: malignancy; and Group 5: positive rheumatoid factor or anti-nuclear antibody (ANA). The values of beta hCG versus IhCG for Group 1 showed statistical significance but no clinical significance, indicating negligible cross-reactivity in the alpha subunit group. beta hCG values, although exceeding the IhCG values, correlated well (r = 0.97) in intrauterine pregnancies; however, these values were not believed to be clinically significant. There is negligible interference by alpha subunits, elevated rheumatoid factor, ANA, or malignancy in the determination of IhCG versus beta hCG. The authors conclude that either the IhCG or beta hCG assays may be used in clinical conditions in which a potential exists for interference of alpha subunits, autoantibodies, or heterophile antibodies or in malignancy.     

Urinary hCG patterns during the week following implantation

BACKGROUND: Human chorionic gonadotrophin (hCG) is used to monitor pregnancy status. Yet the pattern of hCG excretion in the first week following implantation has not been adequately described.Therefore the aim of this study was to describe the average profile of hCG and its variability during the 7 days following estimated implantation in a population of naturally conceived pregnancies. METHODS: We measured daily hCG concentrations in first-morning urine for 142 clinical pregnancies from women with no known fertility problems. Mixed-effects regression models were used to estimate the hCG trajectory and its variability in relation to pregnancy outcomes. RESULTS: hCG rose 3-fold between the day of detection and the next day (95% CI = 2.7-3.4). The relative rate of rise decreased thereafter, reaching 1.6-fold (95% CI = 1.5-1.8) between days 6 and 7. HCG levels followed a log-quadratic trajectory, and the patterns of rise were unrelated to number of fetuses, risk of spontaneous abortion or sex of the baby. Later implantations (after 10 luteal days) produced slower rates of increase. CONCLUSIONS: Although mean hCG follows a log-quadratic trajectory during the first week of detectability, there is high variability across pregnancies. Later implantation may reflect characteristics of the uterus or conceptus that slow hCG production.     

Positive pregnancy tests in a nongravid, premenopausal woman due to hCG beta-chain production by multiple myeloma

Positive pregnancy test results occurred in a nongravid, premenopausal woman while she was receiving chemotherapy for multiple myeloma. We tested 2 hypotheses to account for this finding: (1) Heterophil antibodies caused positive interference in the immunoassays. (2) Genuine human chorionic gonadotropin (hCG) originated from a nonsyncytiotrophoblastic source. Paraprotein was eliminated as a source of positive interference because 3 different instruments with unique capture and signal antibodies gave similar results (83, 90, and 97 mIU/mL [83, 90, and 97 IU/L]). Human antimouse antibodies (HAMAs) were unlikely to cause positive interference because immunoreactivity was maintained after serum was treated to neutralize heterophil antibodies. Immunoassays performed after gel filtration of serum indicated that immunoreactivity was due to genuine hCG. The high-molecular-weight fraction (heterophil antibody) had 6 mIU/mL (6 IU/L) of hCG. The low-molecular-weight fraction (hCG) had 86 mIU/mL (86 IU/L) of hCG. Immunohistochemical stains revealed that myeloma cells expressed immunoreactive hCG. Hence, multiple myeloma caused positive pregnancy test results in a nongravid woman.     

Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies

A two-site sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Monoclonal anti-beta-hCG antibody was used for coating microtitration plates and monoclonal anti-alpha-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or beta-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r = 0.968) with the values obtained by radioimmunoassay.     

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-α) and beta (hCG-β) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-α) but not the combined (hCG) α subunit to hCG-α T cell hybridomas, while hCG-α-primed lymph node cells (LNC) responded to both hCG-α and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-α-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and FcγR, in the processing and presentation of hCG and hCG-a to HAG 5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-α. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-α and hCG, and present in hCG-α-primed mice, were extremely effective in presenting the free as well as the combined a subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the FcγRII-B2 gene to present immune complexes of either hCG-α or hCG. We found that hCG-α and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its a subunit. Using HBG 6, an hCG-β Tcell hybridoma, we performed similar experiments with the FcγRII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of α T cell determinants in the αβ dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of α and β T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.     

Preparation of europium-streptavidin in a time-resolved fluoroimmunoassay for interleukin-3

The preparation and immunoassay performance of europium-labeled streptavidin is described. The Eu-streptavidin conjugate can serve as a general detection reagent in time-resolved fluoroimmunoassays (TRFIA). The usefulness of such a strategy has previously been demonstrated with the Eu3+ chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (Diamandis et al. 1988). In this report the conjugation of streptavidin was accomplished with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)diethylenetriamine-N1,N3,N3(+)-tetraace tic acid, using a 50 M excess of the label. The conjugation ratio of Eu3+/streptavidin was 16. The use of the Eu-streptavidin reagent in a two-site immunometric assay to measure human recombinant interleukin-3 in human plasma, showed that the useful range of the assay was 20-25,000 pg/ml.     

Receptor specific probes for the study of Fc gamma receptor specific function.

Human leukocytes express three distinct families of receptors for the Fc region of IgG (Fc gamma R). We have prepared erythrocytes (E) coated with monoclonal anti-Fc gamma R antibodies for the study of receptor specific phagocytosis using biotin and streptavidin. In this technique, both the E and the monoclonal antibody (mAb) are biotinylated and coupling of the mAb to the E occurs through the use of streptavidin. The same biotin/streptavidin principle was used to prepare E coated with human IgG. Using this technique, receptor specific probes or probes coated with natural ligand (IgG) can be prepared rapidly with the use of small amounts of mAb or IgG. Finally, we have used these receptor specific probes to demonstrate that all three families of Fc gamma R (Fc gamma RI, Fc gamma RII and Fc gamma RIII) expressed on human monocytes and human macrophages are phagocytic receptors.     

Thiol-Click Based Polyglycerol Hydrogels as Biosensing Platform with In Situ Encapsulated Streptavidin Probes

An in situ streptavidin-encapsulated hydrogel based on dendritic polyglycerol (dPG) which is functionalized with either an acrylate, allyl or acrylamide group and dithiolated polyethylene glycol (PEG) is constructed via a thiol-click chemistry approach and is investigated for biosensing applications. The hydrogel platform is screened for the encapsulation and release efficiencies of the model protein streptavidin under varying physicochemical conditions, for example, crosslinking chemistry reactions, the molar ratio between the two gel components, macromonomer concentrations or pH-values. By that, tailor-made hydrogels can be developed, which are able to encapsulate or release the model protein for several days based on its modality. Furthermore, the accessible binding site of encapsulated streptavidin or in other words, the biotin-binding performance is quantified, and the stability of the various hydrogel types is studied by rheology measurements, ¹H NMR, gel permeation chromatography (GPC), and mass loss experiments.     

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.     

Prolongation of follicular phase by delaying hCG administration results in a higher incidence of endometrial advancement on the day of oocyte retrieval in GnRH antagonist cycles

BACKGROUND: Prolongation of follicular phase by delaying hCG administration has been reported to result in a significantly lower ongoing pregnancy rate that did not seem to be due to an embryonic factor. The aim of this prospective randomized study was to assess the effect of delaying hCG administration on endometrial histology. METHODS: Ten oocyte donors underwent endometrial biopsy on the day of oocyte retrieval and endometrial histology was assessed by Noyes' criteria. Ovarian stimulation was performed with recombinant (r)FSH and daily GnRH antagonist starting on day 6 of stimulation. Patients were randomized by a computer-generated list to receive 10 000 IU of hCG either as soon as > or =3 follicles > or =17 mm were present on ultrasound (early-hCG group, n = 5) or 2 days after this criterion was met (late-hCG group, n = 5). RESULTS: When hCG was delayed, endometrial advancement was present in all samples examined (median advancement 3 days, range 2-3 days). On the contrary, no secretory changes were observed when the follicular phase was not prolonged (difference in the proportion of patients with advancement between the early-hCG and the late-hCG group: 100%, 95% CI: 38-100). CONCLUSIONS: Prolongation of follicular phase by delaying hCG administration results in a higher incidence of endometrial advancement on the day of oocyte retrieval in GnRH antagonist cycles.     

Application of immunomagnetic particles to enzyme-linked immunosorbent assay (ELISA) for improvement of detection sensitivity of HCG

This investigation was aimed at using superparamagnetic particles to enzyme-linked immunosorbent assay (SPIO-ELISA) of human chorionic gonadotropin (hCG) to enhance detection sensitivity of hCG. We found that N-(3-dimethyl aminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was the best cross-linking reagent to link anti hCG α antibody to superparamagnetic particle (SPIO-anti hCG α antibody immunomagnetic particle). To improve the specificity of the assay, a horse radish peroxidase (HRP)-labeled anti-hCG beta monoclonal antibody was used to detect captured hCG using double antibody sandwich ELISA assay. SPIO-ELISA application to determine hCG increased the sensitivity to 1 mIU/mL, which is a level of sensitivity enabling the diagnosis of pregnancy during the early gestational period.     

Improved ELISA for the detection of adenovirus antigen in faeces extracts by the biotin/streptavidin interaction

Two enzyme-linked immunosorbent assays for detection of adenovirus antigen in faecal extracts have been established. A conjugate of rabbit anti-(human) adenovirus immunoglobulin and horseradish peroxidase (HRP) prepared by means of a hetero-bifunctional reagent, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), was used in the conventional ELISA, and a biotin-labelled antibody in combination with a streptavidin/peroxidase complex in the other (BS-ELISA). A collection of 60 faecal extracts, in which adenovirus had been demonstrated by immunosorbent electron microscopy (ISEM) in 29 samples, was selected and examined by ELISA and BS-ELISA. The presence of adenovirus was demonstrated in 38 (63%) of the samples by BS-ELISA compared to 34 (58%) by ELISA, showing the advantage of the biotin/streptavidin system for setting up ELISA assays. Comparison of the three different techniques showed that the biotin/streptavidin (BS-ELISA) is the most sensitive assay for detection of adenovirus antigen in faecal samples.     

There is no role for uterine curettage in the contemporary diagnostic workup of women with a pregnancy of unknown location

BACKGROUND: The aim of this study was to generate and evaluate a new protocol that defined non-viability in the pregnancy of unknown location (PUL) population and therefore ensured no viable intra-uterine pregnancy (IUP) would be interrupted if uterine curettage was performed. A secondary aim was to evaluate published biochemical criteria that define non-viability in a PUL population to establish if these criteria could result in inadvertent termination of pregnancy (TOP) if uterine curettage was performed. METHODS: All clinically stable women classified as having a PUL were included in this study. Protocol 1 was developed retrospectively based on data from 500 consecutive PULs. Using this protocol, no cases of viable IUPs would undergo uterine curettage and the potential for TOP was eliminated. This protocol was then validated prospectively on the data from a further 503 consecutive PULs. Results were then compared with three established criteria (Protocols 2-4) for the use of uterine curettage as a diagnostic tool to classify the location of PULs. Protocol 2 defined non-viability when the hCG ratio (hCG at 48 h/hCG at 0 h) was or=2000 U/l or when the initial serum hCG was <2000 U/l with a serum hCG rise of <35% over 48 h (hCG ratio<1.35); Protocol 4 advised uterine curettage with a serum hCG rise of <50% over 48 h (hCG ratio<1.50). The number of uterine curettages performed and viable IUPs that would have undergone an unplanned TOP were recorded for all protocols. RESULTS: A total of 12 572 consecutive women were scanned: 1003 (8.0%) women were classified as PULs. Training set consisted of 500 PULs: 278 (55.6%) failing PULs, 176 (35.2%) IUPs and 46 (9.2%) ectopic pregnancies (EPs). Test set consisted of 503 PULs: 255 (50.7%) failing PULs, 203 (40.4%) IUPs and 45 (9.0%) EPs. Protocol 1 when developed retrospectively on the training set would have resulted in 293 uterine curettages and no potential TOP. Protocol 1 tested prospectively on 503 PULs would have resulted in 272 uterine curettages and no potential TOP. Three established criteria were tested on the entire data set (n=1003). Protocol 2 would have resulted in 114 uterine curettages and 14 (12.3%) potential TOPs; Protocol 3 would have led to 611 uterine curettages and seven (1.2%) potential TOPs; Protocol 4 would have resulted in 617 uterine curettages and three (0.5%) potential TOPs. No harm came to the women whose EP diagnosis was delayed. CONCLUSIONS: Established criteria for the use of uterine curettage in the management of PULs, including those advocated by the American Society for Reproductive Medicine (ASRM), can theoretically result in an inadvertent TOPs. On the basis of these data, a change in contemporary clinical practice should be considered to avoid further damage to wanted pregnancies. We conclude that uterine curettage should not be used in the routine diagnostic workup of women with a PUL.