RESUMO
OBJECTIVE: Human Chorionic Gonadotropin (hCG) plays a crucial role in embryo implantation and in maintenance of pregnancy. An immuno-contraceptive approach involves the use of a recombinant hCGß-LTB vaccine formulated with adjuvant Mycobacterium indicus pranii (MIP), to prevent pregnancy without disturbing ovulation, hormonal profiles, and menstrual cycles in women. The present work in mice was designed to address issues encountered in clinical trials conducted with hCGß-LTB vaccine, with focus on two primary concerns. Firstly, it aimed to determine the optimal vaccine dosage required to induce a high level of anti-hCG antibodies. Secondly, it aimed to assess the safety profile of the vaccine, specifically injection site reactions in the form of nodules, observed in some of the subjects. METHODS AND RESULTS: Studies undertaken indicate that a 2 µg dose of the protein version of the vaccine, administered in mice through the intramuscular route, can induce high anti-hCG titres. Furthermore, administering a booster dose enhances the antibody response. Our findings suggest that the concentration and frequency of administration of the adjuvant MIP can also be reduced without compromising vaccine efficacy. CONCLUSION: The issue of nodule formation at the injection site can be mitigated either by administering the vaccine along with MIP intramuscularly or injecting hCG vaccine and MIP at separate intradermal sites. Thus, protein vaccine administered at a 2µg dose via the intramuscular route addresses both efficacy and safety concerns.
The Phase I/II clinical trials initiated with the recombinant hCG vaccine in women revealed inadequate antibody titres in all subjects, alongside the development of nodules at the injection sites in some participants. Studies were undertaken in mice to propose potential strategies for mitigating injection site reactions and enhancing the antibody response. It was concluded that the optimum dose of the protein version of the vaccine to get high antibody titres, is 2 µg administered intramuscularly while upholding safety standards.
Assuntos
Gonadotropina Coriônica , Vacinas Sintéticas , Animais , Feminino , Camundongos , Vacinas Sintéticas/imunologia , Gonadotropina Coriônica/imunologia , Anticoncepção Imunológica/métodos , Vacinas Anticoncepcionais/imunologia , Formação de Anticorpos/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Adjuvantes Imunológicos , Reação no Local da Injeção , Engenharia Genética , Injeções Intramusculares , Gonadotropina Coriônica Humana Subunidade beta/imunologiaRESUMO
Human chorionic gonadotropin (HCG), a vital protein for pregnancy determination and a marker for trophoblastic diseases, finds application in monitoring early pregnancy and ectopic pregnancy. This study presents an innovative approach employing electrochemical immunosensors for enhanced HCG detection, utilizing Anti-HCG antibodies and gold nanoparticles (AuNPs) in the sensor platform. Two sensor configurations were optimized: BSA/Anti-HCG/c-AuNPs/MEL/e-AuNPs/SPCE with [Fe(CN)6]3-/4- as a redox probe (1) and BSA/Anti-HCG/PPy/e-AuNPs/SPCE using polypyrrole (PPy) as a redox probe (2). The first sensor offers linear correlation in the 0.10-500.00 pgâmL-1 HCG range, with a limit of detection (LOD) of 0.06 pgâmL-1, sensitivity of 32.25 µAâpg-1âmLâcm-2, RSD <2.47 %, and a recovery rate of 101.03-104.81 %. The second sensor widens the HCG detection range (40.00 fgâmL-1-5.00 pgâmL-1) with a LOD of 16.53 fgâmL-1, ensuring precision (RSD <1.04 %) and a recovery range of 94.61-106.07 % in serum samples. These electrochemical immunosensors have transformative potential in biomarker detection, offering enhanced sensitivity, selectivity, and stability for advanced healthcare diagnostics.
Assuntos
Técnicas Biossensoriais , Gonadotropina Coriônica , Técnicas Eletroquímicas , Ouro , Limite de Detecção , Nanopartículas Metálicas , Polímeros , Pirróis , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/análise , Gonadotropina Coriônica/imunologia , Ouro/química , Humanos , Nanopartículas Metálicas/química , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Polímeros/química , Pirróis/química , Imunoensaio/métodos , Imunoensaio/instrumentação , Ferricianetos/química , FemininoRESUMO
Immunochromatographic assay (ICA) is widely applied in various fields. However, severe matrix interference and weak signal output present major challenges in achieving accurate and ultrasensitive detection in ICA. Here, a polydopamine (PDA)-mediated magnetic bimetallic nanozyme (Fe3O4@PDA@Pd/Pt) with peroxidase-like activity was synthesized and used as a probe in ICA. The magnetic property of Fe3O4@PDA@Pd/Pt enabled effective magnetic enrichment of targets, thereby reducing the matrix interference in the sample. PDA coating on the magnetic bimetallic nanozyme was employed as a mediator and a stabilizer. It improved the catalytic ability and stability of the magnetic bimetallic nanozyme by providing more coordination sites for Pd/Pt growth and functional groups (-NH and -OH). In addition, the Pd/Pt bimetallic synergistic effect could further enhance the catalytic ability of the nanozyme. A method was developed by integrating Fe3O4, PDA, and Pd/Pt into Fe3O4@PDA@Pd/Pt as a probe in ICA. With the proposed method, human chorionic gonadotropin and Escherichia coli O157:H7 were successfully detected to be as low as 0.0094 mIU/mL in human blood serum and 9 × 101 CFU/mL in the milk sample, respectively. This method may be readily adapted for accurate and ultrasensitive detection of other biomolecules in various fields.
Assuntos
Gonadotropina Coriônica/sangue , Escherichia coli O157/isolamento & purificação , Contaminação de Alimentos/análise , Imunoensaio/métodos , Indóis/química , Nanopartículas de Magnetita/química , Polímeros/química , Animais , Anticorpos Monoclonais/imunologia , Benzidinas/química , Catálise , Gonadotropina Coriônica/imunologia , Compostos Cromogênicos/química , Escherichia coli O157/imunologia , Limite de Detecção , Leite/microbiologia , Oxirredução , Paládio/química , Platina/químicaRESUMO
PROBLEM: To evaluate pregnancy-compatible phenotypic and functional changes in peripheral blood natural killer (pNK) cells during frozen embryo transfer (FET) cycles. METHOD OF STUDY: Peripheral blood was collected from patients undergoing frozen embryo transfer cycles at three separate time points in the cycle. pNK cell phenotype was analyzed by flow cytometry. Impact of pregnancy status on pNK cell cytotoxicity was characterized by two methods: (1) a three-dimensional endovascular tube formation approach and (2) a NK cell-specific K562 cell kill assay. RESULTS: A total of 35 patients were enrolled, 15 with clinical pregnancies and 20 with negative serum ß-hCG levels. Overall percentage of CD45+ CD3- CD56+ pNK cell did not change during the FET cycle. Pregnancy resulted in an increase in CD45+ CD3- CD56+ pNK cell population on the day of serum ß-hCG. pNK cells from non-pregnant patients caused significant tube disruption when compared to pregnant patients. Addition of serum from pregnant women reduced the tube disruption by pNK cells from non-pregnant patients. pNK cells from pregnant patients showed significantly lower cytotoxicity toward K562 cells in serum-free conditions. The addition of pregnancy serum decreased non-pregnant pNK cell cytotoxicity. Pregnancy status had no impact on VEGF-A and VEGF-C serum levels. Recombinant hCG added to non-pregnant serum resulted in a significant reduction in non-pregnant pNK cell-mediated K562 cell kill. CONCLUSION: There was no difference in pNK cell populations based on timing of the FET cycle. However, pregnancy increased the percentage of CD45+ CD3- CD56+ pNK cells. Additionally, pNK cells from pregnant women have reduced cytotoxicity and this is possibly mediated by hCG.
Assuntos
Gonadotropina Coriônica/imunologia , Transferência Embrionária , Células Matadoras Naturais/imunologia , Gravidez/imunologia , Adulto , Antígenos CD/imunologia , Linhagem Celular , Sobrevivência Celular , Gonadotropina Coriônica/sangue , Criopreservação , Embrião de Mamíferos , Feminino , Humanos , Fenótipo , Gravidez/sangueRESUMO
Women were studied undergoing ICSI for 84 who suffer non-pregnancy at the Fertility Center, Al-Sadr Medical Hospital in Najaf Governorate, Period between January 2019 and March 2020. WBC, Vitamin D3 and ß-hCG were measured, The pregnant women was divided into (Pregnancy Group, and spontaneous miscarriage) and then demonstrate the immunological effect on pregnancy of women after ICSI technique. Current resultsstudy showed a significant increase (p<0.05) in hormone level ß-hCG is evidence of the presence of high success rates for pregnancy in women who performed operations IVF, where the success rate at the beginning of the matter reached 61.9%, after which it decreased to 33.3% after the first three months due to the occurrence of spontaneous miscarriage of pregnant women due to various immunological and physiological reasons, a positive correlation between the level of ß-hCG and other parameters in the study (Vitamin D3 -WBC).Also The current resultsshowed a significant decrease in a groups (pregnancy failure) and the group (spontaneous miscarriage) compared with the control group (continued pregnancy) in relation to the level of vitamin D3 Also, The current results showed a significant increasein (pregnancy failure) and (spontaneous miscarriage) compared with control groups (continuation of pregnancy) in relation WBC numbers, and the present study founds a negative relationship between the level of vitamin D3 and WBC.
Assuntos
Humanos , Feminino , Gravidez/imunologia , Aborto Espontâneo/imunologia , Colecalciferol/deficiência , Injeções de Esperma Intracitoplásmicas/métodos , Gonadotropina Coriônica/imunologia , Leucócitos/imunologiaRESUMO
A composite, reduced graphene oxide (rGO) doped with silver nanoparticles (Ag NPs), was prepared by using binary reductants of sodium citrate and hydrazine hydrate. Carbon quantum dots (CQDs) synthesized by papaya peel combined with silver ions to form a CQDs-loaded silver nanoparticle (AgCQDs) nanocomposite. Polymer nanospheres (PNS) were generated via the infinite coordination polymer of ferrocene dicarboxylic acid and employed as carriers to load AgCQDs. The prepared AgCQDs@PNS-PEI has good biocompatibility and electrical conductivity and can be used as a matrix for the immobilization of a secondary antibody (Ab2). A sandwich-type electrochemiluminescence (ECL) immunosensor using AgCQDs@PNS-PEI nanocomposite as probe has been developed for the detection of human chorionic gonadotropin (HCG). The proposed immunosensor exhibits a linear range from 0.00100 to 500 mIU mL-1 and the detection limit is 0.33 µIU mL-1 (S/N = 3) under optimal conditions. The sensor exhibits excellent selectivity, good reproducibility, and high stability. These features demonstrate that the proposed method has promising potential for clinical protein detection and displays a new strategy to fabricate an immunosensor. Graphical abstract.
Assuntos
Gonadotropina Coriônica/sangue , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Medições Luminescentes/métodos , Nanosferas/química , Pontos Quânticos/química , Adulto , Anticorpos Imobilizados/imunologia , Carbono/química , Carica/química , Gonadotropina Coriônica/imunologia , Feminino , Frutas/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Nanocompostos/química , Polietilenoimina/química , Reprodutibilidade dos Testes , Prata/químicaRESUMO
We aimed to investigate the effect of intrauterine administration of autologous hCG-activated PBMCs in RIF women with low Th-17/Treg cell ratio. 248 women with a history of implantation failure volunteered to receive PBMC-therapy. After immunologic consultation and doing flow cytometry analysis, 100 women with at least three IVF/ET failure who had low Th-17/Treg ratio in comparison with healthy control were enrolled in this study. These 100 patients were randomly divided into two groups as PBMC receiving (n = 50) and controls (n = 50). Then PBMCs were obtained from patients and treated with hCG for 48 h. Afterward, PBMCs were administered into the uterine cavity of the patient in the study group, two days before ET. The concentration of inflammatory cytokines was examined in the supernatant of cultured PBMCs after 2, 24, and 48 h of incubation using the ELISA method. The frequency of Th-17, Treg, and the Th-17/Treg ratio was significantly lower in RIF women than the healthy controls (P < 0.0001). The secretion of inflammatory cytokines was significantly higher after 48 h compared to 2 and 24 h (P < 0.0001). The pregnancy and live birth rate were significantly increased in women undergoing the PBMC-therapy compared to control (PBS-injecting) group (P = 0.032 and P = 0.047, respectively). The miscarriage rate was considerably lower in PBMC-therapy group (P = 0.029). Our findings suggest that intrauterine administration of autologous in vitro hCG-activated PBMCs improves pregnancy outcomes in patients with at least three IVF/ET failures.
Assuntos
Transfusão de Sangue Intrauterina/métodos , Gonadotropina Coriônica/imunologia , Transferência Embrionária/métodos , Infertilidade Feminina/terapia , Leucócitos Mononucleares/transplante , Aborto Espontâneo/imunologia , Aborto Espontâneo/prevenção & controle , Adulto , Coeficiente de Natalidade , Transfusão de Sangue Autóloga/métodos , Método Duplo-Cego , Implantação do Embrião/imunologia , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Idade Materna , Gravidez , Taxa de Gravidez , Resultado do Tratamento , Adulto JovemRESUMO
Human chorionic gonadotrophin (hCG) is the first specific molecule synthesized by the embryo. hCG RNA is transcribed as early as the eight-cell stage, and the blastocyst produces the protein before its implantation. hCG in the uterine microenvironment binds with its cognate receptor, luteinizing hormone/choriogonadotropin receptor (LHCGR), on the endometrial surface. This binding stimulates leukemia inhibitory factor (LIF) production and inhibits interleukin-6 (IL-6) production by epithelial cells of the endometrium. These effects ensure essential help in the preparation of the endometrium for initial embryo implantation. hCG also effects angiogenic and immunomodulatory actions as reported in many articles by our laboratories and other ones. By stimulating angiogenesis and vasculogenesis, hCG provides the placenta with an adequate maternal blood supply and optimal embryo nutrition during the invasion of the uterine endometrium. The immunomodulatory properties of hCG are numerous and important for programming maternal immune tolerance toward the embryo. The reported effects of hCG on uterine NK, Treg, and B cells, three major cell populations for the maintenance of pregnancy, demonstrate the role of this embryonic signal as a crucial immune regulator in the course of pregnancy. Human embryo rejection for hCG-related immunological reasons has been studied in different ways, and a sufficient dose of hCG seems to be necessary to maintain maternal tolerance. Different teams have studied the addition of hCG in patients suffering from recurrent miscarriages or implantation failures. hCG could also have a beneficial or a negative impact on autoimmune diseases during pregnancy. In this review, we will discuss the immunological impacts of hCG during pregnancy and if this hormone might be used therapeutically.
Assuntos
Gonadotropina Coriônica/fisiologia , Aborto Espontâneo/etiologia , Animais , Doenças Autoimunes/etiologia , Autoimunidade , Gonadotropina Coriônica/química , Gonadotropina Coriônica/imunologia , Transferência Embrionária , Feminino , Humanos , Imunomodulação , Células Matadoras Naturais/imunologia , Gravidez/imunologia , Isoformas de ProteínasRESUMO
The process of implantation, trophoblast invasion and placentation demand continuous adaptation and modifications between the trophoblast (embryonic) and the decidua (maternal). Within the decidua, the maternal immune system undergoes continued changes, as the pregnancy progress, in terms of the cell population, phenotype and production of immune factors, cytokines and chemokines. Human chorionic gonadotropin (hCG) is one of the earliest hormones produced by the blastocyst and has potent immune modulatory effects, especially in relation to T cells. We hypothesized that trophoblast-derived hCG modulates the immune population present at the maternal fetal interface by modifying the cytokine profile produced by the stromal/decidual cells. Using in vitro models from decidual samples we demonstrate that hCG inhibits CXCL10 expression by inducing H3K27me3 histone methylation, which binds to Region 4 of the CXCL10 promoter, thereby suppressing its expression. hCG-induced histone methylation is mediated through EZH2, a functional member of the PRC2 complex. Regulation of CXCL10 expression has a major impact on the capacity of endometrial stromal cells to recruit CD8 cells. We demonstrate the existence of a cross talk between the placenta (hCG) and the decidua (CXCL10) in the control of immune cell recruitment. Alterations in this immune regulatory function, such as during infection, will have detrimental effects on the success of the pregnancy.
Assuntos
Quimiocina CXCL10/genética , Gonadotropina Coriônica/imunologia , Decídua/imunologia , Epigênese Genética , Histonas/imunologia , Adulto , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Quimiocina CXCL10/imunologia , Decídua/citologia , Decídua/metabolismo , Feminino , Humanos , Imunomodulação , Metilação , Placentação , Gravidez , Adulto JovemRESUMO
Immunochromatographic assays (ICAs) are mainstream point-of-care diagnostic tools in disease control, food safety, and environmental monitoring. However, the important issue pertaining to the influence of sample addition methods on the detection performance of ICAs has not been addressed, and related information is still lacking. Herein, we selected the well-accepted gold nanoparticles (AuNPs) as visual labels. AuNP-based ICA was then used to explore the effects of three sample addition methods (i.e., dry, wet, and insert) on the analytical performance of ICAs by using competitive and sandwich models. Under optimized conditions, the competitive ICA with clenbuterol as an analyte showed a negligible difference (pâ¯>â¯0.05) in the detection performance of the three methods in ideal phosphate buffered saline solution. However, the wet method demonstrated the worst performance in pork samples (pâ¯<â¯0.05). The sandwich ICA strip with human chorionic gonadotropin as an analyte revealed the significantly different analytical performances of the three approaches in phosphate buffer (PB) solution and spiked serum (pâ¯<â¯0.05). Two independent linear correlations were observed with the increase in target concentration. However, for the wet method in the PB solution and serum, the first linear correlation was at a relatively narrow target concentration range, and the second linear correlation was at a wider concentration range compared with those for the dry and insert methods. Our findings demonstrated that sample addition methods slightly influence competitive ICAs (pâ¯>â¯0.05) but remarkably affect sandwich ICAs (pâ¯<â¯0.05). We believe that this study can further explain the differences in detection results for the same target analyte in actual ICA detection. The results may serve as a reference in the rational selection of the appropriate sample addition method for succeeding ICA works.
Assuntos
Coloide de Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Animais , Anticorpos Monoclonais/imunologia , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/imunologia , Clembuterol/sangue , Clembuterol/imunologia , Contaminação de Alimentos/análise , Humanos , Limite de Detecção , Carne de Porco/análise , SuínosRESUMO
A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH. The sampling concept showed a high degree of performance between 10 and 1000â¯ng/mL (R2â¯>â¯0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100â¯pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.
Assuntos
Gonadotropina Coriônica/sangue , Teste em Amostras de Sangue Seco/métodos , Papel , Sequência de Aminoácidos , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Biomarcadores/química , Gonadotropina Coriônica/química , Gonadotropina Coriônica/imunologia , Cromatografia Líquida , Teste em Amostras de Sangue Seco/instrumentação , Humanos , Limite de Detecção , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteólise , Reprodutibilidade dos Testes , Tripsina/químicaRESUMO
A colorimetric immunoassay is described for the pregnancy marker human chorionic gonadotropin (HCG). The assay is based on the use of MnO2 nanorods acting as a peroxidase mimic. The nanorods were prepared by a hydrothermal method using EDTA as a template. They exhibit excellent peroxidase-like activity and stability. The nanorods were immobilized in a chitosan matrix in the wells of a BSA-modified microplate which then were further modified with streptavidin and biotinylated capture antibodies. The specific recognition between HCG and the antibodies in wells inhibit the peroxidase-like activity of the nanorods. Hence, the substrate 3,3',5,5'-tetramethylbenzidine is less efficiently catalytically oxidized by H2O2 to form a blue colored product. This results in a decrease in the absorbance at 652 nm. Response is linear in the 0.5 to 400 mIU·mL-1 HCG concentration range, and the detection limit is 0.36 mIU·mL-1 under optimized conditions. The method is highly specific, acceptably reproducible and stable. It was applied to the determination of HCG concentration in serum samples, and results were in good agreement with data obtained by the reference method." Graphical abstract Schematic presentation of colorimetric immunoassay for human chorionic gonadotropin (HCG) by using MnO2 nanorods with peroxidase-like activity immobilized in microplate wells. The specific recognition between HCG and the antibodies in wells inhibits the peroxidase-like activity of the nanorods. TMB: 3,3',5,5'-tetramethylbenzidine.
Assuntos
Materiais Biocompatíveis/metabolismo , Gonadotropina Coriônica/sangue , Imunoensaio/métodos , Compostos de Manganês/química , Nanotubos/química , Óxidos/química , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Materiais Biocompatíveis/química , Gonadotropina Coriônica/imunologia , Colorimetria , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Peroxidase/metabolismo , Reprodutibilidade dos TestesRESUMO
Background Heterophile antibody (HAb) interferences in immunoassays can cause falsely elevated hCG concentrations leading to incorrect diagnosis and treatments options. When results are not consistent with the clinical findings, hCG HAb interference investigation may be requested by the physician. A retrospective evaluation of the frequency of HAb interference was performed among cases of physician-requested investigations and the effectiveness of commercially available blocking reagents to detect HAb interference in two immunoassay systems was evaluated. Methods One hundred and thirteen physician requests for hCG HAb investigation from 2008 to 2017 were reviewed. The primary method used to measure hCG was the Beckman Coulter Access Total ßhCG (2008-2010) and the Roche Elecsys HCG+ß (2014-2017). HAb investigation included measurement by two immunoassays before and after treatment of samples with heterophile blocking reagents and serial dilution studies. Results Five cases of HAb and HAb-like interference were identified. The interference frequency was 6.7% for the Beckman assay and 2.9% for the Roche assay. The presence of HAb was detected using heterophile blocking reagents and an alternative method in three cases. The other two cases were detected due to discrepant results with an alternative method and non-linear serial dilutions (HAb-like). Conclusions HAb interference was observed in the Beckman and the Roche assays. The heterophile blocking reagents failed to detect 40% of interference cases. Blocking reagents should not solely be used for these investigations. Multiple strategies including the use of serial dilutions and using an alternative platform are critical when troubleshooting interferences in hCG immunoassays.
Assuntos
Anticorpos Heterófilos/análise , Gonadotropina Coriônica/análise , Ensaio de Imunoadsorção Enzimática , Anticorpos Heterófilos/imunologia , Gonadotropina Coriônica/imunologia , HumanosRESUMO
A novel sampling concept for mass spectrometric bottom-up targeted protein analysis is here demonstrated with polymeric sampling spots integrated with instant immunocapture for analysis of dried matrix spots. The polymers 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) and pHEMA-Tosyl for covalent attachment of antibodies where investigated alongside with adsorption on non-treated filter paper. From performance characterization, the pHEMA-VDM had the best performance. The sampling spots demonstrated fast and easy sampling and preparation of human serum spiked with the biomarker human chorionic gonadotropin. The sampling spots enabled a detection limit of 1â¯ng/mL (26.4â¯pM) within a five point concentration curve from 1â¯ng/mL to 20â¯ng/mL (R2 =â¯0.97). The detection limit was demonstrated to be two times lower than previously demonstrated with standard DMPK-C sampling cards. A five point concentration curve from 100â¯ng/mL to 2000â¯ng/mL was also investigated (R2 =â¯0.998). Intra day precision was within 16% and 23% for concentration range 1â¯ng/mL to 20â¯ng/mL and 100â¯ng/mL to 2000â¯ng/mL, respectively. Inter day precision was within 20%. Accuracy was determined to 10% and 11% for 2.5â¯ng/mL and 20â¯ng/mL, respectively. The sampling spots were also demonstrated in a realistic setting where serum samples from two confirmed patients with testicular germ cell cancer were analyzed. These analyses confirmed an elevated hCG content in the sera of 418.5⯱â¯4.2â¯ng/mL and 21⯱â¯0.02â¯ng/mL hCG for patient one and two respectively.
Assuntos
Gonadotropina Coriônica/sangue , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Gonadotropina Coriônica/imunologia , Cromatografia Líquida , Humanos , Imunoensaio/métodos , Lactonas/química , Limite de Detecção , Masculino , Espectrometria de Massas , Neoplasias Embrionárias de Células Germinativas/sangue , Papel , Poli-Hidroxietil Metacrilato/química , Polivinil/química , Neoplasias Testiculares/sangueRESUMO
Intrauterine infusion of human chorionic gonadotropin (IUI-hCG) has been proposed to improve the outcome of in vitro fertilization-embryo transfer (IVF-ET), since it plays a critical role in synchronizing endometrial and fetal development. As the early mediator from embryo, hCG promotes the decidualization, angiogenesis, maternal immune tolerance, and trophoblast invasion, favoring successful implantation of embryo. Although multiple clinical trials have been conducted to verify the efficacy of IUI-hCG on IVF-ET outcome in recent years, the findings remained controversial. The difference in study design and population might be the cause to the different consequences after administration of hCG. More importantly, the endometrial receptivity, which might affect the efficacy of IUI-hCG, has not been assessed in women receiving this intervention. Selecting the right population suitable for IUI-hCG based on known etiology would be crucial in enhancing its efficacy and minimize any possible complications. Investigation of optimal indications for IUI-hCG should be highlighted in the future.
Assuntos
Gonadotropina Coriônica/imunologia , Transferência Embrionária/métodos , Endométrio/fisiologia , Fertilização in vitro/métodos , Transfusão de Sangue Intrauterina , Gonadotropina Coriônica/administração & dosagem , Ensaios Clínicos como Assunto , Implantação do Embrião , Feminino , Humanos , Imunomodulação , Seleção de Pacientes , GravidezRESUMO
Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high-throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence-activated cell sorting (FACS) for the isolation of antigen-specific chicken-derived antibodies is demonstrated. To this end, yeast-displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target-specific scFv-Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG-binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns.
Assuntos
Gonadotropina Coriônica/imunologia , Citometria de Fluxo/métodos , Genes erbB-1/imunologia , Fragmentos de Imunoglobulinas/isolamento & purificação , Animais , Afinidade de Anticorpos , Antígenos/química , Antígenos/imunologia , Galinhas/imunologia , Gonadotropina Coriônica/genética , Epitopos/imunologia , Genes erbB-1/genética , Humanos , Imunização/métodos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Biblioteca de Peptídeos , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae/genéticaRESUMO
The Ultra Micro Analytical System (SUMA) is an ELISA-based analytical platform, developed andmanufactured by the Cuban Immunoassay Center (IC), which is primarily used in clinical medicine applications. In this article, we describe the validation of the UMELISA HCG kits, which are based on SUMA, as a pre-screening procedure for the detection of human Chorionic Gonadotrophin (hCG) in urine for anti-doping purposes. Validation of assay performance parameters showed satisfactory results, in accordance with the criteria established by the World Anti-Doping Agency (WADA): intra-assay repeatability (6.7-9.7%), inter-assay reproducibility (7.8-10.5%), accuracy (91-98%), limit of detection (2.7 IU/L), and linearity. Relative sensitivity and specificity and Predictive Positive and Negative Values were used to evaluate the Efficacy showing a value of 97.6%. A Kappa Index analysis was applied to check agreement with the commercially available, reference assay COBASe411 (Roche), which is often applied in WADA-accredited anti-doping laboratories for measurement of intact (heterodimeric) hCG in urine. UMELISA HCG kits are considered as fit for anti-doping control purposes.
Assuntos
Gonadotropina Coriônica/urina , Dopagem Esportivo/métodos , Dopagem Esportivo/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Detecção do Abuso de Substâncias/métodos , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Gonadotropina Coriônica/imunologia , HumanosRESUMO
OBJECTIVES: To compare immunohistochemical detection of Human chorionic gonadotropin (hCG) expression in paraffin embedded tissue of squamous cell carcinomas (SCC) and high grade squamous intraepithelial lesions (HSIL). METHODS: The samples in this retrospective study were obtained from the archives of the Pathology Department at Gaziantep University, Gaziantep, Turkey, over the period from January 2012 to September 2016. The study group consisted of 55 cases of SCC and 45 cases of HSIL. Tissue expression of hCG was detected by specific binding of anti-hCG antibody using an automated immunohistochemistry staining device. The categorical variables of intensity and coverage were analyzed statistically using Pearson Chi-Square test. RESULTS: High grade squamous cell lesions cases showed weak (84.4%, n=38/45) to no (15.6%, n=7/45) staining for hCG. None of the HSIL cases showed strong positivity. Strong positivity for hCG was detected in 90.9% (n=50/55) of SCC cases. CONCLUSION: Our study supports the association of ectopic hCG expression in cancer pathogenesis by demonstrating strong hCG immunoreactivity only in SCC cases. This finding can be helpful in supporting the diagnosis of invasive carcinoma in small or fragmented biopsies, which can on their own be confusing for the pathologists.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Gonadotropina Coriônica/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Biópsia , Carcinoma de Células Escamosas/patologia , Gonadotropina Coriônica/imunologia , Feminino , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Estudos Retrospectivos , Lesões Intraepiteliais Escamosas Cervicais/diagnóstico , Lesões Intraepiteliais Escamosas Cervicais/patologia , Turquia , Neoplasias do Colo do Útero/patologiaRESUMO
Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure. Then, changes in the production of MAb in culture medium were evaluated using ELISA. The hybridoma cells after single and combined treatment with ATRA, DHA and vehicles were IP injected to Balb/c mice and the changes in production of MAb in ascites were determined by ELISA. The results showed that single and combined treatment of ATRA and DHA elevated the production of MAb by hybridoma cells in both in vivo and in vitro. The production of MAb following in vitro single treatment with 1⯵M of ATRA and 10⯵M of DHA for 2â¯days was significantly increased. The in vitro effects of ATRA on increase of MAb production was obtained more than DHA. The MAb productions in combined treatment with 0.5â¯µΜ of ATRA plus 5â¯µΜ of DHA were significantly increased in in vivo and in vitro. However, the effect of DHA was obtained more significant in in vivo conditions. The results of this study showed for the first time that in vitro and in vivo treatments of ATRA and DHA could increase the production of MAb in mouse M3C5 hybridoma cells.
Assuntos
Anticorpos Monoclonais Murinos/biossíntese , Ácidos Docosa-Hexaenoicos/farmacologia , Imunoglobulina G/biossíntese , Tretinoína/farmacologia , Animais , Líquido Ascítico/metabolismo , Gonadotropina Coriônica/imunologia , Hibridomas , Masculino , Camundongos Endogâmicos BALB CRESUMO
We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.