RESUMO
Human chorionic gonadotropin (hCG) hormone is required for maintenance of early pregnancy and is a potential marker in the diagnosis and prognosis of both pregnancy and trophoblastic diseases. Murine hybridomas were generated against purified hCG. Seven hybrid clones secreting antibodies against hCG molecule with IgG1/kappa subclass were established. The indirect ELISA result demonstrated that six MAbs (BEL-1 to BEL-6) recognized hCG in both holo and free beta subunit form with negligible cross-reactivity against a closely related hormone, human luteinizing hormone (hLH). In this fusion, only one MAb (ALC-1) showed a cross-reaction with hLH, which designated an alpha subunit specific. The outcome of Western blot ascertained that ALC-1 recognized the conformational epitope on alpha subunit of hCG at Mr 23 kDa band in nonreducing condition (NR). In contrast, epitopes belonging to all MAbs recognized beta subunit of hCG were in linear peptide structure at Mr 34 kDa band (NR) and Mr 26 kDa band (R). These six MAbs were further characterized by using two beta subunit carboxy-terminal synthetic peptides (beta109-119 and beta109-145). Three of them (BEL-1, BEL-3, and BEL-4) recognized only epitope harboring in beta109-145 fragment, the others recognized both types of the synthetic peptide. In order to select the most suitable MAbs specific to beta subunit of hCG for exploiting with ALC-1 in the sandwich-type immunometric assay, competitive ELISA was employed. Six individual MAbs specific to beta subunit of hCG were used to compete with biotinylated ALC-1 to evaluate the proximity of their epitopes on the holo form of hCG molecule. Of all six MAbs, BEL-5 depicted the lowest percent inhibition result, which indicated the bottom-most steric hindrance effect. Consequently, MAb BEL-5 will be the most appropriate antibody to utilize in concert with ALC-1 in place of devising a sandwich-type immunometric assay for measuring holo-hCG level.
Assuntos
Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Ascite/imunologia , Biotinilação , Western Blotting/métodos , Gonadotropina Coriônica Humana Subunidade beta/genética , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/genética , Feminino , Subunidade alfa de Hormônios Glicoproteicos/genética , Humanos , Hormônio Luteinizante/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Homologia de SequênciaRESUMO
Double-particle reversed passive hemagglutination (RPHA) was performed for the detection of an intact heterodimeric form of human chorionic gonadotropin (intact hCG) composed in Profasi hCG (P-hCG). This technique relied on two mixed types of human O RBC, which were individually coated with two distinct monoclonal antibodies that recognized alpha or beta subunit of hCG, i.e. ALC-1 and BEL-5, respectively. The positive hemagglutination result was achieved by this technique. However, in the BEL-5 coated single-particle control system, positive results for both P-hCG and beta subunit hCG solution were realized. The occurrence of beta-multimer hCG was a causative molecule revealed by the hemagglutination inhibition technique. Thereby, the novel method called "combined immunoprecipitation and agglutination" was developed to overcome this problem. The free beta subunit together with the betamultimer hCG were eliminated from other forms presented in P-hCG after using the ALC-1 coated particles. The precipitated particles, which captured the heterodimer hCG molecule, reacted further with soluble BEL-5 to subsequently form a trellis. A positive result was obtained only with P-hCG, but not with beta subunit hCG or hLH. This study is inferable as a model for the detection of heterodimeric molecule by an elementary method.