Deciphering a Conformation-Specific Epitope of hCG-Through Immunokinetics

Proteins and peptides are comprised of both sequence-specific and conformation-specific epitopes. Sequence-specific epitopes are delineated by a peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays, etc. Available methods for deciphering conformation-specific epitopes are cumbersome (X-ray crystallography, etc.), time-consuming, and require expensive equipment. Therefore, it is indispensable to develop a simple method for identification and mapping of conformation-specific epitopes. In the present investigation, the radiolabeled human chorionic gonadotropin- (125IhCG) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC-G1G10.1-125IhCG complex (NCcom) was prepared and the dissociation of radiolabeled hCG was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k-1), association constants (k+1), and affinity constants (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCG involved in interaction with the complementary paratope on MAb G1G10.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of a conformation-specific epitope of hCG consists of Arg (94, 95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Therefore, the results of the present investigation suggested that the dissociation kinetics coupled with SS-SPRIA unequivocally assists in deciphering amino acid residues constituting a conformation-specific epitope of hC

Kinetic analysis of a human chorionic gonadotropin- epitope-paratope interaction

Kinetics of protein-protein or ligand-ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand-ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin- (125IhCG) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC-G1G10.1-125IhCG complex (NCcom) was prepared and the dissociation of radiolabeled hCG was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k-1), association constants (k+1) and affinity constants (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCG involved in interaction with the complementary paratope on MAb G1G10.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCG epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand-ligate interactions and epitope analysis

Molecular dissection of an hCG-β epitope using single-step solid phase radioimmunoassa

Background: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. Method: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of ‘mouse IgG-anti-mouse IgG’ was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) $G_1G_{10.}1$. The extent of competitive inhibition in binding ability of $^{125}IhCG-β$ with chemically or enzymatically modified hCG-β to immobilized MAb $G_1G_{10.}1$ in comparison to hCG-β standards was utilized to identify the epitopic amino acid involved in epitope–paratope interaction. Results: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. Conclusion: The data of SS-SPRIA revealed the 93–100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-β at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field