Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.
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.


METHODS
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(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta 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.


CONCLUSIONS
The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.
Prasad
Pramod V
PV
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi-110067, India.
Chaube
Shail K
SK
Panchal
Manoj
M
Chaudhary
Rajesh
R
Muralidhar
K
K
Rohil
Vishwajeet
V
Kumari
G L
GL
Kumar
Anand
A
Ashish
B
B
Murthy
G S
GS
Shrivastav
T G
TG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry* / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011863:Radioimmunoassay / Q000379:methods*
D000911:Antibodies, Monoclonal; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes