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Monoclonal and polyclonal antibody responses to the myelin basic protein epitope present in human urine
J N Whitaker1, A McKeehan, D W Freeman
1Department of Neurology, University of Alabama at Birmingham 35294-0007.
Abstract:
The myelin basic protein (MBP)-like material (MBPLM) in human urine is expressed in a cryptic epitope(s) present in MBP peptide 80-89 and absent or inaccessible in intact MBP. These features of urinary MBPLM have made it difficult to produce reagents for its further characterization. Using an immunogen of keyhole limpet hemocyanin conjugated to MBP peptide cys 74-89, polyclonal antiserum to urinary MBPLM was prepared. With the same immunogen and screening with urine, the product from one of over 1600 wells from the original fusion, produced monoclonal antibody (mAb) which could detect urinary MBPLM. By radioimmunoassay two rabbit polyclonal reagents recognized a cryptic epitope in MBP peptide 84-89 while the two mAbs recognized another cryptic epitope in MBP peptide 80-85. Both could be used for quantitation of MBPLM in urine. These and previous results indicate the presence of at least three epitopes, one noncryptic and two cryptic, in the decapeptide of MBP 80-89. Of the two cryptic epitopes, the one near the carboxyl-terminal is dominant to that in the amino-terminal portion. The detection of urinary MBPLM with reagents with two different specificities suggests the presence of two or more small MBP peptides in urine.
Insights
Researchers developed new antibodies to detect myelin basic protein-like material (MBPLM) in urine. These antibodies target specific cryptic epitopes, enabling the quantification of MBPLM and suggesting the presence of multiple MBP peptides in urine.
Area of Science:
- Biochemistry
- Immunology
- Urine Analysis
Background:
- Myelin basic protein-like material (MBPLM) in urine presents unique challenges due to cryptic epitopes.
- Characterizing urinary MBPLM requires specific reagents that can detect these hidden epitopes.
Purpose of the Study:
- To develop novel polyclonal and monoclonal antibodies for detecting urinary MBPLM.
- To characterize the epitopes recognized by these antibodies on myelin basic protein (MBP) peptide 80-89.
- To enable the quantification of MBPLM in human urine.
Main Methods:
- Conjugation of MBP peptide cys 74-89 to keyhole limpet hemocyanin to create an immunogen.
- Generation of polyclonal antiserum and monoclonal antibodies using the immunogen.
- Screening of antibodies with human urine.
- Radioimmunoassay for antibody characterization and MBPLM quantification.
Main Results:
- Successfully produced polyclonal and monoclonal antibodies capable of detecting urinary MBPLM.
- Identified two distinct cryptic epitopes within the MBP peptide 80-89 recognized by different antibodies.
- Demonstrated the utility of these antibodies for quantifying MBPLM in urine samples.
- Indicated the presence of at least three epitopes (one non-cryptic, two cryptic) in MBP 80-89.
Conclusions:
- Developed specific reagents for detecting and quantifying urinary MBPLM.
- The findings suggest the presence of multiple, distinct myelin basic protein peptides in urine.
- Characterization of cryptic epitopes advances understanding of MBP in biological fluids.
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