Monitoring of recombinant survival motor neuron protein using fiber-optic surface plasmon resonance.
Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.
Masson
Jean-Francois
JF
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Barnhart
Margaret
M
Battaglia
Tina M
TM
Morris
Glenn E
GE
Nieman
Ronald A
RA
Young
Philip J
PJ
Lorson
Christian L
CL
Booksh
Karl S
KS
D015374:Biosensing Techniques; D005336:Fiber Optic Technology; D006801:Humans; D009046:Motor Neurons / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D055100:Optical Fibers; D011994:Recombinant Proteins / Q000032:analysis; D020349:Surface Plasmon Resonance
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins