A simple method for coating native polysaccharides onto nitrocellulose.
A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.
Feng
S H
SH
Laboratory of Bacterial Polysaccharide, Food and Drug Administration, Bethesda, MD 20892.
Rubinstein
L J
LJ
Stein
K E
KE
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000921:Antibody-Producing Cells / Q000276:immunology; D003101:Collodion / Q000737:chemistry*; D003911:Dextrans; D006825:Hybridomas; D015151:Immunoblotting / Q000379:methods*; D007525:Isoelectric Focusing; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000907:Antibodies, Bacterial; D003911:Dextrans; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D003101:Collodion