James F. Monthony

A solid phase fluorescent immunoassay for the quantitation of the C4 component of human complement

A non-competitive method for the determination of the C4 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C4 in serum in the range of 10 mg/dl to 170 mg/dl within 2 h. C4 levels as low as 150 ng/ml can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C4 levels in healthy adults. Good agreement was found between the two methods.

A solid phase fluorescent immunoassay for the quantitation of the C3 component of human complement

A non-competitive method for the determination of the C3 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C3 in serum in the range of 20 mg/d1 to 195 mg/d1 within 2 h. C3 levels as low as 125 ng/m1 can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C3 levels in healthy adults. Good agreement was found between the two methods.

Cell sorting using a universally applicable affinity chromatography matrix: Solid-phase anti-fluorescein isothiocyanate antibody

Procedures are described for fractionating cells utilizing a universally applicable cellular affinity chromatography matrix. The affinity matrix consists of immunoabsorption purified goat anti-fluorescein isothiocyanate antibody coupled to large derivatized polyacrylamide beads. This matrix may, in principle, be used to isolate any cell subpopulation provided it has a fluorescein-labeled ligand on its surface. In this report the matrix was used to isolate viable purified fractions of mouse surface Ig-positive cells, Lyt1 cells, and mouse lymphocytes that bind the lectin soybean agglutinin. A preliminary experiment using the anti-FITC beads suggested that this technique can provide a fraction of cells enriched in antigen binding cells. Cell populations isolated by this technique retain their ability to respond to in vitro mitogen stimulation, as well as their ability to be maintained in cell culture following fractionation. Additional experiments using a column consisting of goat anti-rabbit Ig antibody coupled to the same support material are also reported.

Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose: Effect of protein charge on immobilization

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.