John E. Herrmann

Immobilization of antibodies on nylon for use in enzyme-linked immunoassay

Antibodies were immobilized by covalent linkage on nylon balls and powder for use in solid-phase enzyme-linked immunoassays. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde or carbodiimides. Up to 0.74 microgram of immunoglobulin G per mm2 nylon could be immobilized, whereas only 0.02 microgram per mm2 could be adsorbed to polystyrene, and the binding to nylon was stable. This eliminated the problem of antibody desorption noted in conventional enzyme-linked immunosorbent assay which are based on simple adsorption to plastics, and gave more reproducible results. The method was also more sensitive, detecting levels of approximately 1 ng per ml of immunoglobulin E in clinical samples. Further, antibodies coupled to nylon balls remained bound under conditions that dissociate antibody-antigen complexes, which permitted reuse of the immobilized antibodies for immunoassays.

Quantitation of immunoglobulin adsorption to plastics

Adsorption of 125I-labelled rabbit IgG to plastics (cellulose nitrate, polyallomer, polystyrene, polyvinyl) was primarily dependent on initial antibody concentration and, to a lesser extent, on time allowed for adsorption. The highest concentration tested (100 mug/ml) gave the highest quantities adsorbed after 18 h at room temperature. This concentration, however, gave the lowest percent adsorption (6.5 to 12.0%) of the initial amount. IgG concentration of 10 mug/ml resulted in 25.0 to 65.1% adsorption over the same time period; at 1 mug/ml, 47.0 to 96.6% of the initial amount was adsorbed. All of the plastics tested adsorbed IgG to approximately the same degree, with the exception of cellulose nitrate. This plastic adsorbed 32 to 49% less than the others, under maximal adsorption conditions.

Enzyme immunoassay and radioimmunoprecipitation tests for the detection of antibodies to Rochalimaea (Rickettsia) quintana.

Summary Bond-phase enzyme lmmu-noassay (EIA) and radioimmunoprecipitation tests were developed for detection of antibodies to Rochalimaea (Rickettsia) quintana in trench fever. Both were equally sensitive, and both detected more positive sera than either passive hemagglutination or immunofluorescence tests. The advantages of EIA are the stability of label and ease and safety of use.

Conjugation of enzymes to anti-poliovirus globulin: Effect of enzyme molecular weight on virus neutralization capacity☆

Abstract Poliovirus type 1 antibody was coupled to s-galactosidase by use of glutaraldehyde. Purification of the crude conjugates by Sepharose 6B column chromatography was effective for isolating the active components. Analysis of crude conjugates by centrifugation in sucrose density gradients showed that they were heterogeneous; purified conjugates migrated as a single band. The virus neutralization capacity of s-galactosidase conjugates was compared to that of conjugates prepared with other enzymes (ribonuclease, alkaline phosphatase, peroxidase and s-glucuronidase). It was found that conjugates prepared with enzymes of mol. wt ⩽ that of IgG retained neutralizing activity to poliovirus type 1; the others (s-glucuronidase and s-galactosidase) had greatly reduced activities.

Detection and identification of influenza virus antigens by nylon-coupled enzyme-linked immunoassay

A direct solid-phase enzyme-linked immunoassay for rapid detection and typing of influenza virus was developed utilizing antibodies immobilized by covalent linkage to nylon beads. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde. For comparison to conventional enzyme-linked immunosorbent assays (ELISA), IgG fractions were adsorbed to polystyrene beads. Influenza type-specific immunoglobulins coupled to nylon beads were used in an enzyme-linked immunoassay to identify influenza A/USSR/77(H1N1), and A/Texas/75 (H3N2). In titrations of viral antigen, antibody coupled to nylon beads detected 1.9 X 10(4) plaque-forming units (PFU) per assay, whereas 2.2 X 10(5) PFU were required in assays utilizing antibody adsorbed to polystyrene beads. Use of fluorogenic or radioactive substrates for alkaline phosphatase-labeled antibodies increased the sensitivity for virus detection 10-fold with this enzyme, but were only slightly more sensitive than chromogenic substrates with peroxidase-labeled antibody.