Quantitation of antibodies immobilized on plastics.
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Antibodies↗
Biomedical subjects
J E Herrmann
Publications and source records attributed to J 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.
Animals↗
Immunochemical and biological properties of the outer membrane-associated lipopolysaccharide and protein of Rochalimaea quintana.
The outer membrane complex of Rochalimaea quintana was isolated by use of ethylene-eiaminetetraacetate and was compared biochemically and biologically both with lipopolysaccharide (LPS) isolated by phenol-water extraction of whole organisms and with lipids isolated by chloroform-methanol extractions of the phenol-water insoluble residues. The outer membrane consisted of protein and LPS components, as distinguished by precipitin tests with sera from patients with trench fever or tests with hyperimmune animal sera. The outer membrane protein component, but not LPS, also reacted with sera from infections with Rickettsia tsutsugamushi. The LPS contained 2-keto-3-deoxy-octonate and heptose. The outer membrane and phenol-extracted LPS were reactive in the chick embryo lethality test, limulus assay, and complement activation. Outer-membrane activity was confined to the LPS component. The lipid extracts were reactive in chick embryo lethality and limulus assays, but did not activate complement.
Animals↗
Factors involved in enzyme-linked immunoassay of viruses and evaluation of the method for identification of enteroviruses.
A quantitative enzyme-linked immunosorbent assay was used for identification of selected enteroviruses: poliovirus type 1, echovirus type 6, coxsackievirus A type 9, and coxsackievirus B types 1 through 6. Partially purified viral antigens or virus-specific antibodies were adsorbed to polystyrene spectrophotometer cuvettes, which permitted the assays to be reported and compared in terms of enzyme units specifically reacting. Both the adsorbed antigen and the adsorbed antibody methods were approximately equal in terms of sensitivity and specificity of reaction. By use [14C]leucine-labeled enteroviruses, the amount of virus that bound to the plastics used was shown to be dependent on the purity of the virus preparation used, but it was higher than the amount that was bound by plastics coated with viral antibody. Diluents which contained 0.15% (vol/vol) Tween 20 and 2.0% (wt/vol) bovine serum albumin in phosphate-buffered saline, pH 7.2, were found to be the most effective in inhibiting nonspecific adsorption of immunoreagents. However, the presence of these inhibitors in phosphate-buffered saline solutions also caused desorption of virus or viral antibody during immunoassays; the amount of virus desorption varied with the type of preparation used, and antibody desorption was dependent on the concentration of antibody initially used for adsorption. For specific identification of a given enterovirus type by the enzyme-linked immunosorbent assay method used, approximately 10(5) plaque-forming units of virus per assay tube were required.
Echovirus 6, Human↗
Enzyme immunoassay of antibody to Rochalimaea quintana: diagnosis of trench fever and serologic cross-reactions among other rickettsiae.
Enzyme immunoassay (EIA) tests were used to diagnose trench fever and to determine cross-reactions of Rochalimaea quintana with other rickettsiae. The results were compared with those obtained by counterimmunoelectrophoresis (CIE). All sera from cases of primary or relapsed forms of trench fever were positive both in EIA, with serum antibody titers of 1:20-1:640, and in CIE, giving one to three precipitin lines. Sera from patients with other rickettsial infections were also tested for reactivity with R. quintana antigen: typhus group (Rickettsia prowazekii, Ricketsia mooseri), 15 sera; spotted fever group (Ricketsia ricketsii, Rickettsia akari), eight sera; and scrub typhus (Rickettsia tsutsugamushi), six sera. Strong reactions occurred with four sera from patients with scrub typhus, giving one or two lines in CIE and EIA titers of 1:40-1:160; these results were extended to guinea pig antisera to R. tsutsugamushi. About 50% of typhus group sera reacted with a single line in CIE and had antibody titers of 1:20-1:80 by EIA. The results show that EIA is accurate for the diagnosis of trench fever and, with the results obtained by CIE, suggest that R. quintana is antigenically related to R. tsutsugamushi and possibly to rickettsiae in the typhus group as well.
Antibodies, Bacterial↗
Enzyme immunoassay and radioimmunoprecipitation tests for the detection of antibodies to Rochalimaea (Rickettsia) quintana.
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Animals↗
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.
Adsorption↗
Conjugation of enzymes to anti-poliovirus globulin: effect of enzyme molecular weight on virus neutralization capacity.
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Alkaline Phosphatase↗
Persistence of enteroviruses in lake water.
Two enteroviruses were inactivated more rapidly in a lake than in sterile lake water; then their coat proteins were degraded and, perhaps, used by microorganisms.
Biodegradation, Environmental↗
Comparison of techniques and immunoreagents used for indirect immunofluorescence and immunoperoxidase identification of enteroviruses.
Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.
Animals↗
Rapid method to determine labeling specificity of radioactive enteroviruses.
The specificity of labeling enteroviruses with (32)P-labeled NaH(2)PO(4) or (14)C-leucine can be determined by comparing the percent adsorption of infective particles and radioactivity to membrane (Millipore) filters.
Adsorption↗
Degradation of coxsackievirus type A9 by proteolytic enzymes.
The means by which coxsackievirus type A9 (CA9) is inactivated by proteolytic enzymes was investigated. After reaction of (14)C-leucine-labeled CA9 with Pronase, free leucine was liberated as measured by radiochromatography. Treatment of (14)C-leucine-labeled CA9 with trypsin or proteolytic filtrates of Pseudomonas aeruginosa caused the release of a variety of labeled substances. The extent of viral ribonucleic acid (RNA) release after exposure of CA9 to Pronase was determined by RNA infectivity tests or trichloroacetic acid solubility tests. Infective viral RNA was found not to be consistently released by reaction of CA9 with Pronase, but further treatment with 1% sodium dodecyl sulfate at pH 7.0 promoted viral RNA release. Sodium dodecyl sulfate treatment of CA9 that had not been reacted with Pronase did not inactivate virus or cause viral RNA release. Reaction of Pronase with (32)P-labeled CA9 resulted in the liberation of virus components soluble in cold trichloroacetic acid, whereas untreated CA9 or CA9 reacted with ribonuclease were precipitated by cold trichloroacetic acid. These results demonstrate that the primary means by which protease-sensitive enteroviruses are inactivated is by degradation of the virus capsid, with subsequent release of viral RNA.
Animals↗
Coupling of peroxidase to poliovirus antibody: characteristics of the conjugates and their use in virus detection.
Antibody to poliovirus type 1 (Po-1) was coupled to peroxidase by use of glutaraldehyde or 4, 4' -difluoro,3, 3' -dinitro diphenyl sulfone. Glutaraldehyde was found to be the superior coupling agent, yielding conjugates that had up to 2.8 x 10(4) enzyme units/ml (75% of total enzyme input). Conjugates migrated as a single band when centrifuged in sucrose density gradients, demonstrating that the purification procedure used was effective in removing both noncoupled enzyme and heterogeneous antibody components. Conjugates were specific for Po-1 and did not adsorb to cells infected with unrelated enterovirus types. Adsorption of conjugates to Po-1-infected cells was demonstrable within 6 h postinfection.
Antibodies, Viral↗
Methods for detecting food-borne enteroviruses.
A method previously reported for detecting virus in a model system composed of cottage cheese contaminated with coxsackievirus type A9 has been adapted to detecting selected strains of enteroviruses in a variety of foods. Bentonite is omitted and serum is added for extracting virus from low-protein foods. Samples of foods, usually 25 g, must contain at least 3 to 4 plaque-forming units for a 50% probability of detecting virus. Sensitivity in detecting echovirus type 6 was lower than that for the other viruses used. After extraction from potato salad, poliovirus type 2 was completely reactivated if it had been neutralized with coproantibody, but it was only partially reactivated if neutralized with hyperimmune rabbit serum.
Aluminum Silicates↗
Food-borne virus:detection in a model system.
The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.
Buffers↗
Rapid methods for the immunodiagnosis of infectious diseases: recent developments.
Current techniques for rapid diagnosis of microbial infections by direct detection of the microbial agent are compared. The techniques include enzyme immunoassay (EIA) tests, immunofluorescence, latex agglutination assays, and nucleic acid hybridization procedures. It is concluded that, for the near future, the preferred methods for rapid diagnosis will be by (1) EIA tests utilizing monoclonal antibodies and improved enzyme detection systems, and (2) improved latex agglutination procedures for certain antigens. Nucleic acid hybridization techniques, as currently performed, will need to be substantially improved to become the methods of choice.
Bacterial Infections↗