Acoustic probe-based ELISA.

The effects of acoustic microstreaming during incubation steps of a prototypical three-step ELISA were studied. Acoustic microstreaming, an orderly mixing of microwell contents induced by linear oscillation of immersible acoustic probes, was shown to be particularly effective when coupled with locating the solid phase on the surface of the probes. Optical densities achieved for acoustic probe-based assays were equivalent to those for uninsonated microwell-based assays with only 20% of the microwell solid phase surface area. Low-level antibody detection was significantly improved and antibody incubation times significantly shortened without loss of signal. Acoustic probe-based assays can enhance assay and laboratory efficiency through testing for multiple analytes in a single sample or increasing available binding surface area (by using probe and well surfaces simultaneously), and by eliminating quenching. Acoustic probe ELISA methodology has significant implications for cost-effective automation.

Lack of effect of pulsed ultrasound on ABO antigens of human erythrocytes in vitro.

The efficacy of pulsed ultrasound in removing ABO blood group antigens from human erythrocytes was investigated in vitro. Cell suspensions were exposed to 5.25 MHz focused ultrasound with 1.23 microseconds pulses, at 0, 20, and 37 degrees C using spatial peak, pulse average intensities of 11, 126, and 1000 W/cm2 and pulse spacings of 10, 100, and 1000 microseconds. A second experiment involved application of 7.5 MHz pulses of 0.77 microseconds duration and 8 W/cm2 SPPA intensity which were spaced 1.25 ms apart. Exposed cells were tested for agglutination by antibody to determine changes in antigen expression. In addition, supernates from exposed cells were tested for the presence of soluble antigen. A sensitive capillary tube agglutination technique was developed for these experiments. No detectable antigen removal occurred as a result of any of the pulsed ultrasound exposures as compared to sham exposures. A positive control, which employed antigenic material prepared from cells disrupted by ultrasonic cavitation, indicated that the assay could detect the soluble antigen equivalent of about one cell in 10,000.

Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by Con-A-stimulated murine lymphocytes in vitro.

Murine spleen cell suspensions stimulated by Concanavalin A (Con-A) were exposed to 1.6-MHz continuous-wave ultrasound at low intensities (spatial-peak values ranging from 16 to 300 mW/cm2) in the presence of a Nuclepore membrane that contained stabilized gas bodies. The ultrasonically activated gas bodies induced cell lysis and reduced the fraction of intact cells that excluded trypan blue. At a spatial-peak intensity of 75 mW/cm2 (spatial-average intensity 15 mW/cm2), Con-A-induced Methyl[3H]thymidine (3H-TdR) incorporation was reduced in cultures exposed at 24 or 48 hr after addition of Con-A but not at 7 or 13 hr. There was no observable effect on cell survival or 3H-TdR incorporation at spatial-peak intensities below 75 mW/cm2.

Whole-bacterial cell enzyme-linked immunosorbent assay for Streptococcus sanguis fimbrial antigens.

A whole-bacterial cell enzyme-linked immunosorbent assay (bactELISA) was developed for detecting fimbrial antigens on Streptococcus sanguis. In this assay, S. sanguis cells were directly adhered to polystyrene or polyvinyl via drying. Use of the assay indicated that consistently high and uniform optical densities could be obtained from well to well. In addition, radioactive assaying indicated increased adsorption to the polystyrene wells over polyvinyl, suggesting that polystyrene may prove superior in the gram-positive bactELISA. Use of the bactELISA may prove valuable to both the clinical and research laboratory involved in the study of bacterial cell surface components or in the evaluation of antisera directed against bacterial antigens, which are difficult to prepare as purified derivatives.

BrucELISA: an enzyme-antibody immunoassay for detection of Brucella abortus antibodies in milk: correlation with the Brucella ring test and with shedding of viable organisms.

An indirect enzyme-antibody immunosorbent assay (BrucELISA) is described for the detection of antibody to Brucella abortus in cow's milk. Three series of milk samples were obtained from an adult-vaccinated dairy herd infected with B. abortus. The BrucELISA system was used as a screening test for individual milks diluted 1:200 (BE 200 test), for undiluted bulk milks, and to determine antibody titer (BrucELISA titration assay). The BrucELISA results correlated highly with positive Brucella ring test reactions and culture positivity, eliminated false-positive Brucella ring test reactions, detected antibody in some samples which were Brucella ring test negative, and distinguished between vaccinated and infected animals. BrucELISA titration assay titers of greater than 1:800 were correlated with shedding, or were prognostic for animals which eventually became shedders. Binding of the enzyme-antibody conjugate to bovine immunoglobulin in the absence of rabbit anti-bovine immunoglobulin occurred with culture-positive or -negative milks showing titers of greater than 1:1,600 (the beta effect); the effect was also of predictive value in identifying eventual shedders. The BrucELISA system is a sensitive, specific, and inexpensive method for screening large numbers of individual or bulk milk samples for the presence of antibody to B. abortus.

Preparation of rhinovirus antisera in the South American hystricomorph rodent, Octodon degus (38507).

Rhinovirus antisera have been prepared for rhinoviruses (RV) 7,9,26,32,67 and 87 in guinea pigs and in degus. Titers achieved were either similar in the 2 animals (RV7) somewhat higher in the degu (RV9 and RV32) or clearly higher in the degu (RV26, RV67 and RV87). Specificity of the antisera was similar in both animals. In special instances where it is difficult to prepare high-titered rhinovirus antisera in the guinea pig, the degu offers an attractive alternative source.