A rapid and sensitive microassay for bacterial collagenase and other proteolytic activities on collagenous substrates
Most of the frequent substrate digestion assays have the drawback of being tedious and costly, thus aggravating serial determinations of collagenase exercise. Within the assay introduced right here, the proteolytic response proceeds on a sheet of chromatographic paper bearing spots of dried gelatin or collagen options. After utility of the enzyme pattern, the sheets are incubated in a water-saturated ambiance for 45 min and the response merchandise are visualized by spraying with chromatographic, ninhydrin-containing peptide reagents. By its particular yellow staining, the exercise of bacterial collagenase will be discriminated from the grey, brown, pink, or violet spots that are produced by eight examined noncollagenolytic proteases.
The detection restrict for collagenase exercise reaches all the way down to 0.002 U in a pattern quantity of 1-2 microliters dropped onto a spot of 25 microliters gelatin resolution (3%), thus corresponding with the sensitivity of a traditional radioassay. The sensitivity for noncollagenolytic proteases is healthier than 2 ng per pattern, herewith exceeding a standard casein-agar diffusion take a look at. The tactic can also be evaluated for native collagen. Inside 1.5 h, 100 samples will be assayed. An instance for the applying in a collagenase purification step (ion change chromatography) can also be introduced.
Detection of human immunodeficiency virus and different retroviruses in cell tradition supernatants by a reverse transcriptase microassay.
A micromethod for the detection of human immunodeficiency virus (HIV) and different retroviruses in cell tradition supernatants is described which applies a DEAE ion exchanger for restoration of polynucleotides synthesized in vitro by the retroviral reverse transcriptase. Cell tradition, pattern preparation, and take a look at efficiency together with the washing step are tailored to microtitre plates. In comparison with the usual methodology this system produced much less non-specific reactions, leading to a greater than 3-fold larger sensitivity, a better reproducibility resulting from decrease intrarun variations and allowed a rise within the every day pattern accomplishment per particular person 3- to 4-fold at decrease prices per pattern.
Microassay for nuclear binding of steroid receptors with use of intact cells from small samples of avian and human tissue
A micro model of a nuclear binding assay to evaluate the organic exercise of receptors for steroid hormones was developed for utility to small (needle) biopsies of human tumors for the aim of predicting responses to steroid remedy.
This simpler assay requires 10-fold much less tissue than the unique nuclear binding assay described for progesterone receptors within the avian oviduct, endometrium, and endometrial carcinomas (Spelsberg TC, et al., Endocrinology 1987;121:631). We describe the applying of this micro assay to regular avian oviduct and cancers of the human breast, and we exhibit a tissue specificity and saturation of nuclear binding.
The micro assay reliably measured as little as 0.5 mg equivalents of tissue per assay tube. Outcomes for breast tumors decided to be estrogen-receptor-positive by the usual dextran-coated charcoal methodology had been additionally decided with this nuclear binding assay. As described beforehand for progesterone receptors in endometrial carcinomas, some receptor-positive breast biopsies displayed negligible capability for nuclear binding. Subsequently, with the current assay we’ve got recognized nonfunctional receptors in these biopsies, which can be helpful for correct prediction of sufferers’ responses to remedy with hormones.