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A semiquantitative microassay for measurement of relative number of blood mononuclear cells infected with human immunodeficiency virus

by Frank Green
A easy semiquantitative microassay was developed for the measurement of relative variety of contaminated peripheral blood mononuclear cells (PBMC) from people contaminated with human immunodeficiency virus (HIV). The assay relies on cocultivation of serially diluted PBMC of a seropositive individual with phytohemagglutinin-stimulated regular PBMC. The microassay has comparable sensitivity with the usual virus tradition methodology in detecting constructive HIV cultures.
Because the microassay makes use of solely 2-Three x 10(5) sufferers’ PBMC, the assay can be most fitted for HIV isolation from HIV-infected infants or from AIDS sufferers with extraordinarily low T-cell counts. The microassay will also be used to measure antiviral results of a drug on persistent HIV an infection in vitro. As a result of the microassay measures the relative variety of contaminated PBMC, it may be readily used for following the quantitative antiviral impact of a drug in a scientific trial.

A microassay to quantitate collagen synthesis by cells in tradition

A technique to quantitate collagen synthesis, complete protein synthesis, and DNA in 24-well tradition plates is offered. Collagen-producing cells equivalent to human intestinal clean muscle cells and dermal fibroblasts have been pulse-labeled with [3H]proline. After incubation, the plates have been heated to 90 levels C to cease isotope incorporation and sonicated to lyse the cells and an aliquot was eliminated for DNA quantitation. Service protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was eliminated by repeated precipitations.
After incubation with purified bacterial collagenase, each the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein have been quantified. Outcomes have been expressed as the quantity of radioactivity integrated into collagen and noncollagen protein per nanogram DNA and in addition as the share of collagen synthesis per complete protein synthesized.
The benefit of this method over earlier makes an attempt to scale down the assay is that your entire assay for DNA, collagen, and non-collagen protein will be carried out in the identical nicely with none switch of fabric. This system additionally supplies a big financial savings of tradition medium, serum, development elements, and cell materials.

A microassay for mammalian folylpolyglutamate synthetase

A brand new assay for the enzyme folylpoly-gamma-glutamate synthetase (FPGS) that gives vital benefits over different printed procedures has been developed. This assay relies on the addition of excessive particular exercise [3H]glutamic acid to (6-S)-tetrahydrofolate adopted by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently certain macromolecular complicated by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase.
This complicated is then separated from extra labeled glutamic acid by centrifugal elution of a 1-ml Sephadex G-50 column. The assay was discovered to be helpful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Sometimes, clean values of 100-200 cpm are seen with a sign usually greater than 10 occasions larger. Evaluation of 20-30 samples will be completed in lower than 90 min. Because of this, this assay has confirmed helpful for detection of enzyme in elution fractions from chromatographic columns.

Microassay for malignant hyperthermia susceptibility: hypersensitive ligand-gating of the Ca channel in muscle sarcoplasmic reticulum causes elevated quantities and charges of Ca-release.

A crude preparation of heavy sarcoplasmic reticulum (HSR) was remoted utilizing 1 gram of muscle obtained from swine prone to malignant hyperthermia (MH) and from management swine. The caffeine and ATP concentration-dependency of Ca-release was decided utilizing suction filtration with radioisotopic 45Ca as a tracer. Charges of launch have been decided utilizing a speedy filtration system. Caffeine and ATP-induced Ca-release from MH-susceptible (MHS) HSR occurred at one-tenth the focus of agonist that was required for management muscle HSR. No variations in charges and quantities of launch have been noticed when agonist concentrations have been used that brought about most launch for controls. Nevertheless, on the threshold focus of caffeine inflicting launch for management HSR, the MHS HSR launched 4-times as a lot Ca and at 3-times the speed of controls. These findings point out that elevated charges and quantities of Ca-release are because of the hypersensitivity of the Ca-release channel of HSR and that this abnormality will be detected utilizing 1 gram of muscle.
This research describes a dot enzyme immunoassay (Dot-EIA) for detecting hepatitis B floor antigen (HBsAg). The outcomes demonstrated that the detection stage of this assay for HBsAg was 1.5 ng/ml; no false-positive or -negative readings have been noticed. Additionally, this Dot-EIA had some benefits over normal EIA: (1) antiserum could possibly be straight and instantly certain on nitrocellulose paper set into microfiltration equipment, (2) the paper could possibly be simply washed underneath decreased strain utilizing a water aspirator, (3) all assay steps could possibly be carried out at room temperature inside 2 h, (4) the well-defined brown spots could possibly be evaluated by each visible remark and densitometric studying. The Dot-EIA reported right here could also be helpful for speedy prognosis and screening of HBsAg in serum.

A brand new delicate microassay for the measurement of erythrocyte glycogen

Typical ethanol precipitation of sub-microgram quantities of glycogen results in low yields (lower than 50%). Quantitative recoveries of 90% have been attained, nevertheless, when the isolation temperature was raised to 50 levels C and ethanol was changed by the much less polar propan-2-ol. This enchancment enabled improvement of an erythrocyte assay for glycogen which was each delicate (0.1 microgram glycogen) and required only one ml of complete blood. 26 paediatric specimens have been analysed and a reference vary of values from undetected to 78 micrograms glycogen/g haemoglobin (Hb) was obtained.
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Preimplantation sexing and prognosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay.

Hypoxanthine phosphoribosyl transferase (HPRT)-deficient male embryos derived from heterozygous (service) feminine mice have been recognized by biochemical microassay of X-chromosome-coded HPRT exercise in a single cell taken from the 8-cell embryo or in 5-10 cells sampled from the blastocyst. Within the latter process, service feminine blastocysts is also distinguished from affected males, and regular women and men, as having intermediate HPRT exercise within the sampled trophectoderm cells. In the course of the assay procedures, the operated preimplantation embryos have been cultured.
They have been then transferred, in batches as recognized, to recipient females. The ensuing fetuses have been sexed by gonad morphology and assayed for HPRT exercise. All these recognized as HPRT-negative embryos by biopsy on the 8-cell or blastocyst levels have been certainly HPRT-negative males. The heterozygous females have been additionally appropriately recognized by the trophectoderm biopsy process.
The intercourse of an embryo will also be recognized by HPRT exercise dosage in a single blastomere taken from 8-cell embryos from a standard mating and cultured for 12 hours earlier than assay. Each X chromosomes are lively in feminine morulae and the blastomeres sampled from feminine preimplantation embryos have twice the X-coded HPRT exercise in comparison with these from the male embryos. The accuracy of this process for sexing was once more verified by switch of the putative male and putative feminine embryos into recipient females.
Frank Green