Semi-automated Assay Research Articles

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265–275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 μl or less, and can detect less than 0.5 μg of protein (equivalent to about 10 3 cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.

A semi-automated assay for plasma catecholamines using high-performance liquid chromatography with electrochemical detection

A semi-automated assay for plasma catecholamines using high-performance liquid chromatography with electrochemical detection

Quantitative semi-automated enzymatic assay for tissue glycogen

1. 1. A simple, rapid, specific and reproducible assay for tissue glycogen is described. 2. 2. The method involves the incubation of a denatured tissue homogenate with amyloglucosidase resulting in the complete hydrolysis of glycogen to glucose. The glucose content of the homogenate supernatant is then determined with the aid of a glucose analyzer. 3. 3. The method has been used to measure the glycogen content of a variety of discrete and complex animal tissues and is particularly convenient for microdeterminations.

An enzymic creatinine assay and a direct ammonia assay in coated thin films.

We developed a thin-film enzymic assay for creatinine that makes use of creatinine iminohydrolase (EC 3.5.4.21) to convert creatinine to N-methylhydantoin and ammonia. The ammonia diffuses through a semipermeable layer and is quantitated by reaction with bromphenol blue. A paired analysis of the sample on a separate coating without the enzymic reaction measures endogenous ammonia and, for samples with normal concentrations of ammonia, allows accurate determination of serum creatinine to 150 mg/L without dilution. Results of this assay (y) compare well with those by a liquid-chromatographic comparison assay (x) by linear regression (slope = 0.935, intercept = 1.13 mg/L, r2 = 0.995). It is insensitive to many substances, such as ketones and keto acids, that interfere with conventional assays. Results of the ammonia assay (y) correlate well with those by a semi-automated enzymic assay (x) based on glutamate dehydrogenase (slope = 1.068, intercept = 17.3 mumol/L, r2 = 0.985).

A semiautomated assay for nonesterified fatty acids in the 0.02 to 0.4 μeq/ml range

A semiautomated assay for nonesterified fatty acids in the 0.02 to 0.4 μeq/ml range

Evaluation of a commercially available semi-automated bioluminescence system for bacteriuria screening.

A commercially available semi-automated bioluminescence adenosine triphosphate assay system for rapid detection of significant bacteriuria was evaluated. Excellent reproducibility of results using the bioluminescence apparatus was noted, and there was no bacterial carry-over using a pre-diluter. The results obtained with 2,000 urine specimens tested by bioluminescence and a routine cultural technique were compared. The bioluminescence system gave no false negative results and a bioluminescence positive/culture negative finding of 13.6% in general specimens and 45.0% in ante-natal and maternity specimens. In the latter group, the majority of urines yielded growth of lactobacilli and/or diphtheroids (less than 10(4) organisms/ml) when subcultured on enriched media. Results indicated that bioluminescence may identify urinary tract infections in patients receiving antimicrobial therapy. The advantages of this 45 minute technique for bacteriuria screening are presented.

Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8–9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. The assay system can be automated by the use of 96-channel automatic pipettors throughout, so large numbers of cultures can be set up for accurate CTL-p frequency determinations. The method is suitable for accurately estimating the total number of functional T cells of the cytotoxic class in developing T-cell populations, and as a future development for scanning their specificity repertoire.

Rapid, quantitative, semiautomated assay for virus-induced and immune human interferons.

An improved human interferon (IF) assay is described. This procedure is based on the ability of encephalomyocarditis virus to replicate in WISH cell microcultures with the production of discrete plaques in the presence of a liquid tissue culture medium. Performance of 50% plaque reduction endpoint assays in micro-culture required only 0.1 ml of specimen for determinations using duplicate dilutions beginning at 1:3. Semiautomated equipment facilitated simultaneous in situ dilution and distribution of multiple IF samples in cultures containing preformed WISH cell monolayers. An incubation period of 5 to 6 h was adequate for development of maximal antiviral activity by both virus- and immune-induced IF. Sensitivity of the encephalomyocarditis microplaque reduction assay was comparable to that of other commonly used techniques. The method is rapid, can be completed within 30 h from the beginning of the IF assay, and is able to accommodate as many as 40 to 50 samples at a single time. Encephalomyocarditis microplaque reduction is suitable for the quantitation of IF as an antiviral agent or a lymphokine.

A semiautomated electron capture gas chromatographic assay of 3-methoxy-4-hydroxyphenylethyleneglycol in urine

A semiautomated method for the assay of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine has been developed. The method incorporates a new efficient (95%) extraction procedure combined with an automated gas chromatographic system. This system (consisting of a pulsed electron capture detector and an automatic sample injector which valves the sample between two columns) will accept continuous, unattended, sequential sample input. The limit of sensitivity of the trifluoroacetic anhydride derivative of pure MHPG is 3 pg. Analysis of samples from the same 24-h urine ( n = 21) over a 2-month period resulted in a mean of 794 ng/ml with a coefficient of variation of 3.5%. MHPG levels from 12 control males and 9 females with no history of psychiatric disorder, were found to contain 1605 and 1034 μg/24 h, respectively.

Aspirin—a national survey I: Semiautomated determination of aspirin in bulk and tablet formulations and salicylic acid in tablet formulations

A semiautomated assay to determine aspirin (acetylsalicylic acid) in aspirin tablets, powdered tablet composites, and pure drug substances is presented. The sample was dissolved in alcoholic buffer, diluted, and extracted with chloroform. The absorbance of the chloroform solution was read at 280nm. Results obtained by the USP XIX and semiautomated methods are compared. The proposed method is accurate and precise, and common excipients do not interfere. Recoveries of 100% were obtained. A semiautomated assay for salicylic acid in aspirin tablets also is presented. The sample was dissolved in alcoholic buffer, ferric nitrate was added, and the absorbance of the resulting purple color was read at 532nm. The method is suitable as a rapid screening procedure for testing the salicylic acid content of aspirin products. Results obtained by the USP XIX and semiautomated methods are compared, and the accuracy and precision of the proposed method are given. One hundred seventy tablet samples and 34 bulk drug substances were analyzed for aspirin and salicylic acid content. Approximately 5% of the tablet samples failed to meet the USP XIX limits for aspirin content, and 10% failed to meet the limits for salicylic acid.

Diagnosis of Postnatal Rubella by the Enzyme‐Linked Immunosorbent Assay for Rubella IgM and IgG Antibodies

A semi-automated enzyme-linked immunosorbent assay (ELISA) was established for the detection of rubella IgM antibodies in non-fractionated sera. A cut-off level between rubella IgM positive and negative sera was determined by a study of sera without rheumatoid factor from 200 blood donors. Testing of 12 donor sera containing rheumatoid factor showed that 5 sera gave a positive result in the rubella IgM assay. Rubella IgM antibodies were quantified by ELISA in a study of 214 serial serum specimens drawn from 16 patients with rubella during a period of up to 10 years after the infection. Peak values of the IgM antibodies were reached approximately 8 days after onset of the rash, and the persistence of the IgM antibodies ranged from 17-90 days, with the exception of one patient with a prolonged IgM response. The rubella IgG antibodies increased slowly after the rash and reached maximum levels about 50-120 days, after which a monor decrease was observed. The results of the present study indicate that ELISA is suitable as a routine procedure for the serodiagnosis of recent rubella.

Enzyme-Linked Immunosorbent Assay For Determination Of Igm Rheumatoid Factor

A semi-automated enzyme-linked immunosorbent assay (ELISA) for determination of IgM rheumatoid factor was established. Human gammaglobulin (Cohn fraction II) was used as an antigen on a solid phase of polystyrene microtitre trays. The results were read on a spectrophotometer. ELISA and the sheep cell agglutination test were compared in a study of sera from 400 blood donors, 53 patients with active rheumatoid arthritis and of 200 sera received for routine determination of rheumatoid factor. The results of the two tests correlated well. The ELISA procedure was precise, objective, inexpensive and well suited for quantitative routine determination of IgM rheumatoid factor.

Studies of F VIII procoagulant activity in plasma and concentrates using a semi-automated thrombin generation assay

A semiautomated thrombin generation assay for the quantitation of F VIII procoagulant activity (VIII:C) in plasma and concentrates is presented. The analytical procedure was not dependent on F VIII deficient plasma and required little specialized technical skill. The assay was found to be extremely reproducible at VIII:C levels of clinical relevance, with a sensitivity of 6. 10 −3 units/ml. Kinetic studies of plasma and concentrates revealed differences suggesting changes in the reaction order of one or several enzyme-ligand systems involving VIII: C in the test system. The kinetic profile of high purity concentrates could be reverted to a plasma pattern by the addition of a component present in the plasma of patients with severe F VIII deficiency states.

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (Ka=1.3x10(9)l mol(-1)). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10(-12) mol ABA, the measuring range extends to 20x10(-12) mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.

A semiautomated system for measurement of glutathione in the assay of glutathione peroxidase

The most commonly used assay procedures for measurement of glutathione (GSH) peroxidase are unsuitable for kinetic studies; therefore, a semiautomated system that is based upon the 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) reaction with sulfhydryls was developed for the assay of relatively purified samples of the enzyme. Sulfhydryl standards in the range of 0–1.5 m m gave a linear response with r 2 > 0.998. In the enzyme assay, unreacted GSH was measured continuously. The -ln[GSH] was plotted vs the time of reaction to obtain first-order rate constants. The standard deviation over a twofold enzyme concentration range was 9%. The relative merits of the semiautomated DTNB, manual DTNB, and GSH reductase-NADPH coupled assays for measurement of GSH peroxidase are discussed. Sulfhydryl utilization by GSH peroxidase in the presence of a fixed amount of GSH and increasing amounts of mereaptoethanol was measured by this semiautomated system. It was determined that the enzyme can utilize mercaptoethanol as one of the two donor substrates but that there is a requirement for GSH as the other donor substrate. This semiautomated assay should be applicable to other reaction systems that consume or produce sulfhydryl groups.

ASSESSMENT OF ROASTING EFFECTS ON VITAMIN B6 STABILITY AND BIOAVAILABILITY IN DEH.YDRATED FOOD SYSTEMS

ABSTRACTDehydrated food systems were employed to study the stability and bioavailability of vitamin B6 as affected by roasting at 180°C for 25 min. The roasting conditions were selected to permit estimation of the maximum effects of the roasting process on the B6 vitamers and are above those which would be normally used for commercial processing. The relative degradation of pyridoxine, pyridoxamine, and pyridoxal phosphate was found to be 50‐70% by both microbiological and semi‐automated fluorometric assay methods. Estimates of biologically available vitamin B6 in the roasted model systems, as determined by rat bioassay correlated closely with microbiological results. Thus, vitamin B, remaining after roasting at 180°C for 25 min was biologically available and active. Semiautomated fluorometric values for total vitamin B6 in untreated and roasted systems were 1.65‐3.63 times higher than corresponding microbiological assay data. Fluorescence spectra studies of samples prepared for the semiautomated assay revealed no detectable interfering compounds. Microbiological assay provided an accurate evaluation of available vitamin B6 in the roasted model food system as shown by the rat bioassay. The consistently high results from the semiautomated fluorometric method indicate that further work is required before this method can be accurately used to quantitatively determine the level of vitamin B6 in foods.

Regional levels of GABA in the brain: rapid semiautomated assay and prevention of postmortem increase by 3-mercapto-propionic acid.

Abstract— A simple and rapid semiautomated assay for GABA in central nervous tissue is described. The method is based on a simple manual procedure of isolating GABA from tissue extracts on small CM Sepharose Cl 6B columns, followed by an automated fluorimetric detection (continuous flow system) with o‐phthalaldehyde (OPA) and β‐mercapto‐ethanol (β‐ME) at an alkaline pH. GABA is separated from other compounds that fluoresce in our detection system. By using low concentration of OPA and β‐ME and allowing only a short reaction time with these reagents, the detection is specific towards GABA. The detection limit of the assay is 1 nmol.A procedure is described for the prevention of postmortem GABA increase in rat and mouse brain by intravenous injection of 3‐mercapto‐propionic acid (1.2 nmol/kg) 2min before decapitating the animal. This treatment and microwave irradiation result in similar GABA levels in mouse brain and substantia nigra tissue from rat brain. We found a great conformity in regional GABA levels in the rat and the mouse brain.

A novel semiautomated method for the estimation of free fatty acid in serum or plasma

A modification of the semiautomated assay method of Antonis (1965. J. Lipid Res. 6:307-312) for free fatty acid is presented. Free fatty acids are extracted from serum or plasma into di-n-butyl ether-2-methoxyethanol; the extract is almost free from phospholipids. The acids are analyzed in a portion of extract by a copper soap method using diphenylcarbazide. The extractant, being less dense than water, is easily separated from an aqueous phase both in the extraction of samples and in the assay of copper soaps. The assay is comparable in accuracy with well-tried titrimetric methods and is quicker and easier to operate.

Acetaldehyde formation during deproteinization of human blood samples containing ethanol

Production of acetaldehyde during deproteinization of ethanol-containing human blood with perchloric acid has been studied using a semiautomated acetaldehyde assay based on vapor-phase transfer followed by enzymic oxidation and fluorimetry. Results obtained compare well with and extend those obtained in similar studies which have used gas chromatography as the method of acetaldehyde analysis. The extent of acetaldehyde production was variable but was related to blood ethanol concentration and could be decreased by increasing the dilution of the blood with the deproteinizing solution. Thiourea (25 m m) was found to have no effect on the acetaldehyde production, and insignificant quantities of acetaldehyde were formed during deproteinization of plasma in the presence of ethanol. It is suggested that more than 90% of the acetaldehyde produced during processing of ethanol-containing human blood for assay originates from reactions occurring when blood cells, as distinct from plasma, are treated with a deproteinizing agent.

Determination of Diphenoxylate Hydrochloride and Atropine Sulfate in Solutions and Tablets

Methods for the determination of diphenoxylate hydrochloride and atropine sulfate combinations in solutions and powdered tablet composites are presented. A semiautomated assay for diphenoxylate hydrochloride in individual tablets (content uniformity) also is presented. The USP XIX assays for these products are cumbersome and, in the case of solutions, inaccurate due to spectral interferences; the proposed methods offer substantial improvements in sensitivity, specificity, and speed. Results obtained by the USP and proposed methods are compared for several lots of commercial products. The accuracy and precision of the proposed methods are shown by standard recovery studies.