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A direct method for quantification of non-transferrin-bound iron.

A direct method for quantification of non-transferrin-bound iron has been developed. This assay relies on the use of a large excess of a low affinity ligand (nitrilotriacetic acid, NTA) which removes and complexes all low molecular weight iron and iron nonspecifically bound to serum proteins. Iron bound to transferrin, ferritin, desferrioxamine, and its metabolites is unaffected. The Fe-NTA complex present in the serum ultrafiltrate is then quantified using an automated HPLC procedure where on-column derivatization with a high affinity iron chelator (3-hydroxy-1-propyl-2-methyl-pyridin-4-one) takes place. The iron complexes of desferrioxamine and its metabolites are unaffected by the above-derivatization procedure. With minor modifications, this method is equally applicable for the quantification of low molecular weight iron in other biological fluids.

Acetates

Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography.

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzyme-linked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG1 with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution compositions, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h.

Animals

Quantification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and simultaneous ultraviolet-visible/radioactive detection.

An HPLC-based method for quantification of desferrioxamine (DFO) and its iron chelating metabolites in plasma has been developed. This assay overcomes stability problems associated with DFO by the addition of radioactive iron to convert unbound drug and metabolites to radio-iron-bound species. A dual detection system utilizing uv-vis absorption and radioactive (beta-particle) detector was used to quantify total and radio-iron-bound species. The use of octadecyl silanol solid phase extraction cartridges permits concentration of samples and allows accurate quantification of drug and metabolites down to 0.1 nmol/ml.

Chromatography, High Pressure Liquid

Quantification of biomineralization: an in-vitro tissue culture system and microanalysis of calcium, phosphorus and trace elements by total-reflection X-ray fluorescence.

An in-vitro tissue culture system with folded periostea of 17-day-old fetal chick calvaria was combined with analytical methods to achieve quantification of biomineralization. A scanning electron microscope with an energy-dispersive X-ray detector was applied to show the distribution of calcium, phosphorus and trace elements. Calcium and phosphorus were concentrated in the zone of the mineralized matrix. Strontium was distributed similar to calcium. Zinc was distributed equally in the soft tissue and the mineralized matrix. Total-reflection X-ray fluorescence was used for quantification. Thyroxine in high concentration reduces the calcium content of the samples. One week after incubation magnesiumchloride (1.8 mM) or zincchloride (.1 mM) were found to reduce the calcium content by 38% or 82%, respectively.

Animals

The direct quantification in whole serum of HDL subfractions.

A method is described for the direct electrophoretic separation in human serum of three HDL subfractions and the quantification of the cholesterol content of these by means of gas-liquid chromatography. Used in conjunction with established procedures for the separation of total HDL, VLDL and LDL on agarose gels (with gas-liquid chromatography quantification of cholesterol in each band) the procedure affords comprehensive information on lipoprotein metabolism in patients. Two technicians can process 10 samples per day, making the method suitable for semi-large scale screening of patients such as may occur in clinical trials or to evaluate in greater detail lipoprotein patterns of patients suspected of having lipoprotein abnormalities on the basis of simpler screening methods. Results obtained by the method correlate well with those obtained by means of ultracentrifugation.

Centrifugation, Density Gradient

Quantification of human serum lipoprotein Lp(a): zone immunoelectrophoresis assay, a new sensitive method as compared to electroimmuno assay.

This study aimed at improving the immunological procedures for the quantification of human serum lipoprotein Lp(a). Lipoprotein Lp(a) was estimated by two immunoelectrophoretic methods: electroimmunoassay and zone immunoelectrophoresis assay. Whereas the electroimmunoassay gave curvilinear calibration lines (in peak height versus concentration plots) the zone immunoelectrophoresis assay revealed linearity in the same concentration range. The results obtained are in good agreement with each other (r = 0.975). The inter-assay coefficient of variation for the zone immunoelectrophoresis assay was 12% as determined by the use of a lyophilised reference serum. Zone immunoelectrophoresis assay is recommended for the routine quantification of lipoprotein Lp(a). Although the levels for total cholesterol (p less than 0.0025), HDL-cholesterol (p less than 0.001) and triglycerides (p less than 0.025) differed significantly between male (n = 24) and female (n = 22) individuals, no difference could be detected comparing the frequency distributions of Lp(a)-levels found for the male and the female group.

Adolescent

Efficacy of latex agglutination and quantification methods for determination of C-reactive protein (CRP) in pediatric sera.

Described is a procedure for serum C-reactive protein (CRP) determination, consisting of a semiquantitative rapid CRP latex agglutination test, using dilutions of the serum, and the quantitating spot immunoprecipitate assay (SIA). These methods are performed with standard laboratory equipment using no more than 30 microliters of serum for both assays. With visual inspection, the SIA results are available one to two hours after blood sampling. CRP levels obtained by agglutination testing with five batches of latex reagents coated with rabbit anti-CRP agree well with the quantification, i.e. essentially 100% for the negative sera and about 80% for positive sera containing greater than 40 mg CRP/l. The remaining 20% of the samples are classified as low positive at 10 to 40 mg/l on agglutination. False positive or negative agglutination findings are below 2.3% with concordance at 88% between SIA and the CRP agglutination with rabbit antibodies. Two lots of CRP-latex reagents coated with sheep antibodies, however, gave 15.3% and 10.1% false positive findings and poor concordance with SIA ratings, particularly for low positive sera at only 20 and 29%. SIA is suggested for CRP quantification because it compares well with radial immunodiffusion in accuracy (less than 91%) and provides results in 2 h rather than 1-2 days. Rocket electroimmunoassay is less reliable with lower ratings than found in SIA, probably due to the electrophoretic heterogeneity of CRP. This is demonstrated for two of three purified CRP preparations, for which varying agglutination is seen. The combination of methods is especially recommended for diagnosis and monitoring of CRP in infectious processes in neonates and infants because of the required small sample volume--0.5 ml heel-prick blood--the rapidity of reliable (greater than 80%) reporting and the possibility of rating sera with moderate levels of CRP.

Animals

Quantification of 3-methylglutaconic acid in urine, plasma, and amniotic fluid by isotope-dilution gas chromatography/mass spectrometry.

A method is described for quantification of the trace metabolite, 3-methylglutaconic acid, by isotope-dilution gas chromatography/mass spectrometry using synthetic 3-[2,4,6-13C3]methylglutaconic acid. Results are shown for quantification of 3-methylglutaconic acid in plasma, urine, cerebrospinal fluid and amniotic fluid for both normal controls and patients with different forms of 3-methylglutaconic aciduria. A simple method for the synthesis and purification of 3-[2,4,6-13C3]methylglutaconic acid is also described.

Adult

An improved method for quantification of very long chain fatty acids in plasma.

Most peroxisomal disorders can be detected via analysis of very long chain fatty acids (VLCFA) and phytanic acid in plasma or serum. Previous methods utilizing gas-liquid chromatography (GLC) alone are time consuming and are hampered by interference from cholesterol derivatives. We describe here a GLC-mass spectrometry method for the simultaneous quantification of VLCFAs and phytanic acid. The method employs single ion monitoring with deuterated internal standards. We studied 38 normal controls and 12 patients with peroxisomal diseases and found complete discrimination between the two groups. Comparison with other methodology is discussed. We believe this to be a practical and accurate method for the quantification of both VLCFAs and phytanic acid in serum or plasma. It should be useful for laboratories involved in the diagnosis of biochemical disorders.

Adrenoleukodystrophy

24 hour continuous tremor quantification based on EMG recording.

A new method is described that allows continuous quantification of parkinsonian and essential tremor over a period of up to 24 h on the basis of surface EMG recording from antagonistic forearm muscles. Tremor extraction from EMG data and all other calculations are performed on a digital computer. The occurrence, intensity and frequency of tremor are calculated. The data evaluation is fully automatic, including quality control and elimination of artifacts. Since tremor changes considerably over a 24 h period, long-term quantification gives more reliable data to study diurnal variations and to control the effects of medical treatment on tremor. As an additional advantage tremor recording is not restricted to a laboratory environment. Tremor can be monitored under everyday conditions and in the presence of moderate voluntary EMG activity.

Electromyography

Trypanosoma cruzi: quantification in tissues of experimentally infected mice by limiting dilution analysis.

A limiting dilution assay (LDA) was developed for the quantification of Trypanosoma cruzi in the heart and blood of infected mice. Three groups of swiss mice were injected ip with "CL", "Colombiana," and "Y" strains. At 1-day intervals after infection, blood and the heart were removed. Serial blood dilutions in LIT medium were performed and distributed in four groups of 24 microplate wells. The growth of parasite was visually checked in an inverted microscope. It was found that curves of parasitemia obtained by parasite counting in a hemocytometer or estimated by LDA were similar. A similar method was used to quantify parasites in the heart of mice. The heart was cut, washed, dried, and its weight was determined. The heart pieces were disrupted by passage through a mesh stainless-steel screen into LIT. Serial dilutions of the heart homogenate were made in LIT and added to at least 24 replicate microplate wells. Parasites were detectable earlier in the heart of mouse infected with Y strain when compared to CL and Colombiana strains. Parasites were detected in the heart of mice of all strains by 6 days after infection. This LDA for quantification of T. cruzi permits a more precise evaluation of the number of living parasites in infected tissues.

Animals

Quantification of enzyme activities in brain sections by microphotometry.

1. Catalytic enzyme histochemistry offers the possibility to demonstrate enzyme activities quantitatively (microphotometry) in brain sections of those sites where they are localized. 2. A prerequisite for quantification are appropriate histochemical procedures for the demonstration of enzymes, which are shortly discussed. 3. For the microphotometric determination of enzymes in brain sections the scanning microphotometry is at present the technique of choice. 4. This is described in the example of an image plane scanning system. 5. Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. kinetic and end-point measurements. 6. Methods for the microphotometric determination of certain important oxido-reductases and further enzymes are presented. 7. It is concluded that quantitative catalytic enzyme histochemistry could be a source of results complementary to those provided by conventional biochemistry.

Animals

Rapid, sensitive high-performance liquid chromatographic method for the quantification of promethazine in human serum with electrochemical detection.

A method of analysis has been developed to quantify promethazine in human serum with a sensitivity that was suitable for bioavailability studies following a 50.0-mg rectal dose. The limit of quantification from 1.0 ml of serum for promethazine using electrochemical detection was 0.200 ng/ml. At this concentration, the total coefficient of variation obtained from seven replicates over the course of three days of validation was 7.53%. The amount of serum required, the ease of sample preparation and the precision of the method at the limit of quantification demonstrated an improvement over previous assays. A validation study was completed that included an evaluation of recovery, ruggedness, linearity of response, accuracy, precision, sensitivity, stability and selectivity. The method was then used to determine promethazine serum levels in a 36-subject bioavailability study following a 50.0-mg suppository dose.

Chromatography, High Pressure Liquid

Vectorcardiographic bites. A method for detection and quantification applied on a normal material.

Vectorcardiographic bites as an expression of small fibrotic or necrotic areas in the myocardium have been discussed for many years. Distinct definitions of bites and normal limits for bites have, however, not been established. Therefore, an algorithm for computer detection and quantification of bites is presented. To find a bite, sectors of the QRS loop rotating opposite the way of the main part of the loop (eg, a clockwise-rotating sector in an otherwise counterclockwise-rotated loop) are detected. The bite is then delineated, using an iterative procedure. Finally the amplitude, duration, and area of the bite are calculated. The method for detection and quantification was applied on a well-defined normal material to obtain normal limits for bites. The commonly used criterion for an abnormal bite (amplitude greater than or equal to 0.1 mV and duration greater than or equal to 10 msec in the horizontal or sagittal plane) results in a specificity of only 87%. A specificity of 95% is yielded with the criterion of bite amplitude greater than 0.15 mV in the horizontal or sagittal plane.

Adult

Quantification of human immunoglobulins by semiautomatic polyethylene glycol precipitation radioimmunoassays. Estimation of circulating immune complexes and immunoglobulin synthesized in vitro.

Simple, reliable semiautomatic radioimmunoassays have been developed for the measurement of human immunoglobulins (IgG, IgA, IgM, IgK and Ig lambda). The assays are based on the separation of free from antibody-bound radiolabelled fragments of immunoglobulin by precipitation with 13% polyethylene glycol. The precipitate is harvested and washed on glass fibre filters by means of a commercial cell harvester. Radiolabelled lambda chain from Bence Jones urine was used for lambda chain assay and Fab' gamma from pooled IgG for the kappa assay. Labelled Fab or Fc fragments of IgG, IgA and IgM were used for the class specific assays. Selected commercial antisera were used throughout. The sensitivity limits of the assays performed according to the standard procedure were about 20 ng Ig per ml. The assays have been used for the quantification of circulating immune complexes and both cellular and secreted immunoglobulin produced in cell cultures. The results demonstrate the importance of the simultaneous quantification by L chain and H chain specific assays.

Animals

Quantification of lymphocyte activation by measurement of DNA polymerase alpha activity.

Measurement of the activity of the enzyme DNA polymerase alpha has been investigated with regard to its potential usefulness as a method for the detection and quantification of lymphocyte activation in vivo. A modified enzyme assay was developed in order to optimise measurement of activity in crude homogenates of cells or tissues, thus allowing the convenient handling of multiple samples. Specificity of the assay for polymerase alpha was ensured by the inclusion in the assay mixture of dideoxythymidine triphosphate, an inhibitor of the other eukaryotic DNA polymerases. The activity of DNA polymerase alpha was found to be closely correlated with [3H]thymidine incorporation in a mitogen-stimulated in vitro system. The usefulness of the polymerase alpha method for the quantification of lymphocyte activation was validated in 3 different in vivo systems of either immune-mediated or drug-induced lymphoid cell response.

Animals

A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b-9 complex enables selective detection and quantification of non-activated C9.

Recently, monoclonal antibodies recognizing epitopes exposed in activation products of complement but hidden in the native components have been characterized and used for selective quantification of the activated protein. We now demonstrate that an epitope in the native component C9 is hidden in the terminal SC5b-9 complex. A monoclonal antibody against this epitope enabled selective detection of C9 without influence of the amount of SC5b-9 present. This antibody recognizing native soluble C9 was used to construct a quantitative double-antibody ELISA with unique sensitivity and specificity. Combination of this assay with an assay previously described for selective quantification of the SC5b-9 complex provides an important tool for evaluating terminal pathway activation of complement.

Animals

An ELISA method for the quantification of anti-streptolysin-O antibodies.

This paper describes a new assay, based on the ELISA technique, for the quantification of antibodies to streptolysin-O (ASLO). We have compared its performances with that of a standard method (inhibition of hemolysis). Using a panel of 137 sera covering the whole range of ASLO titers, the results showed a good correlation between both methods but the ELISA method was more reproducible than the standard technique, thus represents a convenient alternative for the quantification of ASLO.

Antibodies, Bacterial