A rapid, sensitive enzyme-linked immunoassay for human thyrotropin

In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.     

A rapid and sensitive immunoresonance scattering spectral assay for microalbumin

BACKGROUND: Microalbuminuria (MAU) is the earliest clinical finding for renal disease and a risk factor for hypertensive cardiovascular disease. Several methods, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoturbidimetry (IT), immunonephelometry (IN), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA) and time-resolved fluorescence (TRF) have been applied for detection of MAU. However, the resonance scattering (RS) spectral assay, based on the immunoreaction and its resonance scattering effect, has not been reported. METHOD: In the presence of 75 mg/l polyethylene glycol (PEG), the immunoreaction of microalbumin (Malb) and its goat anti-human Malb antibody took place specifically in pH 4.4 buffer solution and aggregated to form immunocomplex particles that exhibit a strongest resonance scattering peak at 488 nm, and it was used to assay of Malb. RESULTS: The RS intensity at 488 nm (DeltaI) was proportional to the Malb concentration (C) in the range of 0.03-0.96 mg/l, the regression equation was DeltaI=116.0C-2.1, the detection limit was 0.02 mg/l. Urine samples from 20 healthy subjects were assayed by this assay. The results were in agreement with those obtained with IT. CONCLUSION: This assay has been applied to detection of Malb in real samples, with simplicity, rapidity, high sensitivity and good selectivity.     

Use of a rapid, sensitive immunoradiometric assay for thyrotropin to distinguish normal from hyperthyroid subjects

Using a new three-site immunoradiometric assay for thyrotropin (TSH), we measured concentration of this hormone in the serum of 47 patients with hyperthyroidism and 46 controls. The mean and range of serum TSH concentration was significantly lower in thyrotoxic than in control subjects, and it was possible to correctly identify 96% of thyrotoxic patients on the basis of a serum TSH concentration less than 0.5 milli-int. unit/L. We conclude that such measurement is highly sensitive for distinguishing hyperthyroid from normal subjects, and that the lower limit of normal for TSH in serum is about 0.5 milli-int. unit/L.     

Development of a rapid and sensitive immunofluorometric assay for glutathione S-transferase A

BACKGROUND: The short half-life of the alpha-class glutathione S-transferases (GSTAs) in plasma combined with their even distribution throughout the liver lobule suggests that they may be useful complements to the more traditionally used liver markers. However, the currently available assays for measuring GSTAs in biological fluids have a poor dynamic range and are cumbersome, requiring multiple steps and prolonged incubation times. METHODS: Hybridomas that secrete monoclonal antibodies to human GSTAs were produced and used to develop a rapid one-step immunometric assay for the determination of GSTA in serum. The assay uses a time-resolved immunofluorometric assay (TR-IFMA) format and requires 35 min of incubation. The reference interval was determined using 208 serum samples from healthy blood donors. We also compared our TR-IFMA with a commercially available enzyme immunoassay (EIA) for GSTAS: RESULTS: The assay had a detection limit of 0.07 microg/L with a measuring range up to 625 microg/L. Within-run imprecision (CV) was 1.8-2.6% over the concentrations of GSTA tested (2.5-311 microg/L), with a between-run CV of <5%. In healthy blood donors, the median values and reference intervals were 2.0 microg/L and 0.6-7.2 microg/L for females and 2.6 microg/L and 0.7-9.8 microg/L for males, respectively. GSTA concentrations determined with the TR-IFMA correlated well with those obtained using a commercially available EIA. CONCLUSIONS: This report describes a new assay for monitoring the concentrations of GSTAs in human serum. The method may be useful in further evaluating the potential of monitoring serum GSTAs in the routine clinical setting.     

Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria

We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.     

A sensitive colorimetric assay for human urinary kallikrein

l-Prolyl-l-phenylalanyl-l-arginine-α-naphthylester (Pro-Phe-Arg-NE) was synthesized as a new substrate for use in the assay of kallikreins. An assay was developed based on the colorimetric determination of α-naphthol released by the enzyme reaction.With Pro-Phe-Arg-NE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001 KU. Thus use of Pro-Phe-Arg-NE provides a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25-μl sample of human urine.Zymograms of human urinary kallikrein were prepared using Pro-Phe-Arg-NE as substrate. Six bands were separated by polyacrylamide disc gel isoelectrophoresis.     

Glycosidases in normal and diseased human muscle

A simple and reproducible method of extracting free tryptophan in plasma by ultrafiltration is described. Tryptophan concentration in plasma and ultranitrate is measured by a modification of the method of Denckia and Dewey.     

A sensitive immunochemiluminescence assay for human serum amyloid A protein

We describe an immunochemiluminescence assay for human plasma serum amyloid A protein (SAA) in which specific rabbit polyclonal antibodies against synthetic peptides are used. The detection of the antigen-antibody reaction at 425 nm is based on a brief emission of light by a luminophor component (signal) in response to chemical energy. The working range of the assay covers plasma SAA concentrations from 5 to 100 micrograms/L. The lower detection limit is 5 micrograms/L, the within- and between-assay CVs are less than 12%. Bilirubin, cholesterol and triglyceride in final concentrations of up to 220 mumol/L, 8.1 mmol/L and 2.68 mmol/L, respectively, do not interfere with the assay. Results were correlated with those obtained by the enzyme-linked immunosorbent assay using the same antibodies (r = 0.95; p less than 0.001; n = 50). This method is inexpensive, simple and easily automated.     

A rapid and sensitive assay for the quantitation of carboxypeptidase N, an important regulator of inflammation

BACKGROUND: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration. METHODS: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine. RESULTS: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140). CONCLUSION: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.     

Arylamidases in normal and diseased human muscle.

Human skeletal muscle homogenates were found to contain enzymes that catalyze the hydrolysis of beta-naphthylamides of leucine, arginine and lysine, known substrates for neutral and basic arylamidases. They also contained a trace of activity towards alpha-aspartyl-beta-naphthylamide. The muscle arylamidases were found to be inhibited by p-chloromercuribenzoate, Hg2+ and puromycin. Leucyl, arginyl and lysysl arylamidases were slightly activated by cobalt ions. When compared to controls, no significant differences in muscle arylamidase activities were observed in patients with muscular dystrophies and certain denervating diseases.     

Acid hydrolases in normal and diseased human muscle

The activities of acid phosphatase and β-acetylglucosaminidase in muscles from normal persons and from patients with various muscular and neuromuscular diseases have been determined. They are normal in all minimally abnormal muscles irrespective of their different disease etiologies. Increased amounts of these enzymes are only found in the severely deranged muscles obtained from patients with Duchenne muscular dystrophy, other dystrophies, polymyositis, spinal muscular atrophy and in other myopathies. These results suggest that these enzymes are involved in muscle degeneration in humans.     

A rapid, sensitive and specific radioimmunoassay for methotrexate

A rapid, sensitive, radioimmunoassay for methotrexate has been developed using tritiated methotrexate and an antiserum raised in rabbits against the immunogen, bovine serum albumin containing 34 methotrexate residues per protein molecule. Separation of bound and free drug was attained using donkey anti-rabbit precipitating serum. The method can accurately measure as little as 50 fmol methotrexate in capillary blood samples and is not interfered with by vast excesses of natural folates nor citrovorum rescue factor. The method can handle 30 samples per day with the results available after 6 h. This makes it suitable for monitoring serum methotrexate levels after high dose therapy when a result is required within 24 h to provide helpful information regarding impending toxicity and the need for extended citrovorum rescue factor or maintenance of urinary pH.     

A highly sensitive chemiluminescent assay for glycerylphosphorylcholine in human seminal plasma

Glycerylphosphorylcholine (GPC), one of the major phosphorus-containing-choline compounds of seminal plasma, is secreted mainly by the epididymal epithelium under androgenic control. This study reports a new method that uses chemiluminescence to determine seminal GPC content, comparing it with a spectrophotometric technique. The results, obtained with both techniques studying 20 fertile patients (as control), 35 infertile patients with normospermia, 23 infertile patients with oligozoospermia and impaired motility and 9 patients with excretory azoospermia, demonstrate that the GPC chemiluminescent assay is more sensitive, simple and rapid than the spectrophotometric assay. Our data confirm that GPC may be used as a marker of vas deferens and ejaculatory duct perviousness, suggesting a possible role of this glycerophosphodiester in sperm motility.     

A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates

BACKGROUND: Diversity in clinical outcome, due to different species of Leishmania, and its presence in asymptomatic blood donors in endemic areas warrant development of methods that are sensitive and can rapidly identify infecting species. STUDY DESIGN AND METHODS: The kinetoplast minicircle DNA is known to have heterogeneity in sequence and is present in many thousands of copies in Leishmania. Fluorescence‐based polymerase chain reaction (PCR) was used to amplify minicircle DNA from six Leishmania species from different geographic locations. The sequences were then used to construct a phylogenetic tree. Speciation of 46 blinded parasite clinical isolates from various geographic regions was validated using the assay. RESULTS: Analysis displayed a distinct cluster for each species or strain. Forty‐three of 46 isolates were correctly assigned to the same species identified by isoenzyme electrophoresis. The three untyped isolates were all either new species or samples from a unique geographic region. The minicircles of the three isolates formed new clusters in the tree analysis. Using minicircle DNA as PCR target, the sensitivity of the parasite detection in the spiked blood samples was five parasites per mL. CONCLUSION: Increased sensitivity and speciation without the need for parasite culture will be useful for diagnosis and treatment in clinical settings.