Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole.

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.

A new sensitive and fast peptide immunoassay based on enzyme amplification used in the determination of CGRP and the demonstration of its presence in the thyroid.

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.

Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAK System.

Twin-site ELISA is a simple technique for quantitation of specific proteins in cell or tissue extracts. The application of this method to fos and myc oncoproteins is described. There are two basic procedures: 1. Extraction of fos and myc proteins from biological material in a form suitable for immunoassay. 2. Determination of the amount of fos or myc protein in the extract by ELISA using specific antibodies, one of which is conjugated to alkaline phosphatase (AP) and is detected by the AMPAK™ amplifier system.

Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAK System.

Twin-site ELISA is a simple technique for quantitation of specific proteins in cell or tissue extracts. The application of this method to fos and myc oncoproteins is described. There are two basic procedures: 1. Extraction of fos and myc proteins from biological material in a form suitable for immunoassay. 2. Determination of the amount of fos or myc protein in the extract by ELISA using specific antibodies, one of which is conjugated to alkaline phosphatase (AP) and is detected by the AMPAK™ amplifier system.

Enzyme-amplified immunoassays.

The sensitivity of enzyme immunoassays may be enhanced by the use of enzyme-amplification. This technique uses the enzyme label in the immunoassay to provide a trigger substance for a secondary system that can generate a large quantity of coloured product. Two examples of enzyme amplifiers are described, using either a substrate cycle with phosphorylated hexose sugars, or a redox cycle involving the coenzyme NAD(+). The redox enzyme-amplifier has a detection limit of less than one attomole for the enzyme label, alkaline phosphatase. The limited dynamic range of enzyme-amplified immunoassays may be overcome by kinetic analysis of the colour development in the enzyme-amplifier, to add at least a further order of magnitude to the range of directly measured analyte concentrations in the immunoassay. This is illustrated in an enzyme-amplified immunoassay for human thyroid stimulating hormone. Amperometric measurement of the enzyme-amplifier provides a method to extend the dynamic range still further and compares favourably with the performance of a gamma counter, a luminometer or a fluorimeter.

Post-traumatic osteochondroma of the calcaneus.

Osteochondroma is the most common bone and cartilage tumor. It is usually congenital but may be related to trauma. A case of osteochondroma affecting the calcaneus, an unusual location for this lesion, is presented. Surgical excision appears to be as effective a cure in this location as in others.

Enzyme amplified immunoassays.

Enzyme amplification has been applied to improve the sensitivity of many immunoassays which use alkaline phosphatase as the label. The activity of the enzyme is amplified by using NAD, derived by hydrolysis of NADP, to activate catalytically a substrate cycle that generates a coloured product. In routine use, an amplification factor of 100-fold is easily achieved and, with longer incubation times, this can be increased to allow the detection of as few as 3,000 enzyme molecules. In order to avoid the limitations of spectrophotometry, an electrochemical version of the amplifier has recently been developed which performs with comparable sensitivity in a prototype device. The many advantages of electrochemical detection may ultimately herald the arrival of a new generation of simple, sensitive and inexpensive diagnostic systems.

Surgical correction of hallux abducto valgus in a cerebral palsy patient. A 5-year follow-up.

This article reviews the causes of foot deformities in patients with cerebral palsy. The authors underscore the necessity for a total evaluation of such patients. It is believed that the patient described in this article showed a recurrence of the hallux deformity due to failure to control the causative factors of this condition. Postoperative management with braces or orthoses may well have prevented recurrence.

A practical approach to determining training-solvable problems.

Training is a necessity to progressive companies, but how do you decide which problems would benefit from it? The authors' model, based on current literature and consulting experience with a large organization, stresses the importance of using existing data for a more exact and cost-effective diagnosis.

Histones H1 and H5: one or two molecules per nucleosome?

We have determined histone stoichiometries in nuclei from several sources by a direct chemical method, with the particular aim of quantitating histone H1 and, in chicken erythrocytes, H5, and of distinguishing between one and two molecules per nucleosome. The four histones H3, H4, H2A and H2B are found in equimolar amounts, as expected for the core histone octamer. The molar ratio of H1 in lymphocyte and glial nuclei is 1.0 per octamer, and in liver nuclei from three species 0.8 per octamer. These results suggest that each nucleosome has one H1 molecule; nucleosomes could acquire two molecules of H1 only at the expense of others containing none. The stoichiometry of H5 in chicken erythrocyte nuclei is similar to that of H1 in other nuclei, being about 0.9 molecules per nucleosome; the H1 also present in these nuclei amounts to 0.4 molecules per nucleosome.

Enzyme immunoassay--a new technique for estimating hemoglobin A1c.

We describe a method for estimating hemoglobin A1c (HbA1c) with a commercially available enzyme immunoassay system. The method is based on microtiter plate technology, utilizing an antibody raised to hemoglobin, the epitope being the Amadori product of glucose plus the first eight amino acids on the N-terminal end of the beta chain of hemoglobin. The enzyme immunoassay displays good within-batch (CV 2.3-2.4%) and between-batch (CV 2.6-5.0%) precision, and the results were not affected by different types of anticoagulant. The method was linear within the expected range of results and showed good correlation (r = 0.88-0.98) with established methods for estimating glycohemoglobin. Using this method, we obtained a reference interval of 2.8-4.9% (central 95%) for HbA1c in a nondiabetic population. The percentages of hemoglobin that were HbA1c in diabetics (6.86% +/- 2.51%) were significantly greater (P < 0.001) than in nondiabetics (3.46% +/- 0.52%).

Calcitonin determination by a fast and highly sensitive enzyme amplified immunoassay.

A colorimetric enzyme amplification system was used to develop an immunoassay for human calcitonin (hCT) with a sensitivity of 6 pmol/l, and intra- and inter-assay CVs of 12% and 11.8% respectively for the low pool, and 10% and 11.2% for the high pool. The mean recovery of added synthetic hCT (58.5 pmol) from the plasma of 10 patients was 110% (64.4 pmol). The correlation coefficient between radioimmunoassay (RIA) and amplified enzymo-immunoassay was found to be 0.96 (p 0.001). The assay was successfully applied to the measurement of elevated calcitonin levels in plasma from patients with medullary carcinoma of the thyroid (MCT). AEIA offered a reliable and sensitive alternative to RIA for calcitonin determination with the added advantage of convenience as the label employed was much more stable.

Neuronal nuclei and glial nuclei from mammalian cerebral cortex. Nucleosome repeat lengths, DNA contents and H1 contents.

We have characterised the histone and DNA contents of neuronal and glial nuclei from ox cerebral cortex which have, respectively, repeat lengths of 162 base pairs and 201 base pairs. Although the neuronal population cannot be obtained completely free of glial nuclear contamination, the degree of contamination is easily determined by counting, and has been allowed for in all the methods used here. By diphenylamine assay and flow cytofluorometry we find that the DNA contents of both nuclear types are essentially equal, and equivalent to the diploid value, contrary to some reports. By quantification of the core histones in known numbers of nuclei with respect to an added external standard, we have shown that the ratio of core histone octamers in the two nuclear types, neuronal and glial, is the inverse of the ratio of repeat lengths. Thus the same proportion of DNA is associated with core histone octamers in the two nuclear types, most simply all of the DNA. By complete radiolabelling of the lysine side chains of the histones with methyl [1-3H]acetimidate we have determined the stoichiometry of H1 relative to the core histones. Neuronal nuclei have a low H1 content of 0.45 molecule H1/nucleosome on average; glial nuclei have the 'normal' 1 H1 molecule/nucleosome. In neuronal nuclei about half of the nucleosomes therefore probably lack H1. Whether there is any relation between the low H1 content and the short DNA repeat length of neuronal nuclei, on the one hand, and their high transcriptional capacity (at least when assayed in vitro), on the other, remains to be established.