Influence of different luminols on the characteristics of the chemiluminescence reaction in human neutrophils.

In search for a luminol with very high output of light, 20 different luminol samples were tested for their ability to enhance the chemiluminescence reaction in phorbol myristate acetate activated human neutrophils. We found that the majority of luminols tested (17 samples) gave almost the same light output from neutrophils, and that the major part of the activity was from an intracellular origin. Owing to the fact that three isoluminol samples were unable to monitor respiratory burst activity taking place intracellularly, a very low level of chemiluminescence was obtained with these samples. Their light output was, however, greatly increased when horseradish peroxidase or myeloperoxidase was added, showing that the light-generating reaction with isoluminol as well as with luminol is peroxidase-dependent. The fact that isoluminol could also use myeloperoxidase as amplifying peroxidase, suggests that that the lack of measurable intracellular activity in the presence of isoluminol is somehow related to a limited or restricted diffusion of the molecule to intracellular sites. The isoluminol system constitutes a sensitive system for measuring release of oxygen metabolites from phagocytic cells.

Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers.

Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.

Enzyme-labeled antibodies in bioassays.

Antibody-enzyme conjugates are widely utilized in all spheres of specific analyte detection and measurement, and several trends are evident that will sustain, or even extend, this in the coming years. Of principal importance are the trends toward the development of simplified formats for the rapid and sensitive quantitation of a wide range of analytes without expensive or cumbersome instrumentation, and the exploitation of different types of enzyme and antibody molecules. Advances in hybridoma and recombinant genetics are enabling the practical manipulation of the theoretical repertoire of these reagents, facilitating their availability for a myriad of applications.

Immunoreactivity of plasma parathyrin-related peptide: three region-specific radioimmunoassays and a two-site immunoradiometric assay compared.

We measured parathyrin (parathyroid hormone)-related peptide (PTHRP) in plasma by three region-specific RIAs and compared them with an established two-site immunoradiometric assay (IRMA) of PTHRP1-86 in samples from control subjects and from patients with primary hyperparathyroidism (PH) and humoral hypercalcemia of malignancy (HHM). The two direct RIAs of PTHRP1-34 and PTHRP37-67 were specific for regions 9-18 and 52-61, respectively. In the extraction RIA of PTHRP1-34 we used an affinity gel containing a monoclonal antibody specific for the 17-27 sequence; cross-reacting PTHRP species eluted from the gel were assayed by the RIA of PTHRP1-34. PTHRP1-86 plasma concentrations by IRMA were less than 0.23 pmol/L in control subjects and patients with PH, and were significantly increased in patients with HHM (mean 6.1 pmol/L, P less than 0.001). In contrast, plasma PTHRP1-34 concentrations were not significantly different in the three groups; in HHM patients, the mean was 190 pmol/L. Plasma PTHRP37-67 concentrations were similar in control and PH groups and, although significantly increased in HHM patients (mean 440 pmol/L, P less than 0.002), showed poor diagnostic discrimination. PTHRP1-34 concentrations after affinity extraction of plasma were also significantly higher in HHM patients (mean 10.7 pmol/L, P less than 0.001), but showed incomplete diagnostic discrimination. We conclude that the diagnostic utility of the direct RIAs for quantifying PTHRP is markedly inferior to the IRMA of PTHRP1-86.

Development and validation of a two-site immunoradiometric assay for human atrial natriuretic factor in unextracted plasma.

This two-site immunoradiometric assay (IRMA) for human atrial natriuretic factor (ANF)99-126 in plasma utilizes a mouse monoclonal antibody raised against rat (r) ANF103-125 with specificity directed towards the ring structure of ANF, and a rabbit antiserum to human (h) ANF99-126. The monoclonal antibody is radioiodinated, and the IgG fraction of the antiserum is coated onto wells of a microtiter plate. Plasma or standard hANF99-126 (150 microL) is incubated with the radioligand in coated wells for 24 h. The detection limit is 0.9 pg per well, corresponding to 2 pmol/L, with a working range (CV less than 10%) from 4.5 to 540 pmol/L. Intra- and interassay precision are 7% and 9%, respectively, and the assay is unaffected by plasma matrix. In humans, the IRMA is specific for hANF99-126, the major circulating form of ANF, and does not cross-react with metabolites having deletions at the carboxy terminus. Plasma IRMA values in normal seated subjects were 6.6 +/- 1.5 (SEM) pmol/L and results correlated with those of an extraction RIA (r = 0.81, P less than 0.001).

An enhanced chemiluminescent enzyme immunoassay for follicle stimulating hormone.

An enhanced chemiluminescent enzyme immunoassay for serum follicle stimulating hormone is described which involves sequential reaction of anti-follicle stimulating hormone antibody immobilised to the inside surface of an opaque microtitre plate with sample, monoclonal anti-alpha thyroid stimulating hormone antibody, and an anti-mouse IgG - horseradish peroxidase conjungate. Bound peroxidase activity was measured using a p-hydroxycinnamic acid enhanced chemiluminescent luminol-hydrogen peroxide reaction. The assay was sensitive (detection limit 0.01 mU/well) precise (intra-assay precision 2.5-8.1%, inter-assay precision 6.7-11.9%) and results obtained with this assay and a competitive radioimmunoassay were in good agreement (correlation coefficient 0.98).

An enhanced chemiluminescence enzyme immunoassay for serum oestradiol.

Oestradiol in serum was determined with a simple enhanced chemiluminescent enzyme immunoassay. The assay is based on oestradiol labelled with horseradish peroxidase and the IgG fraction of an oestradiol antiserum coated on a black polystyrene microtitre plate. The enzyme activity of bound label was determined using a p-hydroxycinnamic acid-enhanced chemiluminescent reaction. The assay was sensitive (1.8 fmol/well), precise (intra- and inter-assay CV 4-10% and 8-12%, respectively for sample concentration in the range 122-1330 pmol/L) and showed good agreement with conventional radioimmunoassays (r = 0.99).

Improvement in the antibody binding characteristics of microtitre wells by pretreatment with anti-IgG Fc immunoglobulin.

A preliminary coating of the surface of polystyrene microtitre wells with anti-IgG Fc immunoglobulin improves the binding capacity of the wells for antibody and leads to a marked improvement in the reproducibility of immunoassays carried out in the wells. In an enhanced chemiluminescent enzyme immunoassay for oestradiol the binding capacity for oestradiol of wells precoated with anti-IgG Fc immunoglobulin and then coated with anti-oestradiol immunoglobulin was more than twice that of wells coated directly with anti-oestradiol immunoglobulin. Intra-assay precision in the precoated wells ranged from 1.3 to 4.0% compared to 2.9 to 6.7% (0-3200 fmol oestradiol) in wells which had not been precoated.

Inhibition of membrane bone formation by vitamin A in the embryonic chick mandible.

Embryonic chick mandibles (Hamburger and Hamilton [HH] stages 17-25) were cultured in the presence of various concentrations of vitamin A to determine the effect of hypervitaminosis A on membrane bone formation. In normal development, the mandible differentiates a centrally located Meckel's cartilage surrounded by membrane bones. Mandibles cultured without added vitamin A differentiated normally, though the timing of differentiation was retarded from that in ovo. Treatment with vitamin A interfered with skeletogenesis to varying degrees depending upon the initial age of the explant and the concentration of vitamin A. At low concentrations of vitamin A (1 microgram/ml), neither cartilage nor membrane bone formed in young explants (HH stage 17), whereas cartilage formed in 78% and membrane bone in 11% of older explants (HH stage 25). Higher concentrations of vitamin A (2-4 micrograms/ml) inhibited membrane bone formation in all explants, and 4 micrograms/ml of vitamin A inhibited chondrogenesis in most (88%) of the older explants. To determine whether tissue interactions influence this effect of vitamin A on skeletogenesis, mandibular mesenchyme was separated from its epithelium and treated with vitamin A. Under normal culture conditions, isolated mesenchyme (HH stage 25) differentiated both cartilage and membrane bone. Hypervitaminosis A inhibited membrane bone formation in the isolated mesenchyme at all levels tested (1-4 micrograms/ml) and inhibited chondrogenesis at levels 2-4 micrograms/ml. Hence, vitamin A can act directly upon the mesenchyme to inhibit both membrane bone formation and chondrogenesis, but its action is mitigated by the presence of the epithelium.

Enhanced luminescence enzyme immunoassay for factor VIII related antigen.

A sandwich enzyme immunoassay for plasma factor VIII related antigen has been developed which exploits a para-iodophenol enhanced chemiluminescent reaction to detect the horseradish peroxidase label. The assay entailed 15 min incubations with sample and with conjugate and had a detection limit of 0.12 mU. It showed good within batch precision (coefficient of variation = 2.95-5.8%) and results on a series of 57 specimens agreed with results obtained by immunoelectrophoresis (correlation coefficient = 0.97).

An enhanced chemiluminescence enzyme immunoassay for serum progesterone.

A competitive enhanced luminescent enzyme immunoassay for serum progesterone is described, which is based on a 11 alpha-hydroxyprogesterone 11-hemisuccinyl-horseradish peroxidase conjugate and a black polystyrene microtitre plate sensitised with anti-progesterone IgG. Bound label was determined using a mixture of 4-iodophenol, luminol and peroxide, and the light emitted from the wells of the plate quantitated using a luminescent plate reader. The assay was sensitive (detection limit 0.5 pg), precise (CV 2.7 - 9.0% in the concentration range 4.3-67.7 nM) and showed good correlation (r = 0.99) with a conventional radioimmunoassay.

The extent and causes of malnutrition in children in the Tsolo district of Transkei.

A survey of children under 5 years old living in a rural Transkei village was carried out. It was found that 36% of the children were below the Boston third percentile (BTP) (including 57% of those children between the ages of 18 and 32 months), and that nearly 30% die before the age of 2 years. The relative importance of education, dietary understanding and socio-economic status in accounting for this situation is assessed.