[The response to vaccination for hepatitis B in chronic hemodialysis patients and in staff groups exposed to hepatitis B].
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Hepatitis B↗
Biomedical subjects
B Dinesen
Publications and source records attributed to B Dinesen.
ELISA for human IgG and IgM anti-lipopolysaccharide antibodies with indirect standardization.
A new attempt to standardize ELISAs for the quantitation of human IgM and IgG anti-lipopolysaccharide antibodies (anti-LPS), without the use of a specific standard material, is described. Sandwich ELISAs for total IgG and IgM were combined with indirect ELISAs for anti-LPS IgG and IgM antibodies on a 96-well microtest plate using identical assay conditions. The concentration of specific IgG or IgM anti-LPS was read on the respective standard curve for total IgG or IgM. The results were corrected for residual immunoreactivity remaining unbound in the wells after one sample incubation in the combined assays. The quantitative results of IgG anti-LPS correlated well with results obtained using an ELISA with direct standardization (r = 0.969). 28 mg/l of IgM anti-LPS was found as median value among 121 blood donors using the described ELISA principle. Binding studies demonstrated a lower apparent affinity of donor anti-LPS IgM than anti-IgG.
Enzyme-Linked Immunosorbent Assay↗
Anti-lipopolysaccharide antibodies measured by enzyme-immunoassay in Danish blood donors.
To explore whether the intravenous administration of naturally occurring high-titer human anti-lipopolysaccharide IgG (anti-LPS) is beneficial in the treatment of human septic shock, it is necessary to select blood donors according to anti-LPS concentrations. The present paper describes an enzyme-linked immuno-adsorbent assay (ELISA) for the screening of blood donors to obtain such antibodies. Human specific IgG antibodies directed against smooth LPS from eleven different bacterial strains and species were quantitated. The ELISA is simple and uses only commercially available reagents. By optimizing assay conditions, a low unspecific background activity (OD below 0.010) was obtained, together with a large specific reading interval. 2021 human plasma samples from volunteer blood donors at six different blood-banks in Denmark were screened by the ELISA. The median anti-LPS antibody concentration was 9 mg/l. Five per cent of the donors had anti-LPS concentrations above 40 mg/l (expected to be therapeutically useful). The age- and sex-related distributions indicated that the highest prevalence of high anti-LPS concentration was in males aged 40-49 years. It is concluded that specific anti-LPS gammaglobulin based on naturally occurring anti-LPS antibodies can be obtained in Denmark following the screening of blood-donors, using the assay developed.
Adult↗
Micro-ELISA for the quantitation of human urinary IgG.
When investigating glomerular changes in the early stages of diabetic renal disease, it is important to be able to estimate low concentrations of urinary immunoglobulins, as well as the albumin/immunoglobulin ratio. For this reason there is a need for highly sensitive and specific routine assays for urinary immunoglobulins. A 'sandwich' enzyme-linked immunoadsorbent assay (ELISA) for the quantitation of urinary immunoglobulin G is described, in which microtest plates are used as the solid phase. The assay is specific, sensitive and simple, and only used commercially available reagents. The assay range was 5-200 micrograms/l. The relative standard deviations within and between assays were 5 and 9%, respectively. Recovery of IgG added to urine was 100-102% (n = 12), and dilution of urine was linear. The assay range allowed for the quantitation of IgG in human urine samples, covering the clinical range from physiological to raised pathological values.
Enzyme-Linked Immunosorbent Assay↗
ELISA for human proinsulin.
A micro enzyme-linked-immunosorbent-assay (ELISA) for monitoring circulating human proinsulin (hPI) was developed. A micro test plate was coated with guinea pig anti-insulin antibody. As labelling system peroxidase-labelled F(ab1)2-fragments of a guinea pig anti-human-C-peptide was used. The detection limit in buffer (95% level) was 0.6 pmol/l corresponding to 0.06 fmol/incubation well and to 1.2 pmol/l in serum, since samples were diluted 50%. Standard operating range was from 0-160 pmol/l. Interassay variation was 9% estimated from two human control materials (assayed within the range 6-9 pmol/l and 9-14 pmol/l, respectively). Insulin in samples did not interfere in concentrations below 400 pmol/l. Human C-peptide, porcine, and bovine proinsulins did not cross-react even at 10 000 pmol/l. In 38 healthy fasting subjects a reference range less than 1.2-13 pmol/l with a median of 4.1 pmol/l was found. Serum from total pancreatectomised patients showed values below the detection limit. The value from a patient with an insulinoma was 263 pmol/l. When stored at -20 degrees C human proinsulin appeared stable in serum or plasma for at least 9 mth. This ELISA, although among the most sensitive immunoassays for human proinsulin, is still not sensitive enough to measure the concentrations expected in samples from IDDM patients in the fasting state. In spite of this the method is useful in characterising beta-cell function in stimulated situations, as well as in the diagnosis of insulinoma.
Animals↗
Beta 2-microglobulin in urine and serum determined by a micro-ELISA technique.
An enzyme-linked immunoadsorbent assay for the determination of beta 2-microglobulin in serum and urine using microtest plates as solid phase is described. All reagents are commercially available. The assay has a high capacity and it is inexpensive using only 3% of the amounts of antibodies required in a previously described ELISA based on tubes as solid phase.
Diabetes Mellitus, Type 1↗
Reactivity of factor VIII inhibitors in a micro ELISA for factor VIII: CAg and in solid phase immunoisolation of VIII: CAg.
Factor VIII coagulant antigen (VIII: CAg) was measured in a sandwich-ELISA. Microplates were used as solid phase and peroxidase conjugated F(ab')2 fragments of IgG isolated from inhibitor plasma was used as label without affinity purification. The capacity of the assay was high and the sensitivity for VIII: CAg was 0.002 U/ml. Using this assay it was possible to measure coagulation inactive VIII: CAg in samples from purification studies. Below 0.05 VIII: CAg U/ml these samples responded in parallel with standard plasma. Seven of 7 inhibitor antibodies tested were able to inhibit binding of peroxidase-conjugate in the VIII: CAg assay, and the inhibitory capacity correlated with coagulation inhibition as measured by the Bethesda method. Using the highest titered antibodies bound to a solid phase, VIII: CAg was isolated and identified in SDS-PAGE as a doublet with a molecular weight of 77-80 kD.
Antigen-Antibody Complex↗
Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy.
An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from normoalbuminuria to overt clinical nephropathy. Intra-assay variation was 2.1% and interassay variation 8.3%. Recovery of added albumin to urine was 95%-106% and dilution of urine was linear. The correlation to urinary albumin determined by immunodiffusion was excellent (n = 80, r = 0.99). Intraindividual variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose excretion, indicating that minor metabolic variations are not responsible for the huge intraindividual day-to-day variations of UalbV. The study shows that more than one UalbV measurement must be done before classifying patients into groups with or without incipient diabetic nephropathy.
Adolescent↗
An enzyme immunoassay (ELISA) for the quantitation of human factor VIII coagulant antigen (VIII:CAg).
For the purposes of prenatal diagnosis and carrier detection of haemophilia A, an enzyme linked immunosorbent assay (ELISA) was developed for the quantitation of plasma Factor VIII coagulant antigen (VIII:CAg). It is based upon a human antibody. Results are compared to assays for Factor VIII coagulant activity (VIII:C) and Factor VIII related antigen (VIIIR:Ag) in plasma from 30 normal female individuals, 5 fetuses from mothers normal with respect to bleeding status, 10 obligate carriers of haemophilia A, 10 patients with haemophilia A, 5 with von Willebrand's disease, and 5 with haemophilia B. The ELISA developed is simpler than previously published VIII:CAg methods owing to its use of total IgG instead of immunologically affinity-purified antibodies. It is specific (as judged from clinical results), sensitive (detection limit: 0.005 units/ml), and sufficiently precise (between-assay coefficient of variation: 11%) for the purposes mentioned. The coefficient of correlation between VIII:CAg and VIII:C results is 0.86. The introduction of ELISA for quantitating VIII:CAg represents an advantage as compared to existing immunoradiometric assays (IRMA) mainly due to the stable and non-radioactive reagents used in the ELISA.
Antigens↗
The development of a rapid ELISA for IgE utilizing commercially available reagents.
An enzyme-linked immunosorbent assay (ELISA) for human immunoglobin E (IgE) has been developed. To coat the polystyrene tubes (the solid phase) an antibody concentration of 6 mg/l in sodium carbonate buffer, pH 9.8, at 37 degrees C for 24 h was found superior to other conditions. The maximum amount of globulin adsorbed to the polystyrene surface was estimated to be 2.7 mg X m-2. This is consistent with a monolayer being adsorbed. While some of the anti-IgE molecules are inactivated during adsorption, the remaining molecules, once adsorbed, appear to retain activity for extended periods, and the coated tubes were stable for several weeks. Kinetic studies of the association of analyte, of antibody-enzyme conjugate and enzymic catalysis were used for the optimisation of the assay and allowed us to reduce assay time to 6 h. The association of analyte to the coated tube and the association of anti-IgE-peroxidase conjugate to bound IgE, in the present design, had T1/2 of approximately 0.5 h. Equilibrium is not obtained. The dissociation of analyte, surprisingly, was nearly undetectable. At least one of the two antibodies employed in the sandwich assay, preferably the anti-IgE-peroxidase conjugate, should be free of non-specific antibodies in order to eliminate non-specific reactions. The influence of serum matrix was eliminated by diluting serum samples and standards 1:21 in buffer. For the assay of very low concentrations (i.e. less than 10 kIU/l) standards produced in IgE-free serum are required to compensate the influence of matrix. Comparison with a commercial kit (Hoechst-Behringwerke, Frankfurt/Main, FRG) showed good agreement. All reagents are commercially available and the assay is well suited for routine use.
Enzyme-Linked Immunosorbent Assay↗
Secretion of IgM-rheumatoid factor and IgM by blood lymphocytes in rheumatoid arthritis.
A direct plaque forming cell (PFC) assay for the detection of complement fixing IgM-rheumatoid factor (RF) secreting lymphocytes was evaluated. The specificity of the RF-PFC assay was demonstrated by the inhibitory action of exogenous human IgG. The sensitivity of the RF-PFC assay was similar to that of a reverse PFC assay detecting all cells secreting IgM (IgM-PFC). In blood from 75% of seropositive patients with rheumatoid arthritis (RA) RF-PFC were demonstrated, median value 6.8 (range: 0.4-1477) RF-PFC/10(6) mononuclear cells. Practically no RF-PFC were detected in seronegative RA patients and controls. Despite the fact that most IgM-RF undoubtedly is produced outside the blood, a positive correlation was found between the number of RF-PFC and the Waaler-Rose titer in serum. The number of circulating IgM secreting cells did not differ significantly between seropositive RA patients, seronegative RA patients and controls. Comparison of RF-PFC and IgM-PFC in seropositive patients revealed that in mean 7% of IgM-secreting cells in blood secrete IgM-RF.
Antibody Specificity↗
Production and secretion of immunoglobulins by in vitro-activated human B lymphocytes.
Activation induced by pokeweed mitogen in cultures of mononuclear cells from human blood was followed sequentially by simultaneous quantitation of live cells, thymidine incorporation, cells displaying cytoplasmic, IgM, IgG, IgA or IgD, cells secreting IgM, IgG or IgA and cumulated IgM secretion. Maximal cellular activity was found after 7 days of cultures, with means of 16000 IgM-, 20700 IgG- and 9900 IgA-secreting cells per 10(6) originally cultured cells. The cumulated IgM secretion after 21 days of culture averaged 10400 ng per 10(6) originally cultured cells. A close correlation was found between the number of IgM-secreting cells and the cumulated IgM secretion.
Antibody-Producing Cells↗
[30-minute ACTH test. Reproducibility and interpretation].
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Adrenocorticotropic Hormone↗
Comparison of pituitary-adrenocortical response to hypoglycaemia and surgery.
The hypothalamic-pituitary-adrenocortical (HPA) responses to the hypoglycaemic stimulus and to operation (laparotomy) are compared. The studies were carried out in eight patients who were subjected to insulin-induced hypoglycaemia on the day before elective abdominal surgery. Plasma corticotrophin (ACTH) and plasma cortisol concentration increases were virtually indentical during the two procedures, which suggests that hypoglycaemia and surgical trauma are comparable stress factors and that the hypoglycaemia test is reliable in predicting the HPA responses to major surgical trauma.
Adrenocorticotropic Hormone↗
Plasma corticotropin and cortisol in patients with pituitary adenomas.
Plasma ACTH and cortisol were measured during insulin-induced hypoglycemia in 39 patients with a pituitary adenoma or after hypophysectomy and in 26 healthy persons. Adrenocortical insufficiency was defined as failure to increase plasma cortisol to at least 500 nmol/liter (18 microgram/dl) during hypoglycemia. No proper distinction was possible between patients with normal and impaired cortisol response to hypoglycemia on the basis of the ACTH results. When all 39 patients were compared to the 26 controls, the cortisol results differed significantly, but those of ACTH did not. These results demonstrate a limited value of ACTH measurements in secondary adrenocortical failure.
Adenoma↗
A sensitive enzyme immunoassay for the quantitation of human IgM.
A highly sensitive and specific two-site solid-phase enzyme immunoassay for the quantitation of human immunoglobulin M (IgM) in biological fluids is presented. Detection limit is 20 micrograms/l (12 ng/tube). Routine working range is 30--1000 micrograms/l. Between-assay coefficient of variation is 10%. The assay allows quantitation of IgM in human cerebrospinal fluid.
Body Fluids↗
[30-minute ACTH test to determine integrated hypothalamo-hypophyseal adrenocortical function].
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Adrenocorticotropic Hormone↗
Discrepancy between ACTH and cortisol responses to insulin induced hypoglycaemia.
The purpose of this study was to assess the normal relationship between plasma ACTH and cortisol during insulin induced hypoglycaemia. Twenty-six healthy persons were studied. Peak plasma concentration and the amounts of ACTH and cortisol secreted during hypoglycaemia were compared. There was no significant correlation between the ACTH and cortisol values. The ACTH responses varied significantly more than those of cortisol. This probably signifies an over-capacity of ACTH secretion in comparison to the secretory capacity of the adrenal cortex.
Adrenocorticotropic Hormone↗