First direct assay for intact human proinsulin Houssa, Paule; Dinesen, Bo; Deberg, Michelle ...
Clinical chemistry (Baltimore, Md.),
07/1998, Letnik:
44, Številka:
7
Journal Article, Web Resource
Recenzirano
Odprti dostop
We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 microL of serum or plasma. The assay is based on the use of two ...monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2-100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1-6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2-18 pmol/L) and 153 pmol/L (98-320 pmol/L), respectively.
We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity ...(directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (approximately 10(10) L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.
Insulin autoimmune hypoglycemia is characterized by recurrent hypoglycemia and high levels of immunoreactive insulin in the presence of insulin autoantibodies. The mechanisms inducing hypoglycemia ...are largely unknown. An 123Iinsulin scintigraphic scanning was performed to directly demonstrate the effect of antibodies on insulin biodistribution in one patient with this syndrome both before and after treatment. The patient had insulin autoantibodies IgG3 lambda, which had a single site dissociation constant (Kd = 10(-7) mol/L, by Scatchard analysis), a very fast dissociation rate of immune complexes, and a very rapid association of 125Iinsulin. Insulin receptors on red blood cells were down-regulated. The 123Iinsulin scintigraphic study imaged the buffering effect of antibodies on insulin bioavailability. 123IInsulin was not removed from the blood, and no liver or kidney uptake of the hormone occurred. The frequency and severity of hypoglycemic episodes required treatment. Insulin antibody levels decreased and 123Iinsulin biodistribution improved after treatment with plasmapheresis and prednisone. Improved hormone bioavailability was further evidenced by the reduction in the hypoglycemic delay after i.v. insulin from 90 min before any treatment to 60 min after plasmapheresis and 30 min after steroid administration. Glucose tolerance was normal after treatment. Plasmapheresis followed by steroid treatment can lower the insulin antibody concentration, abolish severe hypoglycemia, and improve insulin biodistribution and glucose tolerance in insulin autoimmune hypoglycemia.
Visualization of viral clearance in the living animal Verdin, E.M; Maratos-Flier, E; Kahn, C.R ...
Science (American Association for the Advancement of Science),
04/1987, Letnik:
236, Številka:
4800
Journal Article
Recenzirano
The early events in viral dissemination via the bloodstream were identified by monitoring the fate of $^{123}$I-radiolabeled reovirus after it was injected intravenously in rats. Continuous ...scintillation camera imaging showed that reovirus serotypes 1 and 3 were cleared from the circulation in less than 10 minutes by specific and distinct target organs. Reovirus serotype 1 accumulated predominantly in the lungs and the liver, whereas serotype 3 accumulated in the liver and the spleen with very little virus uptake by the lungs. Incubation of reovirus serotype 1 with a monoclonal antibody directed against the viral hemagglutinin before injection totally inhibited the clearance of the virus by the lungs. Similar results were obtained when viruses biolabeled with $^{35}$S were used. These results demonstrate that viruses can be rapidly transported through the bloodstream to specific target organs and that the localization of the viruses depends on the interaction between specific viral surface components and the target organ.
Scintigraphic scanning with 123Iinsulin provides a direct and quantitative assessment of insulin uptake and disappearance at specific organ sites. Using this technique, the biodistribution and ...metabolism of insulin were studied in type 1 diabetic patients and normal subjects. The major organ of 123Iinsulin uptake in both diabetic and normal subjects was the liver. After iv injection in normal subjects, the uptake of 123Iinsulin by the liver was rapid, with peak activity at 7 min. Activity declined rapidly thereafter, consistent with rapid insulin degradation and clearance. Rapid uptake of 123Iinsulin also occurred in the kidneys, although the uptake of insulin by the kidneys was about 80% of that by liver. In type 1 diabetic patients, uptake of 123Iinsulin in these organ sites was lower than that in normal subjects; peak insulin uptakes in liver and kidneys were 21% and 40% lower than those in normal subjects, respectively. The kinetics of insulin clearance from the liver was comparable in diabetic and normal subjects, whereas clearance from the kidneys was decreased in diabetics. The plasma clearance of 123Iinsulin was decreased in diabetic patients, as was insulin degradation, assessed by trichloroacetic acid precipitability. Thirty minutes after injection, 70.9 +/- 3.8% (+/- SEM) of 123Iinsulin in the plasma of diabetics was trichloroacetic acid precipitable vs. only 53.9 +/- 4.0% in normal subjects. A positive correlation was present between the organ uptake of 123Iinsulin in the liver or kidneys and insulin degradation (r = 0.74; P less than 0.001). Both decreased insulin uptake in the liver and kidneys and decreased insulin degradation were inversely correlated to the binding capacity of antiinsulin antibodies in plasma of diabetics (r = -0.87 to -0.92; P less than 0.001). In summary, type 1 diabetic patients have altered patterns of insulin biodistribution and metabolism, with decreased organ uptake and slow degradation of 123Iinsulin, which correlated with the insulin antibody-binding capacity of their serum. Thus, antiinsulin antibodies, even at subclinical concentrations, may modulate the metabolic effects of insulin in the diabetic by prolonging the biological half-life of the hormone as well as by altering its distribution and uptake at specific organ sites.
Superiority of radiobinding assay over ELISA for detection of IAAs in newly diagnosed type I diabetic children.
C Levy-Marchal ,
M P Bridel ,
F Sodoyez-Goffaux ,
M Koch ,
J Tichet ,
P Czernichow and
...J C Sodoyez
Department of Diabetology, Robert Debré Hospital, Paris, France.
Abstract
OBJECTIVE: Liquid- or solid-phase assays have been used for insulin autoantibody (IAA) determination, and the method of IAA
measurement has not been standardized. RESEARCH DESIGN AND METHODS: IAAs were determined by radiobinding assay (RBA) and enzyme-linked
immunosorbent assay (ELISA) in two large age-matched groups of nondiabetic and newly diagnosed insulin-dependent (type I)
diabetic children. RESULTS: Positivity for IAA by RBA (greater than or equal to nondiabetic mean + 3SD) was 2 of 178 (1.1%)
and 55 of 173 (32%) in nondiabetic and diabetic children, respectively. Prevalence of IAA by RBA was significantly higher
in the youngest age-group (63% between 0-4 yr). Positivity for IAA by ELISA was 1 of 178 (0.6%) and 8 of 169 (4.7%) in nondiabetic
and diabetic children, respectively. Concordance rates between both assays were 0 of 3 (0%) in control subjects and 5 of 58
(8.6%) in diabetic children. CONCLUSIONS: We conclude that RBA is more appropriate than ELISA for IAA detection at the onset
of the disease. In addition, because available data suggest that IAAs detected by RBA only are high-affinity antibodies, it
is tempting to speculate that IAAs reflect a mature immune reaction against endogenous insulin.
Immunogenicity of long-term intraperitoneal insulin administration with implantable programmable pumps. Metabolic consequences.
V Lassmann-Vague ,
P Belicar ,
D Raccah ,
B Vialettes ,
J C Sodoyez and
...P Vague
Centre Hospitalier Régional et Universitaire Timone, Marseille, France.
Abstract
OBJECTIVE--To assess immunogenicity of intraperitoneal insulin infusion via implanted pumps by two methods and to evaluate
the possible influence of an increased antibody level on metabolic and clinical parameters. RESEARCH DESIGN AND METHODS--We
studied insulin antibody levels in 17 type I diabetic patients before and until 24 months after implantation of a programmable
pump delivering insulin intraperitoneally. Antibody levels were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent
assay (ELISA). They were correlated with HbA1c, insulin requirements, free insulin, and the incidence of hypoglycemia. RESULTS--Insulin
antibodies increased as soon as the 3rd month after implantation. This increase was sustained throughout the study period
(month 0, 25.4 +/- 16.2%; month 3, 41.2 +/- 23.5%; month 12, 45.9 +/- 26%; month 24, 48.7 +/- 25%). The data was correlated
with the two assay methods (RIA and ELISA). Postimplantation level was correlated with preimplantation level, which could
indicate a predictive value of the latter . No correlation was observed with any metabolic parameters, particularly the number
of hypoglycemic episodes. CONCLUSIONS--Our results indicate that intraperitoneal insulin administration by implantable programmable
pumps leads to an increase of insulin antibodies, which are probably high-affinity antibodies (recognized by both RIA and
ELISA). This increase in insulin immunogenicity did not induce significant metabolic consequences, which is reassuring for
the future of programmable insulin pumps.
The circannual rhythms of plasma 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), total and free cortisol have been documented on a circadian basis in January, March, June and October in seven young ...men (24 years old), six elderly men, six elderly women and six elderly demented subjects, both men and women, in their eighties. Blood samples were drawn every 4 h over a 24-h period at each sampling session and urine samples were collected at 4-h intervals only from the young men. A circadian rhythm of 17-hydroxy-corticosteroids (17-OH-CS), 17-ketosteroids (17-KS), urinary free cortisol and 18-OH-DOC was defined for each of the four seasons with stable acrophases throughout the year and the same excretory profiles. A circannual rhythm was validated in young men for 17-OH-CS, urinary free cortisol and 18-OH-DOC but not for 17-KS. A circadian rhythm of plasma free cortisol, the active form of the hormone, plasma total cortisol and plasma 18-OH-DOC was validated in all groups and at all the seasons at which samples were taken. The secretory profiles of 18-OH-DOC, free and total cortisol were very similar, with no differences attributable to age, sex or mental condition except for the levels of plasma free cortisol and 18-OH-DOC which were higher and lower respectively in the elderly subjects. Whereas a circannual rhythm of plasma 18-OH-DOC was validated for all groups, a circannual rhythm of both free and total cortisol in the plasma was validated in young men but not in any group of elderly subjects. This loss of the circannual rhythmicity of cortisol in the elderly may reflect the decrease with age of the capacity to adapt to seasonal external factors.
Circadian changes in plasma 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), total and unbound cortisol were studied in four groups: seven healthy young men, six elderly men, six elderly women and six ...elderly demented patients of both sexes. The daily activities of the subjects were synchronous; blood samples were taken every 4 h and 4 hourly urine samples were collected only from the young men. A circadian rhythm was defined for plasma 18-OH-DOC, total and unbound cortisol in all groups; the secretory patterns of these steroids were parallel, as were the profiles of urinary 18-OH-DOC and unconjugated cortisol. When compared with respect to sex, the 24-h mean level of total cortisol was higher in women; that of unbound cortisol was higher in the three groups of elderly patients than in the young men. No major changes in plasma steroids were observed between elderly demented patients (mainly women) and healthy elderly women. The phasing of total and unbound cortisol showed no major modifications with age, sex or senile dementia. Acrophases of 18-OH-DOC were earlier in elderly patients than in young men. Amplitudes were not modified with sex in elderly patients but were always lower in the demented patients. A circadian rhythm was defined for 18-OH-DOC, unconjugated cortisol, 17-hydroxycorticosteroids (17-OH-CS) and 17-ketosteroids in the urine of the young men. The acrophases of 18-OH-DOC and unbound cortisol were close, as were those of 17-OH-CS and 17-ketosteroids. The lag was short between the acrophases of 18-OH-DOC in plasma and urine and between those of plasma unbound cortisol and urinary unconjugated cortisol; it was much larger between the acrophases of plasma total cortisol and 17-OH-CS. Thus, the process of ageing, and the possible alterations in the central nervous system which are often seen in normal ageing, induced no major modifications in the temporal organization of adrenocortical function, even in subjects who were very advanced in age.
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