Radioimmunological Assay Research Articles

LH-RH ANALOGUE TREATMENT OF CENTRAL PRECOCIOUS PUBERTY: MONITORING BY BIOLOGICAL ASSAY FOR LH

Five children (2 M, 3 F) aged 3.5-9.5 yrs, with central precocious puberty and 1 boy (12 yrs old)with corpus callosum agenesis and sexual aggressive behaviour, have been treated with LH-RH Analogue (Buserelin): a)First 15 days:30±10% mcg/Kg/die subcutaneously; b)Later:1200 mcg/die intranasal, via nebulizer in 3 doses. Each child was assessed for a complete hormonal picture, particularly: a)LHRH test and sexual steroids(Ria method);b)Biological and radioimmunological assay for LH(-30′, -15′, 0)and the mean of the biological/immunoreactive LH ratio(B/I;prepubertal values <0.6) (Fraioli et al.J.Endocrinol.Invest.8:513, 1985). Our results, both clinical and biochemical, returned to normalprepubertal condition at 1 month until the 3rd mo. After this time, they showed an escape of B/I ratio, raising at 2.8(6 mo);2.16(4 mo); 3.1 (4 mo);2.7(12 mo) in spite of persisting normalization of clinical findings and sexual steroid levels. These results can be attributed to an inadequate or discontinuous absorption during the intranasal administration or to a hypothetical change of LH native molecule, occurring during long-term treatment. The elevation of B/I seems to represent an early useful monitor for a new assessment of dosage or route of administration.

Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias

Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in "serum-free" cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM-CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two "de novo" leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.

Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Erythropoietin on Leukemic Erythroid Colony Formation in Human Early Erythroblastic Leukemias

AbstractErythroid colonies from five patients with an early erythroblastic leukemia were obtained in “serum-free” cultures in the presence or absence of recombinant granulocyte-macrophage colony–stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM-CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth.In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two “de novo” leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients’ (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.

Estimation of urinary 6β-hydroxycortisol by "on-line" liquid chromatography

Urinary excretion of 6β-hydroxycortisol is widely used as a simple, non-invasive index of enzyme induction in man and as a biochemical marker for chronic hyperglucocorticoid states. Beside radioimmunological assays, liquid Chromatographie methods for its estimation have been described. Because of the immense challenge to eliminate interfering urinary chromogens, these techniques are very troublesome and time-consuming. Using a previously described principle of sample clean-up, we developed a fully automated, liquid Chromatographie method for 6β-hydroxycortisol estimation in human urine.

56. PHOSPHOLIPIDS AND TRYPSIN GASTRIC OUTFLOW: A MARKER OF DUODENO-GASTRIC REFLUX (DGR) IN CHILDREN

Potential toxicity of bilio-pancreatic secretions on oesophageal and gastric mucosa is wellknown in adults. Nevertheless DGR had never been assessed in children. After a 6 hours fast, we measured the following parameters by their one hour gastric outflow, collected with a naso-gastric tube : trypsin (TR), marker of pancreatic reflux, by radio-immunologic assay; phopholipids (PLP), i.e.lecithin and lysolecithins, markers of biliary reflux, by colorimetry; sialic acid (SA), marker of gastric mucus erosion, by Aminoff method. The measurements were done in 38 controls, divided into 3 groups : 0-2 months (n=8), 2-12 months (n=20), 1-4 years (n=10)As in adults, DGR was defined by a value above mean plus 2 SD for one or the 2 bilio-pancreatic markers. In a preliminary study DGR could be demonstrated in 3 children, with an antral gastritis. The levels of the 3 parameters were then much higher than the definite standards. This non invasive method seems to be a very usefull approach for the diagnosis, mechanisms and treatment of gastric and oesophageal pathologies.

A monoclonal antibody (Po66) directed against human lung squamous cell carcinoma immunolocalization of tumour xenografts in nude mice.

Po66, a mouse IgG1 monoclonal antibody, was produced by immunization against a patient lung squamous cell carcinoma. The tissue reactivity of the antibody was measured by a radioimmunological assay with enzymatically dissociated cells, by an immunofluorescence test on frozen tissue sections and by peroxidase-staining of paraffin sections. The antibody bound to lung squamous cell carcinoma, oesophagus carcinoma and, inconsistently to lung adenocarcinoma but not to the other tumours tested. Some normal tissues also reacted positively, in particular bronchial serous glands, oesophagus epithelium and renal distal and collecting tubules. In normal and malignant tissues showing epithelioid differentiation, Po66 bound to the intermediate maturation area. The antigen immunoprecipitated by Po66 from lung squamous cell carcinoma appeared as a single band with a molecular weight 47,000 to 50,000 daltons. Purified monoclonal antibody Po66 and an unrelated IgG1 immunoglobulin were labelled with radioactive iodine and injected i.v. into nude mice bearing subcutaneous xenografts of human lung squamous cell carcinoma. The localization index in the tumour was 3.3. Antibody labelled with 131I allowed gamma-scintigraphic imaging of the xenografts which were clearly outlined by days 9 to 11.

Clinical experience in an in vitro fertilization and embryo replacement program of a new method for analysis of urinary estrogens.

The clinical experience in an IVF/ER program of a newly developed commercial kit (Estradiol enzymatique; BioMerieux) to determine urinary estrogens enzymatically, is reported. The study included 157 cycles, and the main indication for treatment was infertility due to tubal factor. Follicular development was stimulated in most cases by a combination of clomiphene citrate, human menopausal gonadotropin and chorionic gonadotropin. In addition to the previously used method to monitor follicular growth, ultrasound, daily urinary estrogens (estradiol + estrone) levels were assayed. A correlation between the pattern of urinary estrogen and the clinical results was found. In cycles with continuously rising estrogen up to the time of hCG administration a higher rate of in vitro fertilization and clinical pregnancies were obtained as compared to cycles where urinary estrogen declined or levelled off. It is concluded that this enzymatic assay of urinary estrogens is a good alternative to increase the precision of follicle monitoring when radioimmunological assay of serum estradiol levels cannot be obtained on a daily routine basis.

Myelin basic protein in brains of rats with low dose lead encephalopathy

Postnatal exposure of rats to lead has been shown previously to cause CNS hypo-myelination. Since rats intoxicated with lead often show retarded growth, the superimposed malnutrition, which as such can cause hypomyelination, may contribute to myelin deficit. In the present study control rats and lead exposed rats which did not have any retardation of growth were examined by radioimmunological assay of myelin basic protein (MBP) of homogenates of cerebrum and cerebellum at 30, 60 and 120 days of age. Lead was administered on postnatal days 1-15 by daily intraperitoneal injections of 10 mg lead nitrate/kg body weight. This lead dose results in light microscopically discernible hemorrhagic encephalopathy in the cerebellum of 15-day old rats, but does not induce growth retardation (Sundström et al. 1983). The controls were injected with vehicle only. The amount of lead in the blood and brain homogenates of lead-exposed and control rats 15-200 days old was estimated by atomic absorption spectrophotometry. Significant differences between the lead-exposed and control rats were not found in the cerebral or cerebellar content of MBP. Considering the results of previous investigations, the findings do not exclude a hypo-myelinating effect of lead, but they suggest that exposure to lead without concomitant malnutrition does not cause hypo-myelination in the cerebrum and cerebellum of the developing rat.

A method for measuring testosterone-estradiol-binding globulin — new analysis of experimental data and comparison with two commercially available kits

Some binding assays have been suggested to measure serum testos-terone-estradiol-binding globulin (TeBG) concentration. They have led to discrepant results. This study brought to surface the lack of these methods and the interest of a method using an accurate analysis of the binding isotherm. These observations led us to propose an absolute reference method especially suitable for the radioimmunological assays standardization. Our method used Con A Sepharose as a solid phase and DHT as a radioligand. We succeeded in solving the difficulties related to binding methods by evaluating the non-specific binding of the ligand and ligand-albumin binding. A careful analysis of experimental data, by setting equations specific to this DHT-TeBG system permitted the systematic errors to be corrected and allowed a precise determination of the serum TeBG concentration (27.3 ± 6.0 nmol/l for 30 healthy men; 40 ± 13 nmol/l for 15 healthy women). Moreover, the affinity constant of the ligand for the protein was accurately evaluated K DHT = (0.7 ± 0.1)10 9 ( mol/ l −1) at 20°C, which was not permitted by other methods. Two lines of evidence supported our assumptions and results. On the one hand, using estradiol as a radioligand instead of dihydrotestosterone, we found the same value for serum TeBG concentration; on the other hand, our assay favourably compared with two different kits commercially available. The correlation ( r = 0.89) with 3H-SBP kit Merieux was good though this kit was not suitable for correctly assaying serum TeBG concentrations between 30 nmol/l and 60 nmol/l. An excellent correlation ( r = 0.97) with SHBG IRMA Kit Farmos was found. This antibody-based assay and our binding assay yielded quite similar values that mutually validated both methods.

INCREASED JEJUNAL PROSTAGLANDIN PGE2 CONCENTRATIONS IN CHILDREN WITH COELIAC DISEASE

The purpose of the present study was to evaluate PGE2 levels in chronic enteropathy. 98 children (3 months-19 years) were studied: intreated coeliac disease (UCD) with total villous atrophy (TVA) (n=12), treated coeliac disease (TCD) with normal mucosae (n=3); untreated cow's milk protein intlerance (CMPI) with partial villous atrophy (PVA) (n=10); giardiasis (n=19); idiopathic chronic enteropathy (ICE) with PVA (n=22). 32 normal biopsies were used as controls. PGE2 concentrations (ng/g tissue) in jejunal samples (per-oral suction biopsies) were measured by radio immunological assay. A significant difference was observed between UCD and all the other groups p<0,01 but not between the latter. PGE2 concentration was significantly different in TVA versus PVA (p<0,001) or ontrols (p<0,001) and significantly correlated to the number of IEL (p<0,001). These data showed that the increase of intestinal PGE2 seems to be related to VA and to the number of intra epithelial lymphocytes (IEL).

Antibodies to histamine: Specificity studies and radioimmunological assay

Antibodies against conjugated histamine were raised in rabbits. This amine was coupled to different protein carriers by a bifunctional agent, hexamethylene diisocyanate. The specificity of the antibodies was determined with radioimmunological tests in equilibrium dialysis using an iodinated ligand: 125I-labelled histamine-hexamethylene diisocyanate-glycyl-tyrosine. The latter mimicked the antigenic determinant present in immunogens. Competition experiments were established between the radiolabelled ligand and conjugated histamine, conjugated analogs or unconjugated histamine. Cross-reactivity ratios and affinity constants were calculated from displacement curves, thereby allowing the antibody site to be characterized. The antibodies were found to be highly specific and were used for the assay of histamine in biological samples. For this, polystyrene beads coated with purified antiserum were used to establish a simple and reproducible test.

Cardiac-glycoside-like activity measured by means of radioimmunological (RIA) and radioreceptor (RRA) assays

Cardiac-glycoside-like activity measured by means of radioimmunological (RIA) and radioreceptor (RRA) assays

Evidence for the presence of cAMP, cAMP receptor and transcription termination factor rho in different gram-negative bacteria.

Cyclic AMP has been shown to be present in 12 different Gram-negative bacteria and the regulation of its concentration, as a function of growth conditions, is similar to that described for Escherichia coli K12. Antibodies raised against catabolite activator protein (CAP) and Rho protein of E. coli K12 were used to check for the occurrence of cross-reactive antigens. Using radioimmunological assays, immunoblotting techniques and biochemical criteria we showed a wide distribution of CAP and Rho, structurally and functionally closely related to the corresponding E. coli K12 proteins. These results suggest that transcription is similarly regulated by these factors in Gram-negative bacteria.

Actin participates in the structure of liver intermediate filaments

A dominating protein fraction (p45) having molecular weight of 45000 and pI 5.45 was found in the intermediate filaments pellet obtained from rat liver besides the present cytokeratins. Peptide mapping and radioimmunological assays with antibodies against this protein and muscle actin proved that the p45 protein belongs to the actin group. Immunoelectron microscopy revealed that this protein is located on the liver intermediate filaments. By melting of the cytokeratin complexes in urea it was established that p45 protein is complexed with the low molecular weight cytokeratin.

Metabolism and pharmacokinetic studies of misoprostol.

Absorption, metabolism and excretion of radiolabelled misoprostol were studied in laboratory animals and in humans. Dog and man were similar in terms of key parameters examined. Misoprostol itself was not present in plasma after its oral administration to humans. Misoprostol was rapidly converted by de-esterification to its free acid. This metabolite possesses significant desired pharmacological activity. Further metabolic conversion occurs over time via beta-oxidation of the alpha side chain, omega-oxidation of the beta side chain and reduction to the prostaglandin F analogs. The serum protein binding of the free acid metabolite of misoprostol was similar in young (81-88%) and elderly (81-89%) people. Binding was concentration-independent and was not altered by drugs which one would expect to be co-administered with misoprostol. In the rat, misoprostol neither inhibited nor induced drug metabolizing enzymes. A radio-immunological assay for measurement of the free acid metabolite in human plasma has been developed. This method has a sensitivity of 23 pg/ml and appears to be sufficiently sensitive for use in clinical trials.

CAMP levels in reactive tissues around dimethylpolysiloxane solid implants.

By the aid of radioimmunologic assay, the authors measured the cAMP content of the reactive capsules around solid dimethylpolysiloxane implants in mice at different times; they also measured the same substance in some human connective tissues (granulation tissue and normal dermis) and compared together all the values they obtained. Different concentrations of cAMP in different tissues seem to reflect correspondent histologic findings, since they vary according to them. These values also seem to indicate a close correspondence between the development of the process of wound healing and of the foreign-body reaction following the implantation of alloplastic materials. On the basis of these data, the authors suggest an experimental therapeutic trial to enhance peripheral cAMP synthesis in order to control the process of reactive capsule constitution.

A quantitative Western Blot method for protein measurement

A radioimmunologic assay method for the quantitation of small amounts of protein in recombinant vaccines at the level of 20-150 ng is evaluated which uses the techniques of SDS-PAGE and electrophoretic protein transfer ("Western Blot'). Known amounts of the protein being determined are included on the same gel as the unknown. After protein blotting, the nitrocellulose membrane is treated with antibody specific for the protein being determined and subsequently with [125I] Protein A. An autoradiogram is produced which corresponds directly to the nitrocellulose blot. It can, therefore, serve as a template to locate the labeled protein which is excised from the blot and measured in a gamma counter. The technique is found especially useful for evaluating cell lysates of recombinant bacteria and yeast for the percentage of the recombinant protein in the total protein mixture.

TYace Analysis of Chloramphenicol Residues in Eggs, Milk, and Meat: Comparison of Gas Chromatography and Radioimmunoassay

A radioimmunological assay (RIA) to detect chloramphenicol (CAP) residues in eggs, milk, and meat is described. For tissues and other edible products of chloramphenicol-treated animals (chickens, cows, and pigs), the limit of detection is about 200 ng/kg. Residue levels above 1 microgram/kg can easily be quantitated. When highly specific antisera produced in sheep were used, cross-reactivity was insignificant except for metabolites deviating from the parent compound in the acyl side chain only. Thiamphenicol fails to bind to the antisera; hence, it does not interfere with the assay. In the procedure described, the role of cleanup is merely to remove lipids. Thus, skim milk can be analyzed following appropriate dilution without cleanup. The results obtained by RIA were confirmed by gas chromatography with electron capture detection. The new RIA allows rapid, sensitive, and specific screening of large numbers of samples.

Comparison of DHT-binding and monoclonal immunoradiometric methods for the measurement of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) is conventionally measured by saturation of binding sites with 5cu-dihydrotestosterone (DHT) of known specific activity followed by separation of the bound and free steroid fractions. The techniques used for separation cf these fractions vary and the reference ranges obtained for normal subjects are not in agreement (1, 2 and refs. cited therein]. More recently, antibodies raised against purified SHBG have been used in radioimmunologic and immunodiffusion assays [2-71. Although some antisera contained immunoglobulin G and transferrin antibodies [5,8-lo], several workers have produced monospecific antisera [3,5,7]. A partially characterized murine monoclonal antibody has been reported [ll]. These antibody-based assays are not without problems. Highly purified SHBG may progressively lose binding capacity [3] and immunological activity [2] and can be extensively damaged by radioiodination [4]. Recently, a commercial kit using a radiolabelled monoclonal antibody to SHBG which recognizes a discrete determinant on the molecule has been produced. We have compared this kit with the commonly used binding assay, using ammonium sulfate to separate bound from free steroid.

Cholecystokinin innervation of the ventral striatum: A morphological and radioimmunological study

Immunocytochemistry, radioimmunological assay after surgical cuts, anterograde degeneration and retrograde tracing of fluorescent dyes were used in order to elucidate the cholecystokinin-containing afferents to the ventral striatum (nucleus accumbens, olfactory tubercle and ventral part of the caudate-putamen). In agreement with the report by Hökfelt et al., 37 midbrain cholecystokinin-containing cells supply the posteromedial parts of the nucleus accumbens and olfactory tubercle, as well as the subcommissural part of caudate-putamen. Brainstem cholecystokinin afferents also reach more rostral parts of the ventral striatum including the rostrolateral olfactory tubercle. The ascending cholecystokinin axons enter the medial forebrain bundle at the meso-diencephalic border and maintain a rough medial to lateral topography at the caudal diencephalon. A second major cholecystokinin pathway, with possible origin in the piriform and medial prefrontal cortices and/or the amygdala, projects to the subcommissural caudate-putamen, the olfactory tubercle, the lateral part of the nucleus accumbens and the dorsal part of the bed nucleus of stria terminalis. Finally, the rostral part of the dorsal caudate-putamen receives a substantial cholecystokinin innervation from the basolateral amygdala and possibly from the neocortex. According to radioimmunological data, the descending telencephalic cholecystokinin system accounts for about 60% of all cholecystokinin in the rostral forebrain. The combined use of morphological and biochemical methods provided evidence for a partially overlapping distribution and possible interaction between an ascending brainstem and descending telencephalic cholecystokinin fiber systems within the striatum and related rostral forebrain areas.