Sensitive Radioimmunoassay Technique Research Articles

The Use of Nuclear Medicine Invitro Technology to Study the Effect of Malnutrition on children's Thyroid Function

In this study, forty-nine – under five years, male & female Sudanese children, presented with protein energy malnutrition (PEM, Kwash) according to the WHO criteria have been studied for their thyroid function and other biochemical Parameters. Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH), as a thyroid function indicators, the study included determination of Thyroglobulin (TG), Ferritin, Protein Moreover, Hemoglobin levels in serum. The study was done in different hospitals in Khartoum state. Patients being assessed & examined by pediatricians using a unified clinical protocol and a questionnaire for patient’s characteristics. Thyroid hormones and Ferritin were measured by a sensitive radioimmunoassay (RIA) Technique in 49 children with malnutrition as well as a control group of 20 healthy children. When the results of malnourished children compared to normal children showed that; 22% of malnourished children have severe hypothyroidism (T4 < 30 mol/l) and (T3 < 0.5 mol/l) as the reference range (50 – 150mol/l) and (0.8 – 3 mol/l) respectively. The mean level of TSH for patients was within the normal range but it was lower than the mean of TSH levels for controls. The mean values of TG and Ferritin for patients was higher than that of controls. Protein and Hemoglobin values showed low levels in Patients and negative clinical correlation between Ferritin and Hemoglobin has been found. Our conclusion of this study states that, malnutrition has severe effects on thyroid function.

Identification and Measurement of Carbonic Anhydrase-II Molecule Numbers in the Rat Carotid Body

Carbonic anhydrase (CA) in the carotid body (CB) plays an important role in the maintenance of blood PO2 and PCO2/pH homeostasis by regulating ventilation. It has been observed that the activity of CA in the rabbit CB is stronger under hypoxic conditions than under normoxic and hyperoxic conditions. In conditions of chronic hypoxia, the volume of the CB increases significantly because the number of type I and II cells increases. So far, the number of CA molecules in the CB has not been assessed. We develop a technique to quantify the number of CA molecules in the CB. The CBs were dissected out from 8 rats, immediately frozen with liquid nitrogen, pulverized and centrifuged. The proteins extracted from CB tissue were heat-denatured and separated by electrophoresis on a 12.5% denatured-polyacrylamide gel (SDSPAGE); a 31 kDa protein band was determined which reacted with a rabbit polyclonal antibody specific for rat CA-II in Western blot analysis. The immunoreactive 31 kDa CA-II protein was detected and quantified by laser scanner densitometry using 125I-rProtein A as a tracer. The mean 125I radioactivity emitted by the antibody bound CA-II was 31277 cpm. This value corresponds to 4.57 ng CA-II. When compared with a rat CA-II calibration curve, an average of number of 3.54 x 107 CA-II molecules were quantified for 1 µg of whole CB tissue. This is a sensitive and accurate radioimmunoassay technique and may be useful in future studies on the role of CA-II in different pathophysiologic conditions.

Atrial natriuretic peptide levels in children with habitual epistaxis

Objective: By virtue of no identifiable causes in the majority of children with habitual epistaxis, it continues to be problematic in pediatric clinical practice. The purpose of this study is to explore the possible change of atrial natriuretic peptide (ANP) levels in the children with epistaxis. Methods: Both the plasma and nasal mucus ANP levels have been determined in 30 sick children by a sensitive radioimmunoassay (RIA) technique. Results: Our results revealed that the plasma and nasal mucus ANP levels were considerably decreased in 24 children with habitual epistaxis when compared with control group ( P<0.05), making up 80%, and amongst the interest of these are the nasal mucus ANP levels changing inversely as the times bled from the nose. Conclusion: Although the plasma and nasal mucus ANP levels will not establish the diagnosis of its etiology, it is helpful for us to know the cardiovascular status compensating for chronic blood loss in the children with habitual epistaxis.

Myometrial steroid concentration and oxytocin receptor density in parturient women at term

The aim of this study was to measure oxytocin receptor concentration in myometrial tissue from term pregnant women with normal and dysfunctional labor and to relate this concentration to the progress of labor and to the levels of estradiol and progesterone in the same myometrium. Myometrial biopsies were obtained from 50 term pregnant women undergoing cesarean section. The patients were categorized as follows: not in labor, normal labor, successful oxytocin-augmented labor, and oxytocin-resistant labor. Specific binding of [ 3H]oxytocin to high-affinity sites in membrane preparations from myometrial tissues was determined. Estradiol and progesterone were assayed using tritiated steroids with a sensitive radioimmunoassay technique. Oxytocin receptor density was significantly lower in oxytocin-resistant labor compared to successful oxytocin-augmentated labor ( P < 0.04) and to spontaneously active normal labor ( P < 0.02). Oxytocin receptor concentration was also significantly lower in non-labor patients compared to normal spontaneous labor ( P < 0.01), and successful oxytocinaugmented labor ( P < 0.02). There was a positive relationship between the progress of cervical dilatation (cm/h) and oxytocin receptor density in the myometrium ( r = 0.408, P < 0.025). The concentration of progesterone and estradiol in the pregnant myometrium did not differ in patients with different types of labor or with the state of uterine contractile activity. Our results suggest that individual myometrial sensitivity is an important determinant of the response to administered oxytocin in humans. Furthermore, myometrial oxytocin receptor expression in vivo seems not be related to ovarian steroid concentration in the myometrium. The low oxytocin receptor density in oxytocin-resistant dystocia needs further investigation.

Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection.

In this report we investigated whether induction of IL-12 occurs in response to herpes simplex virus (HSV) infection of the mouse eye, which may serve to regulate the nature of the subsequent immune response. The data show early induction and continued maintenance of IL-12 (p40) mRNA in the cornea and draining lymph node upon ocular infection with HSV. Using a very sensitive radioimmunoassay technique, IL-12 (p40) protein also was detected in the cornea upon ocular HSV infection. Unfractionated splenocytes and enriched populations of dendritic cells, macrophages, and neutrophils all responded to HSV infection in vitro by up-regulating the expression of IL-12 (p40) mRNA. However, cultured corneal cells failed to generate IL-12 (p40) upon exposure to HSV. The data indicate that inflammatory cells that infiltrate the cornea in response to HSV-1 infection are the main source of IL-12 in the eye. Our results are consistent with the hypothesis that IL-12 induction in the eye acts as a triggering event that biases HSV-specific immunity to a type 1 T cell response, which, in the environment of the eye, culminates in an immunopathologic disease.

Atrial Natriuretic Peptide (ANP) increases in the mangrove crab Ucides cordatus when exposed to increased environmental salinity

1. 1. Haemolymph taken from the basal joints of the walking legs of the mangrove Brazilian crab Ucides cordatus, was analysed using a very sensitive radioimmunoassay (RIA) technique, before and after specific extraction and purification methods (Vycor-Glass) for immunoreactive atrial natriuretic peptide (IR-ANP) determinations. 2. 2. The mangrove crabs ( N = 20) were randomly distributed in four groups, exposed to four different salinities, all of them included in the ecological Brazilian mangrove range: 9, 17, 26, 34%. of seawater. 3. 3. The smallest IR-ANP value, found in the 26‰ salinity (1.25 ± 0.2 pg/ml) was coincident with the isosmotic gradient for haemolymph and external medium, where the crab is not submitted to osmoregulatory disbalances. 4. 4. The IR-ANP values found in the 9 and 17‰ salinity, did not show any significant differences when compared with values obtained at the isosmotic point 26‰. 5. 5. IR-ANP level increased significantly with the external salinity level, from 26 to 34‰ (7.5 ± 5 pg/ml; N = 5). There was a significant positive Kendall Correlation Coefficient ( P = 0.035) between the IR-ANP values found at 26 and 34‰ salinity. 6. 6. ANP could participate in the osmoregulation of the Brazilian crab. U. cordatus, contributing towards NaCl extrusion in hyperosmotic environmental salinity.

Effects of dopamine autoreceptor stimulation on the release of colocalized transmitters: In vivo release of dopamine and neurotensin from rat prefrontal cortex

The in vivo release of dopamine and neurotensin from the rat medial prefrontal cortex was studied using perfusion microdialysis coupled with sensitive radioimmunoassay and HPLC techniques. Following stimulation of dopamine autoreceptors with either apomorphine (30 μg/kg, s.c.) or EMD-23448 (10 μM in the perfusion buffer) a decrease in dopamine and an increase in neurotensin release was observed. The release of both substances was measured in the same dialysis sample. These data suggest that activation of dopamine autoreceptors in the prefrontal cortex produces opposing effects on the release of dopamine and neurotensin.

Formation of cysteinyl-leukotrienes by human brain tissue

Using sensitive radioimmunoassay techniques, the formation of cysteinyl-leukotrienes (LT) and prostaglandin (PG) F 2α was investigated in human brain tissue slices in vitro. Under basal conditions spontaneous release of considerable amounts of LTC 4-like material and PGF 2α could be detected from slices of both human grey and white matter. Ionophore A23187 stimulated the release of large amounts of LTC 4-like material while leaving unaffected the formation of the cyclo-oxygenase product PGF 2α. Incubation of grey or white matter in the presence of the cyclo-oxygenase inhibitor indomethacin inhibited the release of PGF 2α but did not affect that of LTC 4-like material. Preincubation of brain tissue in the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) abolished the ionophore A23187-induced release of LTC 4-like material from both grey and white matter and also significantly reduced the spontaneous release of LTC 4-like material from white matter slices. Formation of PGF 2α was not affected by NDGA. Using the isolated guinea pig ileum the LTC 4-like material was shown to possess biological activity which could be antagonized with the slow-reacting substance of anaphylaxis (SRS-A) antagonist FPL 55712. By reversed phase HPLC the immunoreactive LTC 4-like material from human grey matter was shown to coelute with authentic LTC 4, LTD 4 and LTE 4, while material from human white matter coeluted with authentic LTD 4 and LTE 4. The capacity of human brain tissue to synthesize cysteinyl-LT may be important for cellular functions in the central nervous system.

Insulin and insulin-receptor autoantibodies in children with newly diagnosed IDDM before insulin therapy.

Twenty-nine children, aged 1-15 yr, with newly diagnosed insulin-dependent diabetes mellitus (IDDM) had sera taken before insulin therapy to be examined for the presence of insulin-receptor antibodies by measuring the inhibition of binding of radiolabeled insulin to IM-9 lymphocytes in both whole serum and purified IgG fractions. Groups of children with long-standing IDDM and autoimmune endocrine disease as well as a normal control group were studied. A positive result, defined as binding greater than or equal to 2 SD below the mean zero standard, was found in 3 (10.3%) of the 29 newly diagnosed diabetic patients. As a group, they showed significantly greater binding inhibition than the normal control group for both whole serum and purified IgG (one-tailed t test, P less than .05 and P less than .002, respectively). Insulin autoantibodies were also measured by a sensitive radioimmunoassay technique. A positive result, defined as binding greater than 3 SD above the normal control pooled sera, was found in 9 (37.5%) of 24 of the newly diagnosed IDDM group tested. All 3 subjects positive for insulin-receptor antibodies were also positive for insulin autoantibodies, whereas 6 of the 21 receptor-antibody-negative subjects were positive for insulin autoantibodies (Fisher's exact test, P = .0415). This suggests the possibility that the presence of insulin autoantibodies is a prerequisite for the development of insulin-receptor antibodies, i.e., as an anti-idiotypic response. Insulin-receptor antibodies and insulin autoantibodies may play a currently undefined pathophysiologic role in the development of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin Basic Protein and Demyelinating Diseases

Myelin basic protein (MBP) is a major protein component of myelin sheath. Primarily because of its ability to induce experimental allergic encephalomyelitis (EAE) in animals, this protein has been considered to play an important role in the pathogenesis of multiple sclerosis (MS), which is one of the most common demyelinating diseases. Its precise measurement in the cerebrospinal fluid (CSF) has been uncommonly difficult, mainly due to immunoheterogeneity of MBP or MBP-like material. More recently, highly sensitive radioimmunoassay techniques utilizing well-characterized antisera have been developed that facilitated its use in the management of MS. The clinical course of MS is highly variable, and the disease is characterized by periods of remission and relapses. Many studies have demonstrated the release of MBP during relapses and elevated levels of MBP in the CSF can be detected if lumbar puncture is performed within 7 days of the onset of neurologic symptoms suggestive of MS. However, the presence of MBP is not an absolute indicator of MS, as elevated MBP levels are also frequently observed in other demyelinating diseases.

Development of a sensitive solid-phase radioimmunoassay technique for quantification of class specific Escherichia coli antibodies

A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions. Preparation of affinity-purified anti- E. coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 μl of sample. This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection. RIA also compares favourably with ELISA in sensitivity and sample size required. Affinity-purified standards may also be used to quantify the ELISA test.

Relative Bioavailability of a Commercial Trifluoperazine Tablet Formulation using a Radioimmunoassay Technique

The relative bioavailability of a new conventional tablet formulation (5 mg) of trifluoperazine dihydrochloride was studied in 24 healthy volunteers. Using a sensitive radioimmunoassay technique, plasma trifluoperazine concentrations were measured up until 24h following ingestion of single 5‐mg doses of trifluoperazine. The mean ± SD for the peak concentration (Cmax), time to Cmax, area under the curve from 0 to 24h (AUC240), and terminal elimination half‐life following the administration of the test formulation were 2.15 ± 1.07 ng/mL, 4.10 ± 1.38 h, 21.04 ± 11.92 ng‐h/mL, and 9.5 ± 7 h, respectively. Following the ingestion of the original trifluoperazine tablet formulation (5 mg) these same parameters were estimated to be 1.92 ± 0.88 ng/mL, 4.02 ± 1.10 h, 18.03 ± 10.11 ng‐h/mL, and 9.3 ± 7 h, respectively. Large intersubject variations in Cmax and AUC240 were observed. The relative bioavailability of the test formulation was calculated to be 106.5 ± 25.5%.

Thromboxane B 2, 6-keto-PGF 1α, PGE 2, PGF 2α, and PGA 1 plasma levels in arteriosclerosis obliterans: Relationship to clinical manifestations, risk factors, and arterial pathoanatomy

Current concepts of atherogenesis based on animal and human investigations indicate prostaglandins as a key factor in atherosclerotic lesions. The plasma profiles of thromboxane B 2 (TXB 2), 6-keto-PGF 1α, PGE 2, PGF 2α, and PGA 1 were investigated by means of a sensitive radioimmunoassay technique in 40 patients with arteriosclerosis obliterans and in 30 healthy control subjects. Abnormally high levels of TXB 2 and PGE 2 (222.97 ± 320.86 pg/ml, mean ± SD, vs 20 ± 2.1 and 352.66 ± 235.54 vs 24.4 ± 3, p < 0.01) were detected in arteriosclerosis obliterans patients. The ratio between TXB 2 and 6-keto-PGF 1α was increased from 1.2 in control subjects to 6.0 in patients. In arteriosclerosis obliterans TXB 2 increased in relation to clinical manifestations and to the extension of the vascular damage. In addition, TXB 2 was positively related to serum triglyceride content ( r = 0.562, p < 0.05) and inversely related to platelet count ( r = 0.727, p < 0.001). The marked imbalance between the stable metabolites of thromboxane and prostacyclin in arteriosclerosis obliterans patients provides biologic evidence which fits well with the thrombogenic theory of atherosclerosis. These redults further support the theory that prostaglandins may be heavily involved in atherosclerosis.

Studies on the retention of passively transferred antibodies in man. II. Antibody activity in the blood after intravenous or intramuscular administration of anti-HBs human immunoglobulin.

Catabolism of intravenously or intramuscularly administered human immunoglobulin in man was studied by the administration of HBs antibodies at a high titer and analysis with a highly sensitive radioimmunoassay technique. The half-life in the blood of the antibodies in the aggregate-reduced antibody preparation administered intravenously was found to be 17.7 days and of those in the antibody preparation administered intramuscularly to be 23.8 days.

Cervical and serum IgA and serum IgG antibodies to Chlamydia trachomatis and herpes simplex virus in threatened abortion: a prospective study

The aetiological role of Chlamydia trachomatis (CT) and herpes simplex virus (HSV) was investigated in 189 patients with threatened abortion. Assessment of infection was based on isolation, and on determination of serum immunoglobulin (Ig)G and IgA antibodies as well as cervical IgA antibody levels with new sensitive radioimmunoassay (RIA) techniques. One third of the women were delivered of a healthy infant and two thirds aborted, but the two groups were otherwise clinically similar. By isolation, only 2.7% of the patients were CT-positive, but increased cervical IgA antibody level to CT was detected in 41.3%. The mean level of these local antibodies was similar in both study groups, but the mean levels of serum IgA and IgG antibodies were somewhat higher in the patients who aborted although the difference was not significant. None of the cervical specimens was positive for HSV by isolation but the cervical IgA antibody level to HSV was raised in 47.1% of the patients. Both cervical and serum IgA antibody levels to HSV were significantly raised among the patients who aborted, but there were no differences between the patients with spontaneous abortion and those with a blighted ovum. There was no clear association between CT and abortion, but an association between HSV and abortion is possible. The incidence of raised levels of both CT and HSV IgA antibodies in the cervix was surprisingly high in both groups and the significance of this finding remains to be investigated.

Detection of varicella-zoster virus-specific IgA antibodies in varicella and zoster patients and in healthy adults of various ages by solid-phase radioimmunoassay.

The varicella-zoster virus(VZV)-specific serum immunoglobulin A(IgA) response was studied in varicella and zoster patients and in healthy adults of various ages by a sensitive solid-phase radioimmunoassay technique. Significant rises in VZV-specific IgA titer were found for both varicella and zoster patients, while VZV-specific IgA was also detected in greater than 90% of healthy adults but at lower titers. No significant decrease in VZV-specific IgA titer with increasing age was found. The role of humoral versus cell-mediated immunity in VZV reactivation is discussed.

Measurement of airborne antigens

Measurement of potentially harmful materials in the appropriate place in the environment is essential in industry, both to identify the toxic material and the workers who may be affected by it and to follow the effectiveness of measures designed to reduce exposure. This principle applies no less to occupational allergy than to toxicology. Indeed, the same equipment used for collecting and assaying for airborne dusts and chemicals can be used to collect and assay airborne antigens. Three examples can serve as models for application to other situations. Assay procedures for measuring concentrations of diisocyanates in the air of the workplace permitted definition of the concentrations that are toxic and the lower concentrations that may induce asthma. Widespread use of these assays accumulated enough data to permit NIOSH to publish recommended procedures to minimize exposure, limits of allowable exposure, and techniques of air sampling and analysis. A high-volume air sampler that filters about 1700 m 3 of air per day and retains particles larger than 0.1 μm in diameter was used to monitor levels of an antigen from a spray-wash air conditioner that caused hypersensitivity. pneumonitis in a textile factory. The antigen eluted from the filter is measured by a sensitive radioimmunoassay technique using the patients' sera to detect the antigen. Coupled with epidemiologic data, it was possible to estimate concentrations associated with sensitization and concentrations required to elicit episodes of the disease. More importantly, it was possible to follow the result of changes in the factory undertaken to reduce the exposure and to determine whether the goals of levels of airborne antigen were being met. This same air sampler was used to identify and quantitate mouse antigens that reacted with IgE antibody and caused allergic rhinitis and asthma in some animal handlers in animal quarters and in an immunology research laboratory. After elution from the filter sheets, the mouse antigens were measured by the BAST inhibition technique with sera from mouse-allergic patients. As had previously been suspected from indirect evidence, the major airborne antigen proved to be mouse urinary protein. These three examples illustrate a chemical assay for a well-defined low molecular weight substance, radioimmunoassay employing human IgG antibody to an undefined antigen associated with hypersensitivity pneumonitis, and radioimmunoassay employing IgE antibody for occupational asthma. Thus we now have the methods to allow for developing criteria for recommended standards for occupational exposure to any antigen.

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM anti-microbial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of non-absorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.

Studies of rat antibodies specific for myelin basic protein (MBP) antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro

Rats immunized with rat myelin basic protein (MBP) in an encephalitogenic regimen produce antibodies which cooperate with spleen cells from unimmunized rats in the specific lysis of MBP-sensitized chicken erythrocytes (MBP-CRBC) in vitro. Antibodies against MBP capable of participating in antibody-dependent cell-mediated cytotoxicity (ADCC) were detected in serum 9 days post immunazation, and prior to the onset of experimental allergic encephalomyelitis (EAE). Measurements based on a sensitive radioimmunoassay technique had previously established that anti-MBP activity emerges by day 9, early enough to participate in events leading up to EAE. The ADCC assay was designed to further characterize functions of immunoglobulins associated with the humoral response to MBP, with emphasis on the cytophilic properties of early antibodies. The assay described was found to be nearly as sensitive as the RIA and could detect specific syngeneic rat anti-MBP at concentrations of about 0.03 pmoles/ml serum. Whether ADCC-type reactions take place in the central nervous system during acute EAE is not known; however, the presence of early cytophilic antibody in serum suggests that humoral immunity may play an ancillary role to that demonstrated for T-cell immunity in EAE.

Role of cervicovaginal antibody in the pathogenesis of recurrent urinary tract infection in women.

Introital colonization with Enterobacteriaceae is considered to be one of the principal predisposing factors to recurrent urinary tract infections (UTI) in adult females. One proposed mechanism allowing introital colonization in these patients is the absence of local cervicovaginal antibody. To test this hypothesis, we examined cervicovaginal washings from 22 patients with a history of recurrent UTI and 29 normal controls with no history of UTI for specific local antibody by using indirect immunofluorescence. No significant difference in antibody was found in these populations. Fourteen percent (3/22) of the patients had antibody to their introital Escherichia coli and 34% (10/29) of the controls had antibody to their fecal E. coli. Consequently, a sensitive radioimmunoassay technique was developed to detect cervicovaginal antibody. A solid phase was prepared by coupling to Sepharose 4B a pool of eight serogroups of E. coli which are frequently implicated in UTI. Serial dilutions of cervicovaginal washings were reacted with the solid phase, and the absorbed anti-E. coli antibodies were detected by the uptake of (125)I-labeled anti-human immunoglobulin G (IgG) or anti-human IgA. The antibody levels were quantitated by interpolation on a standard curve prepared by using immunospecifically purified human anti-E. coli antibodies. IgG and IgA levels were measured in washings from 10 colonized patients, 13 non-colonized patients, and 12 controls. There were no significant differences in IgG and IgA levels in cervicovaginal washings among the three groups. In these studies, introital colonization was not related to cervicovaginal antibody.