A simple and sensitive double-antibody radioimmunoassay for human growth hormone (HGH) was developed, optimised and validated. The anti-hGH sera raised in 2 rabbits were highly specific with low cross-reactions of 0.19% and 0.3% with human placental lactogen and 0.21% and 0.13% with human prolactin. The mean sensitivity of the assay determined from 28 assays was found to be 0.4 +/- 0.2 mIU/L. Mean recovery of added exogenous hGH was 98.8 +/- 6.8%. Linearity studies of samples diluted at 1:2, 1:4 and 1:8 gave values of 101.3 +/- 5.3%, 109.6 +/- 13.4% and 97.3 +/- 13% respectively of those expected. The reproducibility of the assay was good; within assay coefficient of variation for serum samples with GH concentrations of 2.7, 13.6 and 28.2 mU/l ranged from 5.1 to 8.3% while the inter-assay precision varied from 4.9 to 10.3%. The in-house assay showed good correlation (r = 0.96, p less than 0.001) with a commercial HGH RIA kit (Dainabot, Japan). A reference normal adult fasting GH level of less than 7 mIU/l was established from 95 samples assayed by this method.
Raised prolactin levels have been implicated as a cause for infertility in patients with endometriosis. This study was done to investigate if serum prolactin levels were significantly raised in infertile patients with endometriosis. Serum prolactin levels were studied in 43 infertile patients with endometriosis. For controls, 36 infertile patients with normal pelvic findings were used. For standardization, blood samples were drawn on day 21 of the menstrual cycle. Analysis was done by radioimmunoassay using reagent kits. The mean prolactin level in the endometriotic group was 372 mIU/l (range 187-752) while that in the controls was 333 mIU/l.(range 124-767). There was no statistical difference (t = 1.12). Furthermore the accepted normal level for serum prolactin in our population is less than 540 mIU/l. These results show that there is no evidence to implicate raised prolactin levels as a cause for infertility in patients with endometriosis.
The direct assay of serum progesterone after denaturation of the binding proteins was investigated. 50ul of patients' serum was diluted with 750ul phosphate buffer (0.05M, pH 7.4) and heated to 65 degrees C for 20 minutes. After cooling, 300ul of the treated serum was reacted with a rabbit antiserum to progesterone-11 alpha-hemicuccinyl-bovine serum albumin conjugate (Bioclin, U.K) and 1,2,6,7, tritium labelled progesterone. Separation of bound and free fractions was achieved with dextran coated charcoal. The method correlated well (r = 0.98) with an established method involving ether extraction of progesterone prior to assay. The mean sensitivity was 2.01 nmol/L (range 1.90-2.23nmol/L). The proposed method considerably shortens assay time and removes a tedious and imprecise stage in the conventional method involving extraction of serum.
Radioimmunoassay technique has been used to investigate the variation in the serum human prolactin (hPRL) levels at different stages of menstrual cycle and during a 24-hours period in Malay women. The results showed that the serum hPRL concentrations during menstrual (24.5 ± 4.3 ng/ml), preovulatory (36.6 ± 7.4 ng/ml), ovulatory (29 ± 5.3 ng/ml) and postovulatory (26.6 ± 5.2 ng/ml) periods were not significantly different among each other. The hPRL level was highest during night-sleeping, at 0200hr and was significantly different from the values obtained at 1400, 2000, 0800 and 1400 (last sampling) hours (P < 0.01).
The total serum IgE levels in infants and children was quantitated by the radioimmunoassay technique. The serum levels increased from about 300 IUlml in the 2-3 month old infants to about 8000 1U/m1 in the 10-year-old children who were probably infected with intestinal helminths. The total serum IgE levels in infants with cow's milk protein-sensitive entero-pathy were similar in level to those in normal infants. Infants and children with acute gastroenteritis, giardia infection and malnutrition had elevated levels of serum IgE levels. The high serum I gE levels noted in Malaysian children are probably indicative of the pattern in the tropics. (Copied from article).
A simple, non-isotopic in-house enzyme-linked immunoabsorbant assay (ELISA) for human growth hormone (GH) was developed. The assay involved using in-house polyclonal anti-GH adsorbed onto 96-well microtitre plates, commercially prepared mouse monoclonal anti-GH, and goat anti-mouse IgG horseradish peroxidase detection system. Results of recovery and parallelism studies ranged from 95%-106% and 98%-101% respectively, of the expected values. The detection limit of the assay was 0.008 mIU/well or the equivalent to 0.4 mIU/L of undiluted serum. Intra- and interassay coefficients of variations were 4.8%-7.9% and 6.5%-8.7% respectively. Serum GH levels measured in this assay correlated well with those measured in established in-house radioimmunoassays (r = 0.985, p < 0.001) and immunoradiometric assay from NETRIA (r = 0.984, p < 0.001).
Serum ferritin and haemoglobin estimates were carried out on 78 first time blood donors with a view to determining iron store status. Of these 30 were Malays, 20 were Chinese and 28 were Indians. The ferritin level in Malay donors ranged from 16-160 mg/ml (mean 83 +/- 49.4 mg/ml in chinese donors is ranged from 36-500 mg/ml (mean 242.8 +/- 132 mg/ml), and in the Indian donors it ranged from 5 - 270 mg/ml (mean 94.6 +/- 67.9 mg/ml). The haemoglobin concentration for the whole group was 14.9 +/- 1.49 g/dl. There was no correlation of haemoglobin concentration with serum ferritin levels.
A radioassay for the measurement of cortisol binding capacity in human serum was developed and the results validated by radioimmunoassay of serum corticosteroid binding globulin. The mini-scale method is simple, reliable and rapid, and avoids separation of partitioned phases. The linear correlation between the free cortisol index and free cortisol concentration as calculated by the two methods utilizing serum samples with normal and abnormal protein concentrations was r = 0.962 (n = 114, p less than 0.001) and the regression equation was y = 3.516 x - 2.632. The technique requires no preparation of reagents, and the assay can be performed in 45 minutes using 0.05 ml of serum or plasma.
Since conventional radioimmunoassays (RIA) for measurement of 17-hydroxyprogesterone (17-OHP) in serum samples require a laborious solvent extraction step, a direct and rapid in-house RIA was developed for early diagnosis and management of congenital adrenal hyperplasia (CAH). In-house rabbit anti-17-OHP antiserum, tritium labelled 17-OHP and dextran-coated charcoal were used in assay buffer with low pH 5.1 and preheated serum samples. Both inter- and intra-assay CVs were < 10% and the sensitivity was 1.2 nmol/l or 12 fmol/tube. Results from the direct assay correlated well with values from an extraction assay, r = 0.88 in samples from CAH patients, r = 0.85 in adults and children, 0.69 and 0.40 in term and preterm neonates respectively, 0.66 and 0.63 in luteal phase and third trimester pregnancy; p < 0.001 in all groups except p < 0.05 in preterm neonates. However, results from the direct assay were two to three times higher in serum samples from CAH patients, normal adults and children, but were five to seven times higher in pregnancy and term neonates and thirty times higher in preterm neonates. The markedly elevated levels measured by the direct assay are probably due to cross-reactivities with water-soluble steroid metabolites such as 17-hydroxypregnenolone sulphate and dehydroepiandrosterone sulphate (DHEAS). Although the direct assay is only useful as a screening test for preterm babies, it can be used for both diagnosis and monitoring of treatment of CAH in all other age groups.
In recent years, environmental concerns over ultra-trace levels of steroid estrogens concentrations in water samples have increased because of their adverse effects on human and animal life. Special attention to the analytical techniques used to quantify steroid estrogens in water samples is therefore increasingly important. The objective of this review was to present an overview of both instrumental and non-instrumental analytical techniques available for the determination of steroid estrogens in water samples, evidencing their respective potential advantages and limitations using the Need, Approach, Benefit, and Competition (NABC) approach. The analytical techniques highlighted in this review were instrumental and non-instrumental analytical techniques namely gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), enzyme-linked immuno sorbent assay (ELISA), radio immuno assay (RIA), yeast estrogen screen (YES) assay, and human breast cancer cell line proliferation (E-screen) assay. The complexity of water samples and their low estrogenic concentrations necessitates the use of highly sensitive instrumental analytical techniques (GC-MS and LC-MS) and non-instrumental analytical techniques (ELISA, RIA, YES assay and E-screen assay) to quantify steroid estrogens. Both instrumental and non-instrumental analytical techniques have their own advantages and limitations. However, the non-instrumental ELISA analytical techniques, thanks to its lower detection limit and simplicity, its rapidity and cost-effectiveness, currently appears to be the most reliable for determining steroid estrogens in water samples.
An in-house radioimmunoassay (RIA) for the measurement of androstenedione levels in serum was established and validated. Levels of androstenedione were measured by RIA using serum samples from various normal population groups and patients with congenital adrenal hyperplasia (CAH). Analytical recovery and linearity results were > 95%, while intra- and inter-assay CVs were < 10% and < 22% respectively. The assay sensitivity was 0.5 nmol/l or 25 fmol/tube. In normal population groups, the highest androstenedione levels were found in preterm neonates (1.6-12.4 nmol/l), followed by adult females (1.5-10.2 nmol/l), adult males (1.6-8.0 nmol/l) and term neonates (0.8-8.8 nmol/l), while the lowest values were observed in prepubertal children (0.5-3.4 nmol/l). There were no significant differences in diurnal variation and between follicular and luteal phases. The range of androstenedione levels in untreated or poorly controlled CAH patients (7.6-355.0 nmol/l, median 42.5 nmol/l, n = 20) were significantly higher (p < 0.001) than the upper normal limit of 3.4 nmol/L for prepubertal children. The normal androstenedione reference ranges for paediatric and adult groups have thus been established.
This report describes the proliferation and transmission patterns of Pasteurella multocida B:2 among stressful goats, created through dexamethasone injections. Thirty seven clinically healthy adult goats were divided into three groups consisted of 15 goats in group A, 11 goats in group B and the remaining 11 in group C. At the start of the study, all goats of group A were exposed intranasally to 1.97 x 10(10) CFU/ml of live P multocida B:2. Dexamethasone was immediately administered intramuscularly for 3 consecutive days at a dosage rate of 1 mg/kg. The exposed goats were observed for signs of HS for a period of 1 month. At the end of the 1-month period, 11 goats from group B were introduced into and commingled with the surviving goats of group A before all goats from both groups were immediately injected intramuscularly with dexamethasone for 3 consecutive days. The treatment with dexamethasone was then carried out at monthly interval throughout the 3-month study period. Goats of group C were kept separately as negative control. Three surviving goats from each group were killed at 2-week interval for a complete post-mortem examination. Two (13%) goats of group A were killed within 24 hours after intranasal exposure to P multocida B:2 while another two (13%) goats from the same group were killed on day 40, approximately 10 days after the second dexamethasone injection. All four goats showed signs and lesions typical of haemorrhagic septicaemia. Bacteraemia was detected in 3 goats of group A that were having rectal temperature higher than 41degrees C. The P. multocida B:2 isolation pattern was closely associated with dexamethasone injections when significantly (p < 0.05) higher rate of isolations from both groups were observed after each dexamethasone injection. Transmission of P multocida B:2 from goats of group A to group B was successful when P multocida B:2 was isolated from goats of group B for a period of 28 days. There was a strong correlation between dexamethasone injections, rate of bacterial isolation and serum cortisol level. The IgG level showed an increasing trend 2 weeks after exposure to P multocida B:2 and remained high throughout the study period.
The lymphocyte subsets in the peripheral blood of healthy Malaysian adults (212 subjects, age 18-71 years) were analysed using a flow cytometer FACScan in an effort to establish a reference range for the lymphocyte subsets. The lymphocyte subsets studied were T cells (CD3), B cells (CD19), natural killer (NK) cells (CD3- CD16+/CD56+), helper/inducer cells (CD4), cytotoxic/suppressor cells (CD8) and the helper/suppressor ratio (CD4/CD8). The distributions of T cells, CD4 cells and CD8 cells were symmetric about their means while B cells, NK cells and CD4/CD8 ratio followed a skewed distribution. Differences in race were observed for T cells, NK cells, CD4 cells and CD4/CD8 ratio where the Indians were significantly different from the Malays and the Chinese (higher T cells, CD4 cells and CD4/CD8 ratio and lower NK cells). The B cells were significantly lower in the Chinese than the Malays and the Indians. Age differences were seen only in the Chinese where increased CD4 cells and CD4/CD8 ratio, and decreased CD8 cells were observed. A sex difference was observed only in the Chinese where the CD4/CD8 ratio was significantly higher in females than males.
The clinical applications of salivary cortisol measurements were evaluated by radioimmunoassay of time-matched saliva and plasma samples. Salivary cortisol levels of normal subjects exhibited a significant (p less than 0.001) diurnal variation with a mean (+/- SD) concentration of 8.7 +/- 4.8 nmol/L at 0800-1000 h and 2.4 +/- 1.1 nmol/l at 1500-1700 h. After an overnight dexamethasone suppression test, morning salivary cortisol levels decrease to 2.7 +/- 0.7 nmol/L (p less than 0.001 vs normal). An excellent correlation (r = 0.805) of cortisol measurements with time-matched saliva and plasma samples was obtained (y = 0.03x + 0.88, p less than 0.001, n = 91). Hypercortisolism was confirmed by raised salivary cortisols in only half of patients with elevated total plasma levels, thereby indicating that salivary cortisol measurements is a better index of adrenal status.
HBeAg and anti-HBe were determined in the blood of 189 male blood donors. The incidence of HBsAg was 6.9% while that for HBeAg and anti-HBe was 1.6 and 18%, respectively. Of the 13 samples positive for HBsAg, two (15.4%) were positive for HBe while six (46.2%) were positive for anti-HBe. One specimen was negative for HBsAg but was positive for HBeAg and anti-HBe. The observations are discussed.
The effects of stress and corticosterone on testicular 11beta-hydroxysteroid dehydrogenase (11beta-HSD) oxidative activity have been controversial, whilst that of adrenocorticotrophic hormone (ACTH) have not been investigated before. Hence, the aim of the present study was to determine the in vivo effects of stress due to injection and sham operation, ACTH and corticosterone on testicular and hepatic 11beta-HSD oxidative activity and plasma testosterone levels in normal and adrenalectomized (ADX) rats and their possible mechanism of actions. Adrenalectomy reduced both testicular 11beta-HSD oxidative activity and plasma testosterone levels. The effects of injection and sham operation significantly increased plasma corticosterone levels with decreased testicular 11beta-HSD oxidative activity and plasma testosterone levels in normal but not in ADX rats. Likewise. ACTH or corticosterone treatment for 7 days decreased both testicular 11beta-HSD oxidative activity in a dose dependent manner and plasma testosterone levels in normal rats; but the values in ADX rats remained unchanged. However, none of the above values were significantly lower than that of the ADX levels. Corticosterone seems to maintain testicular 11beta-HSD oxidative activity within the range between normal and ADX rats. These changes are not attributable to diurnal rhythms, as the time of sacrifice has been fixed between 8:30 and 10:30 am. In the liver, no significant change in 11beta-HSD oxidative activity was observed with sham operation, ACTH or corticosterone treatment; but adrenalectomy significantly decreased it. In conclusion, in the intact normal rats, stress, ACTH or corticosterone modulates testicular (but not hepatic) 11beta-HSD oxidative activity indirectly through the adrenal glands and the physiological level of corticosterone is ideal for normal reproductive functions.
Eighty-nine patients who had hydatidiform moles evacuated at the General Hospital, Kuala Lumpur, were followed with serum beta hCG determinations from October 1988 to June 1991. A regression curve for serum beta hCG, as measured by RIA, was derived from the results of 47 of the patients who demonstrated spontaneous regression of serum beta hCG titres. All 47 patients had normal serum titres at 135 days after evacuation. The mean time taken to reach normal level was 82.6 days, while the range was 39 to 135 days (5 to 19 weeks).
The diverse clinical syndromes characterized by asthmatic symptoms, transient pulmonary infiltrates, and eosinophilia have tended to obscure the specific association of one such entity with filarial infections. Serum IgE levels were determined before and after therapy in a group of well-characterized patients with tropical eosinophilia (TE), studied earlier in Singapore. The mean serum IgE level in 14 cases before treatment with diethylcarbamazine was 2,355 ng. per milliliter, with a trend but statistically nonsignificant decrease in levels to 600-1,000 ng. occurring 8 to 12 weeks after therapy. Leukocyte and eosinophil counts showed a rapid reduction after treatment, and although mean complement-fixing (cf) titers to Dirofilarial antigen tended to decrease, they were not significantly reduced until 5 to 6 weeks. The historical development of evidence supporting the filarial etiology of TE was reviewed. Many basic questions engendered by the clinical syndrome of tropical eosinophilia make it an excellent model for study of the immunopathology of parasitic infections.
BACKGROUND: Circulating antibodies to glutamic acid decarboxylase (GADab) and tyrosine phosphatase-like molecule IA-2 (IA-2ab) are major indicators for auto-immune destruction of pancreatic islet cells. They identify a majority of Caucasians with type 1 diabetes and approximately 50% of Asians, providing evidence of an idiopathic aetiology in the latter. The present study investigated these autoantibodies in a mixed ethnic group.
METHODS: Hospital clinic patients with clinically defined type 1 (n = 93) and type 2 (n = 300) diabetes and representing Singapore's major ethnic groups--Chinese, Indians and Malays--were studied. GADab and IA-2ab frequencies, and association of autoimmunity status with clinical and biochemical profiles were analysed.
RESULTS: Radio-immunoprecipitation assays detected either or both antibodies (seropositivity) in 41.9% of subjects with type 1 diabetes. GADab was detected in 36.6% and IA-2ab in 23.7% of type 1 diabetics. Prevalence of IA-2ab showed a reduction in frequency with disease duration (P = 0.026). In clinical type 2 diabetics, seropositivity was 10.0% with higher frequency in Malays (17.5%) than Chinese (9.7%) and Indians (4.5%). Multivariate analysis revealed that low fasting C-peptide was associated with seropositivity (odds ratio (OR) = 0.15; 95% confidence interval (CI) = 0.04-0.58). A significant relationship (OR = 13.5; 95% CI = 5.0-36.7) between insulin requirement and duration (>5 years) was also revealed. In patients with type 2 diabetes there was a trend of gradual progression to insulin dependency. However, there was considerable variation in body mass index between ethnic subgroups of type 2 diabetics, particularly for Chinese (mean (SD) = 26.0 (4.7)) and Malays (mean (SD) = 29.2 (5.9); P < 0.001).
CONCLUSIONS: Presence of both antibodies in our mixed ethnic group of type 1 diabetes patients was much lower than in Caucasians. Significant numbers of patients were seronegative for antibodies. Influences due to ethnicity and adiposity would require further investigations.