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We developed a direct microtiter plate enzyme immunoassay to measure estradiol-17 beta in saliva. The assay has a commercially available monoclonal antibody, raised against estradiol-17 beta-6-carboxymethyloximebovine serum albumin, and a homologous horseradish peroxidase conjugate measured colorimetrically. The detection limit (equivalent to B0-3 SD) is 365 amol/well or 7.3 pmol/L when 50-microL samples are assayed. Cross-reactivity with estrone and estriol, testosterone, or progesterone is0.2%. Estradiol-17 beta was measured in daily samples over five natural menstrual cycles and eight cycles stimulated as a preliminary to in vitro fertilization, and the concentrations and fluctuations found agreed with previously published data. This method gives results in approximately 3 h and may be useful for fertility monitoring and management.

The spread of the human immunodeficiency virus (HIV) and the resulting morbidity and mortality are major public health challenges with adverse social and economic implications. The pregnant population serves as an important marker of the extent of the problem in the sexually active low risk categorized population. Furthermore, a high HIV prevalence among women of childbearing age contributes to neonatal and infant mortality through perinatal transmission and also a large number of uninfected children become orphans. The objective of the present study was to determine the HIV prevalence rate and the risk factors in pregnant women attending antenatal care clinics in the Greater Harare area of Zimbabwe. Women presenting for antenatal care in four maternity clinics between May 1994 and June 1995 were tested for HIV-1 and HIV-2 antibodies following informed consent, counselling and completion of a questionnaire. Of the 1.168 women, 30.4 pc tested HIV-1 positive, with prevalence rates ranging from 23.6 pc at a lower density clinic, 28.6 pc in a medium density clinic, 30.7 pc in a higher density clinic and 33.2 pc at the referral maternity hospital. HIV-2 was present in 7.6 pc of the women. The 20 to 29 years age group had the highest HIV prevalence of 35 pc, (Odds Ratio (OR) = 2.4; 95 pc CI-1.33 to 4.32). Single pregnant women were more likely to test positive, (OR = 2.1; 95 pc CI = 1.2 to 3.7). Thirty five pc of the women reported previous use of condoms and in those where condom use was reported in casual relationships, there was a higher risk of HIV (OR 6.1; 95 pc CI = 2.1 to 17.3). Reported use of intravaginal herbs was associated with HIV risk (OR 1.4; 95 pc CI = 1.1 to 1.8; p0.03). History of genital ulcer (OR = 2.3), discharge (OR = 2.4), rash (OR = 2.7), genital ulcer with PID (OR = 5.8) was significantly associated with HIV infection. Present findings indicate a 30.4 pc HIV prevalence rate for a sample of 1,168 pregnant women in Harare. This rate is much higher than the 18 pc HIV prevalence rate reported for 1,008 pregnant women in the same Greater Harare area in 1990. We conclude that there is need for further innovative and aggressive community based as well as institutional interventions aimed at reducing HIV risk. Prevention strategies should include a wide range of socially contextualized initiatives.During May 1994-June 1995 in Zimbabwe, after informed consent and HIV counseling, 1168 pregnant women attending their first prenatal care visit at Harare Maternity Hospital and three Harare-based maternity clinics had blood taken to test for HIV-1 and HIV-2. The study aim was to determine the HIV prevalence and risk factors for HIV infection among pregnant women. 30.4% tested positive for HIV-1 (compared to 18% for a similar group in 1990). The HIV-1 prevalence ranged from 23.6% at the lowest density clinic to 33.2% at the referral maternal clinic of the hospital. 7.6% tested positive for HIV-2. Risk factors for HIV-1 infections included being 20-29 years old (odds ratio [OR] = 2.1), unmarried (OR = 2.1), unemployed (OR = 2.1); use of condoms in casual relationships as opposed to with husband or boyfriend (OR = 6.1); having a partner who frequented the bar everyday, every weekend, or twice per month (OR = 2); and history of sexually transmitted disease (STD) (OR = 2.3). Medical conditions associated with HIV-1 infection were palpable lymph glands (OR = 10.8), weight loss (OR = 4.5), and chronic diarrhea (OR = 2.2). STDs associated with HIV-1 infection included genital ulcer (OR = 2.3), genital discharge (OR =2.4), genital rash (OR = 2.7), and genital ulcer with pelvic inflammatory disease (OR = 5.8). The researchers recommend multisectoral and multilevel interventions to prevent HIV infections at all socioeconomic levels.

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.

Catechol estrogens such as 2-OH estrone are interesting estrogen metabolites formed in several human tissues and excreted in urine. We developed and thoroughly validated a radioimmunoassay for urinary 2-OH estrone that has several advantages over published RIAs. Because we developed a relatively specific antiserum, we did not include a preliminary chromatographic step to eliminate cross-reacting urinary steroids. We hydrolyzed urinary steroid conjugates with beta-glucuronidase from Helix pomatia because recoveries after acid hydrolysis were only 49.6% compared with 73.8% after enzyme hydrolysis. Published RIAs for urinary 2-OH estrone use acid hydrolysis. Our RIA measured 2-OH estrone independently of the volume of sample, and the detection limit was between 100 and 240 ng/L (10-24 pg per tube). The ED50 was 370 ng/L, and inter- and intraassay CVs for low, medium, and high concentrations were 22.5%, 22.8%, and 19.9%, and 17.4%, 14.3%, and 10.8%, respectively. Median concentrations measured in 14 controls and 33 postmenopausal patients with breast cancer were 0.96 and 1.55 micrograms/g creatinine, respectively, but there was considerable overlap between values from controls and patients.

We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV wasor = 8% in the range 2.7-52 micrograms/L; the interassay CV was usuallyor = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

We investigated the effects on plasma osteocalcin concentrations of different anticoagulants used to collect the blood samples. Plasma osteocalcin concentrations measured by enzyme immunoassay and radioimmunoassay are influenced by the nature of the anticoagulants used. The most significant difference between concentrations found in plasma and serum was seen with oxalate/fluoride anticoagulant, which reduced osteocalcin concentrations to 37.3% of serum values. This is probably related to increased hemolysis with this anticoagulant compared with osteocalcin concentrations in plasma prepared with other anticoagulants. Samples prepared with sodium citrate (0.105 mol/L) or lithium heparin gave values 92.4% and 83.6% of those obtained with matched serum samples. Osteocalcin concentrations were relatively stable in plasma and serum at -20 degrees C for two freeze/thaw cycles. In blood from 100 patients there was a good correlation between osteocalcin concentrations in serum and plasma (lithium heparin) (r2 = 0.831); the slope and intercept (+/- SE) were 0.924 +/- 0.04 and 4.92 +/- 1.25 micrograms/L, respectively. However, in 10 patients, serum osteocalcin concentrations were two- to threefold higher than those in matched plasma samples.

Following in vitro stimulation with phytohaemagglutinin, lymphocytes from coeliac patients transformed less than those from control subjects. Neither gliadin nor detoxified gliadin stimulated lymphocyte transformation in patients with adult coeliac disease, but depressed transformation in lymphocytes from normal subjects and from patients on a gluten-free diet.

In this solid-phase enzymoimmunoassay for estrone in saliva, microtiter plates are used after extraction of the sample with diethyl ether. No chromatographic step is involved. The detection limit of the assay is 450 fg per well (33 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in saliva were respectively 4.2, 12.7; 5.2, 8.7; and 2.7, 5.8%. Using this assay, we found a highly significant correlation (P less than 0.001) between estrone concentrations in time-matched serum and saliva samples. The analytical procedure is rapid and relatively simple. One person can assay 50-60 saliva samples during a normal working day. We conclude that the assay is very suitable, even in small laboratories, for saliva estrone measurements, which, in facilitating serial sampling, enables dynamic observations of estrone concentrations and ovarian activity to be more easily made.

Electrophoretic studies showed that 3-hydroxybutyrate dehydrogenase and aspartate aminotransferase patterns may prove useful for characterising Rhizobium strains. Although a relatively small number of strains was tested in this study, it was interesting thatR. trifolii andR. leguminosarum had similar patterns of both enzymes. Some preliminary studies on the possible roles of the three forms of aspartate aminotransferase in soybean nodules are also presented.

SUMMARY: The concentrations of three enzymes active in ammonia assimilation by Rhizobium japonicum (glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase) were influenced considerably by the N-source in the culture medium. Relatively high concentrations of these enzymes were present when R. japonicum was grown in a medium containing NH4Cl + 2-oxoglutarate. Relatively low amounts of these enzymes were found in bacteria grown in the presence of various amino acids. Active protein and RNA synthesis were necessary for the induction of the enzymes by NH4Cl + 2-oxoglutarate.

A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.

We have developed and thoroughly validated radioimmunoassays (RIAs) for osteocalcin in plasma and serum, demonstrating independence of volume and determining the recovery of added standard and within- and between-assay precision. Antibodies were raised in rabbits and mice by immunization with either osteocalcin adsorbed to polyvinylpyrrolidone or osteocalcin conjugated to bovine serum albumin. In both species, use of the conjugated osteocalcin was more efficacious. Correlation of the results obtained when groups of plasma or serum samples were analyzed by RIAs with different antibodies (two polyclonal and one monoclonal) indicated that the normal range observed for osteocalcin concentrations in serum depends on the antibody used. These findings may account for the wide range of "normal" osteocalcin values reported by different groups.

Small intestinal lactase, sucrase and alkaline phosphatase activities were measured in histologically normal peroral intestinal biopsies from 477 individuals. Enzyme activities varied with age, sex, site of biopsy, and were lowest in post-weaning children and highest in young adults. Lactase activity does not decrease with advancing age.

This rapid solid-phase enzymoimmunoassay for estrone in serum or plasma is done on microtiter plates after the serum is extracted with diethyl ether. No chromatographic or centrifugation steps are involved. The detection limit of the assay is 380 fg per well (28 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in plasma were respectively 4.4, 9.3; 2.3, 9.1; and 2.0, 6.3 percent. There was a good correlation (correlation coefficient 0.95) between estrone concentrations measured with this assay and with a commercial radioimmunoassay. The analytical procedure is simple, and one person can assay 80 serum samples per working day. We conclude that the assay is very suitable for serum estrone measurements and is more convenient than published radioimmunoassays.

This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

The absorption of xylose following an oral load of 15 g D-xylose has been studied by serial blood levels in 17 untreated adult coeliac patients, 21 treated coeliac patients, and 30 non-coeliac patients. A statistically significant difference in xylose blood levels was found between untreated coeliac and non-coeliac patients at all the times studied, but a complete separation between these two groups occurred only at the 75 minute stage. The reproducibility of absorption was assessed by repeating the test in 16 subjects. The 95% confidence limits of the standard error of estimate are narrowest at 75 and 90 minutes.

In this solid-phase enzymoimmunoassay for estrone in saliva, microtiter plates are used after extraction of the sample with diethyl ether. No chromatographic step is involved. The detection limit of the assay is 450 fg per well (33 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in saliva were respectively 4.2, 12.7; 5.2, 8.7; and 2.7, 5.8%. Using this assay, we found a highly significant correlation (P less than 0.001) between estrone concentrations in time-matched serum and saliva samples. The analytical procedure is rapid and relatively simple. One person can assay 50-60 saliva samples during a normal working day. We conclude that the assay is very suitable, even in small laboratories, for saliva estrone measurements, which, in facilitating serial sampling, enables dynamic observations of estrone concentrations and ovarian activity to be more easily made.

Lactase, sucrase, and alkaline phosphatase activities were measured in 833 peroral small intestinal biopsies from 373 patients with coeliac disease. Enzyme activities decreased with increasing degrees of mucosal damage. Enzyme activities in mucosae of patients with coeliac disease in remission were lower than in control groups matched for age, sex, and site of biopsy. Enzyme activities were measured in 81 patients when the mucosa was severely damaged and later when considerable improvement had occurred. Lactase activity remained low in 13% of patients under the age of 18 and in 33% of those over 18 years. Sucrase activity usually improved with histological recovery, but alkaline phosphatase activity tended to remain depressed in patients in whom lactase activity failed to improve.

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

The potential applications of measuring reproductive steroids in saliva were examined for investigating the basis of the lower prevalence of breast cancer and cancers of the reproductive system among former college athletes compared to non-athletes. Daily salivary progesterone measurements during the menstrual cycle by a rapid cost effective enzyme-immunoassay provided a more accurate method than established procedures for detecting abnormal ovarian function such as luteal phase defects. These abnormalities are common in athletes who have a later menarche, a higher incidence of oligomenorrhoea and amenorrhoea than non-athletes. A control corridor was established of upper and lower limits for normality of daily salivary progesterone values during the complete menstrual cycle in 42 volunteers. It is concluded that salivary steroid assays provide an accurate and non-invasive procedure suitable for long term comparative studies on ovarian steroids in female athletes and non-athletes before and after menarche.

We have developed a rapid enzyme immunoassay for progesterone in saliva. This solid-phase assay is carried out on microtitre plates with no extraction or centrifugation steps. The detection limit of the assay is 200 fg per well (3.2 pmol/L). Intra- and interassay coefficients of variation for low, medium, and high concentrations of progesterone were 7.5, 16.0; 9.1, 8.3; and 8.7, 6.7%, respectively. Correlation between total plasma progesterone (assayed by enzyme immunoassay with extraction) and salivary progesterone concentrations was good (r = 0.848, p less than 0.001, n = 56). We found the assay useful for monitoring ovarian function. The analytical procedure is convenient, and one person can assay more than 200 saliva samples per working day. The turnaround time for 36 samples is 2 h, including 1.5 h of incubation time, when previously coated plates are used. We conclude that such assays are very suitable for measuring progesterone in serial saliva samples and could become the preferred method for monitoring ovarian function.

SUMMARY: 3-Hydroxybutyrate dehydrogenase was studied in 14 Rhizobium strains representing six species. Cell-free extracts of the bacteria were subjected to electrophoresis in starch or polyacrylamide gels and 3-hydroxybutyrate dehydrogenase was located in situ on the gels. Also, the rate of reaction of the enzyme, from different Rhizobium strains, with NAD was compared with its rate of reaction with various analogues of NAD. Most of the Rhizobium strains had characteristic 3-hydroxybutyrate dehydrogenase patterns on gel electropherograms and these patterns were highly reproducible. Multiple forms of 3-hydroxybutyrate dehydrogenase were detected in many of the Rhizobium strains.