Double-antibody Enzyme-linked Immunosorbent Assay Research Articles

Infectivity of Maize Chlorotic Mottle Virus from Contaminated Maize Seeds

Seeds have been identified as major sources of introduction and spread of pathogens, with viruses being detected in the seed and also on the seed coat. In this study, the infectivity of Maize chlorotic mottle virus (MCMV) through seeds was investigated. Maize seeds that had tested positive for MCMV previously using double antibody enzyme-linked immunosorbent assay (DAS-ELISA) and real-time reverse transcription polymerase chain reaction (real time RT-PCR) were obtained from various sources. The seeds were soaked in phosphate buffer overnight and the solution used to inoculate maize seedlings. The whole seed was also ground and mixed with the buffer and used for inoculation of seedlings by hand rubbing. Visible MCMV symptoms were observed on less than 2% of the 547 seedlings inoculated with the seed soak and seed extract from contaminated seed 28 days after inoculation and this was confirmed using DAS-ELISA. Use of real time reverse transcription polymerase chain reaction revealed infectivity of MCMV from one of the seed sources used. The mean cycle threshold (Ct) values of samples that showed infectivity ranged from 28.21 to 29.40 cycles. The means were significantly different (P<0.001) from the other samples tested, the healthy and negative controls. When compared to seedlings inoculated with MCMV-infected leaf sap, there was visible development of symptoms associated with MCMV infection, with a severity score of three and Ct values as low as 11.53. The results show evidence of infection of MCMV on maize seedlings caused by virus present in seed extract. Despite rare occurrence of infectivity, the presence of viable virus may cause spread of the virus in the field, leading to development of maize lethal necrosis disease where a cereal potyvirus is present.

Prevalence and factors associated with latent autoimmune diabetes in adults (LADA): a cross-sectional study

BackgroundThe Latent Autoimmune Diabetes in Adults (LADA) is a slowly progressive Type 1 diabetes subgroup with onset during middle age. Studies report that about 10% of adults initially diagnosed with clinical Type 2 diabetes (T2D) have LADA. Inappropriate diagnosis and mismanagement of the LADA can increase the risk of diabetic complications, which affect the quality of life and is the cause of increased mortality. In low-income countries setting, data regarding the magnitude of LADA is limited. We carried out this study to estimate the burden of misdiagnosed LADA among T2D patients in selected health facilities in Dar es Salaam and to bring awareness to the use of Glutamic Acid Decarboxylase (GAD) autoantibody in screening for LADA.MethodologyWe enrolled 186 phenotypically T2D patients in this cross-sectional study, through a standardized data collection tool we obtained participants’ demographic and clinical information. For testing GAD levels, we used a double-antibody Enzyme-Linked Immunosorbent Assay (ELISA). The Fisher’s Exact and student t-tests were used to test the significance of the statistical associations of the glycaemic control and diabetes complications between T2D and LADA.ResultsOut of 186 patients, 156 gave conclusive GAD Ab ELISA reading with LADA accounting for 5.1% (95% CI: 2.5 - 10.0). The mean age of subjects was 54.3 years (Range: 33-85 years). The parameters such as mean age, family history of diabetes mellitus status, Fasting Blood Glucose, clinical characteristics, and complications did not show significant statistical differences between patients with LADA and Type 2 diabetes. However, all LADA- Human Immunodeficiency Virus (HIV) comorbid patients had retinopathy, which was statistically insignificant in 20 (87%) T2D-HIV comorbid patients (p = 0.669). Neither neuropathy, nephropathy, nor Diabetic Mellitus (D.M.) foot syndrome was observed among LADA-HIV comorbid patients. Nevertheless, 22 (95.7%), 3 (13%), and 2 (8.7%) of T2D-HIV comorbidity had neuropathy, nephropathy, or D.M. foot syndrome, respectively.ConclusionsThe study established a LADA prevalence of 5.1% among T2D patients and has shown the role of GAD autoantibody in the screening for LADA. The study calls for a well- designed larger longitudinal study to generate strong evidence on the association of risk factors and complications associated with the LADA. This will develop robust evidence on the association of risk factors and complications associated with the LADA and T2D.

Evaluation of the role of ischemia modified albumin in neonatal hypoxic-ischemic encephalopathy.

BackgroundBirth asphyxia is a leading cause of neonatal mortality. Ischemia-modified albumin (IMA) levels may have a predictive role in the identification and prevention of hypoxic disorders, as they increase in cases of ischemia of the liver, heart, brain, bowel, and kidney.PurposeThis study aimed to assess the value of IMA levels as a diagnostic marker for neonatal hypoxic-ischemic encephalopathy (HIE).MethodsSixty newborns who fulfilled 3 or more of the clinical and biochemical criteria and developed HIE as defined by Levene staging were included in our study as the asphyxia group. Neonates with congenital malformation, systemic infection, intrauterine growth retardation, low-birth weight, cardiac or hemolytic disease, family history of neurological diseases, congenital or perinatal infections, preeclampsia, diabetes, and renal diseases were excluded from the study. Sixty healthy neonates matched for gestational age and with no maternal history of illness, established respiration at birth, and an Apgar score ≥7 at 1 and 5 minutes were included as the control group. IMA was determined by double-antibody enzymelinked immunosorbent assay of a cord blood sample collected within 30 minutes after birth.ResultsCord blood IMA levels were higher in asphyxiated newborns than in controls (250.83±36.07 pmol/mL vs. 120.24±38.9 pmol/mL). Comparison of IMA levels by HIE stage revealed a highly significant difference among them (207.3±26.65, 259.28±11.68, 294.99±4.41 pmol/mL for mild, moderate, and severe, respectively). At a cutoff of 197.6 pmol/mL, the sensitivity was 84.5%, specificity was 86%, positive predictive value was 82.8%, negative predictive value was 88.3%, and area under the curve was 0.963 (P<0.001).Conclusion IMA levels can be a reliable marker for the early diagnosis of neonatal HIE and can be a predictor of injury severity.

Correlation between serum interleukin-38 and acute exacerbation of chronic obstructive pulmonary disease with pulmonary embolism

Objective: To investigate the correlation between serum interleukin-38 (IL-38) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) with pulmonary embolism (PE). Methods: The 94 patients with AECOPD admitted to Tianjin Chest Hospital from August 2015 to April 2018 were suspected of PE. They were divided into two groups based on CT pulmonary angiography (CTPA) and color Doppler ultrasound of lower extremity veins: 39 cases in PE group and 55 cases in Non-PE group. The general data and laboratory examination results of these subjects were recorded. Serum IL-38 was measured by double antibody enzyme-linked immunosorbent assays (ELISA). The between-group differences of above parameters were analyzed. Pearson correlation or Spearman rank correlation was used to analyze the association of IL-38 with each variable in AECOPD patients. Binary Logistic regressions were conducted to determine the risk factors of AECOPD with PE. ROC curve was used to assess the value of serum IL-38 in predicting AECOPD with PE. Results: The serum level of IL-38 was lower in PE group than in Non-PE group [46.3 (33.1, 58.1) vs 61.5 (46.6, 72.5) ng/L, P<0.05]. Correlation analysis showed that serum IL-38 levels were negatively correlated with C reactive protein and fibrinogen in patients with AECOPD (r=-0.38,-0.29, all P<0.05). Binary Logistic regressions showed that lower serum IL-38 level was a risk factor of AECOPD with PE (OR=0.78, 95%CI: 0.61-0.94, P<0.05). The area under the ROC curve was 0.74 (95% CI: 0.63-0.84, P<0.05). When cutoff value of serum IL-38 was 52.1ng/L, the sensitivity was 70.9% and the specificity was 69.2% respectively. Conclusion: IL-38 could relieve the hypercoagulability by inhibiting inflammation in patients with AECOPD and could act as a predictor of AECOPD with PE.

Comparison of Serological and Molecular Methods With High-Throughput Sequencing for the Detection and Quantification of Grapevine Fanleaf Virus in Vineyard Samples.

Grapevine fanleaf virus (GFLV) is the main causal agent of fanleaf degeneration, the most damaging viral disease of grapevine. GFLV is included in most grapevine certification programs that rely on robust diagnostic tools such as biological indexing, serological methods, and molecular techniques, for the identification of clean stocks. The emergence of high throughput sequencing (HTS) offers new opportunities for detecting GFLV and other viruses in grapevine accessions of interest. Here, two HTS-based methods, i.e., RNAseq and smallRNAseq (focusing on the 21 to 27 nt) were explored for their potential to characterize the virome of grapevine samples from two 30-year-old GFLV-infected vineyards in the Champagne region of France. smallrnaseq was optimal for the detection of a wide range of viral species within a sample and RNAseq was the method of choice for full-length viral genome assembly. The implementation of a protocol to discriminate between low GFLV titer and in silico contamination (intra-lane contamination due to index misassignment) during data processing was critical for data analyses. Furthermore, we compared the performance of semi-quantitative DAS-ELISA (double antibody enzyme-linked immunosorbent assay), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), Immuno capture (IC)-RT-PCR, northern blot for viral small interfering RNA (vsiRNA) detection and RNAseq for the detection and quantification of GFLV. While detection limits were variable among methods, as expected, GFLV diagnosis was consistently achieved with all of these diagnostic methods. Together, this work highlights the robustness of DAS-ELISA, the current method routinely used in the French grapevine certification program, for the detection of GFLV and offers perspectives on the potential of HTS as an approach of high interest for certification.

Interleukin-38 expression and clinical significance in serum of patients with chronic obstructive pulmonary disease

Objective: To investigate the serum level of interleukin-38 (IL-38) and its clinical significance in patients with chronic obstructive pulmonary disease (COPD). Methods: Totally 72 patients with acute exacerbation of COPD (AECOPD group) and 65 patients with stable COPD (S-COPD group) were recruited from Tianjin Chest Hospital from June 2016 to August 2017. In the same period 40 elderly healthy subjects were selected as control group (C group). The general data and laboratory examination results of these subjects were recorded. Serum IL-38 was measured by double antibody enzyme-linked immunosorbent assays (ELISA). The inter-group differences of above parameters were analyzed. Pearson correlation or Spearman rank correlation analysis was used to analyze the correlation between IL-38 and each variable, and multivariate stepwise regression analysis was used to determine the influencing factors of serum IL-38 in COPD patients. Results: The serum level of IL-38 was higher in AECOPD group than in S-COPD group[(57.88±13.72) vs (51.75±14.06) ng/L, P<0.05], and was higher in either of the two COPD groups than in C group[(46.37±13.18) ng/L](both P<0.05). Correlation analysis of single factor showed that serum IL-38 levels were positively correlated with body mass index (r=0.190, P<0.05), and negatively correlated with C reactive protein (CRP), fibrinogen (FIB), forced expiratory volume in one second as a percentage of estimated value (FEV1%pred) and the number of acute exacerbations in the past 1 year (r=-0.344, -0.176, -0.195, -0.229, all P<0.05). The CRP level and the number of acute exacerbations in the past 1 year were independent factors affecting the serum level of IL-38 (β=-0.204, -0.183, both P<0.05) in patients with COPD. Conclusion: IL-38 is compensatory increased in serum of patients with COPD and may be used as one of the serological markers for evaluation of COPD.

A novel small molecule immunoassay to detect the mycobacterial siderophore carboxymycobactin

Background: To diagnose tuberculosis (TB), it is necessary to demonstrate the presence of Mycobacterium tuberculosis in a clinical specimen such as sputum. A simple, low-cost rapid test for blood or urine is urgently needed but remains elusive. Tests for host response-derived biomarkers or secreted bacterial compounds have so far failed to provide sufficient sensitivity or specificity and alternative approaches are needed. Carboxymycobactins are amphiphilic siderophores unique to the mycobacteria that have the ability to transverse mammalian cell walls. They have insufficient mass to induce an antibody response and there are currently no simple sensitive tests for their detection. Methods: We report the development of an enzyme-linked immunosorbent assay (ELISA) for carboxymycobactin. Polyclonal antibodies were raised in rabbits following conjugation to a carrier protein, bovine serum albumin allowing development of a sensitive indirect double antibody ELISA. A second keyhole limpet hemocyanin conjugate was manufactured to adhere the hapten to the wells of the microplate. We established a limit of detection for the assay of 1 pg/ml. Carboxymycobactin was detected in culture supernatant from mycobacteria including clinical isolates of M. tuberculosis , but not from cultures of Rhodococcus, Nocardia, Streptomyces, Bacillus cereus, Klebsiella oxytoca, Staphylococcus aureus, Escherichia coli , and Pseudomonas aeruginosa . Sample concentration was achieved following extraction with chloroform. Results: We have demonstrated for the first time the direct detection of carboxymycobactin in culture supernatant by immunoassay. Conclusions: We recommend testing samples from humans and animals to establish the potential utility of carboxymycobactin as a diagnostic marker of active mycobacterial disease.

Chemokine CXCL-1: activity in the vitreous during proliferative vitreoretinopathy.

The aim of this study was to investigate CXCL-1 chemokine levels in the vitreous during rhegmatogenous retinal detachment (RRD) with and without proliferative vitreoretinopathy (PVR) and identify possible correlations with clinical parameters (extent and duration or RRD and PVR grade). Vitreous samples from patients with primary RRD with or without PVR were collected and assayed using a double antibody enzyme-linked immunosorbent assay (ELISA). Eleven vitreous samples from organ donors were employed as a control group. CXCL-1 levels were measured in 35 vitreous samples from 35 RRD patients. Mean CXCL-1 levels (64·82 ± 6·47 pg/ml) were significantly higher (P = 0·048) compared to controls. There was a significant positive correlation between CXCL-1 levels and the extent of the detachment (r = 0·794, P = 0·006). Peak CXCL-1 levels coincided with 3+ quadrant RRD, an interim of 29-60 days' duration and PVR grade B. Increased CXCL-1 levels may be indicative of mild inflammation in the detached retina and the adjacent vitreous. The results of the present study may provide novel insight into the complex interactions taking place during the early and late stages of RRD complicated by PVR.

Effect of miR-29a inhibition on ventricular hypertrophy induced by pressure overload.

To investigate whether inhibition of miR-29a functioning prevents the hypertension-induced ventricular hypertrophy and fibrosis. Patients diagnosed with hypertension and left ventricular hypertrophy were recruited for the study. Serum levels of miR-29a were determined by RT-PCR. Levels of serum matrix metalloproteinase-9 (MMP-9), collagen type I and III (PINP and PIIINP) were determined by double-antibody enzyme-linked immunosorbent assay. Mouse model of transverse aortic constriction (TAC) was established. 7 days after surgery, TAC mice were injected intraperitoneally with antagomir miR-29a or vehicle once a day for 3 days. After 4 weeks of surgery, animals were sacrificed and cross-sections of the hearts were stained and evaluated for hypertrophy and fibrosis. The expression of the protein markers of hypertrophy and fibrosis was determined by immunoblotting. The serum level of miR-29a in hypertensive patients with left ventricular hypertrophy was significantly higher than those in patients with hypertension alone (p < 0.05). The levels of serum miR-29a were positively correlated with those of PINP, PIIINP, and MMP-9 (r = 0.58, 0.45, 0.66, respectively, p < 0.05). In mouse model of pressure overload, the antagomir miR-29a was found to significantly suppress the hypertrophy of cardiomyocytes and the expression of ANP and β-MHC, the hypertrophy indices. Also, the ventricular fibrosis and expression of the marker proteins were blocked in antagomir treated mice. The inhibition of miR-29a was found to be effective in improving the ventricular remodeling and hypertrophy caused by pressure overload.

Early Expression of Tumor Necrosis Factor α and Nuclear Factor Kappa B After Descemet Stripping Endothelial Keratoplasty in Rabbits

To investigate immune rejection after Descemet stripping endothelial keratoplasty (DSEK) compared with that after penetrating keratoplasty (PKP) in New Zealand rabbit eyes and to identify the relationship between tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) and evaluate their involvement in graft rejection. Sixty-nine New Zealand rabbits were divided into 3 groups: group A comprised the control group, which did not undergo either of the procedures; group B underwent DSEK; and group C underwent PKP. Twenty-three Chinese rabbits (46 eyes) were used as donors. The corneal transparency, graft placement, and anterior chamber were observed for 28 days. Blood serum, aqueous humor, and corneal samples were obtained on days 7, 14, 21, and 28 after the procedures. TNF-α levels were measured weekly by double-antibody enzyme-linked immunosorbent assay. NFκB in the cornea was checked by immunofluorescence. There were no graft rejections in group B. Group C showed a 50% rejection frequency with an increase in both the TNF-α concentrations and NFκB expression observed at 7 days and peak levels observed at 28 days after PKP. TNF-α concentrations did not differ significantly (P > 0.05) between groups B and A. NFκB expression was slightly increased at 7 days and returned to normal levels by day 14 after DSEK. Rabbits with DSEK had a lower rejection rate than rabbits with PKP. NFκB and TNF-α may contribute to the early rejection of corneal allografts.

Culex annulirostris (Diptera: Culicidae) Host Feeding Patterns and Japanese Encephalitis Virus Ecology in Northern Australia

Japanese encephalitis virus (JEV) transmission in northern Australia has, in the past, been facilitated by Culex annulirostris Skuse feeding on domestic pigs, the primary amplifying hosts of the virus. To further characterize mosquito feeding behavior in northern Australia, 1,128 bloodmeals from Cx. annulirostris were analyzed using a double-antibody enzyme-linked immunosorbent assay. Overall, Cx. annulirostris obtained > 94% of blood meals from mammals, comprising marsupials (37%), pigs (20%), dogs (16%), and cows (11%), although the proportion feeding on each of these host types varied between study locations. Where JEV activity was detected, feeding rates on pigs were relatively high. At the location that yielded the first Australian mainland isolate of JEV from mosquitoes, feral pigs (in the absence of domestic pigs) accounted for 82% of bloodmeals identified, representing the first occasion that feeding on feral pigs has been associated with JEV transmission in Australia. Interestingly, < 3% of Cx. annulirostris had fed on pigs at locations on Badu Island where JEV was detected in multiple pools of mosquitoes in a concurrent study. This suggests that either alternative hosts, such as birds, which comprised 21% of blood meals identified, or infected mosquitoes immigrating from areas where domestic pigs are housed, may have contributed to transmission at this location. Because Cx. annulirostris is both an opportunistic feeder and the primary JEV vector in the region, environmental characteristics and host presence can determine JEV transmission dynamics in northern Australia.

Determination of Mosquito (Diptera: Culicidae) Bloodmeal Sources in Western Australia: Implications for Arbovirus Transmission

A double-antibody enzyme-linked immunosorbent assay was used to determine the bloodmeal sources of adult mosquitoes (Diptera: Culicidae) collected in encephalitis vector surveillance mosquito traps in Western Australia between May 1993 and August 2004. In total, 2,606 blood-fed mosquitoes, representing 29 mosquito species, were tested, and 81.7% reacted with one or more of the primary antibodies. Aedes camptorhynchus (Thomson) and Culex annulirostris Skuse were the most common species tested, making up 47.2% (1,234) and 35.6% (930), respectively. These species obtained bloodmeals from a variety of vertebrate hosts but particularly marsupials and cows. In contrast, Culex pullus Theobald (72.7%; 24/33), Culiseta atra (Lee) (70.0%; 7/10), Culex globocoxitus Dobrotworsky (54.5%; 12/22), and Culex quinquefasciatus Say (39.3%; 22/56) often obtained bloodmeals from birds. Although Ae. camptorhynchus and Cx. annulirostris are well established vectors of arboviruses, other mosquitoes also may have a role in enzootic and/ or epizootic transmission.

Increased Levels of Lipoprotein (a) Are Related to Family Risk Factors of Cardiovascular Disease in Children and Adolescents From Maracaibo, Venezuela

The aims of this study were to determine Lipoprotein (a) [Lp(a)] concentrations in a sample of subjects from Maracaibo, Venezuela, and to determine the relationship of family risk factors for cardiovascular disease and their Lp(a) levels. Two hundred twenty-seven healthy individuals between 5 and 19 years of age of both genders and multiethnic origins were selected. A complete background clinical chart and laboratory test was conducted for each patient to discard cardiovascular diseases and confirm their healthy state. The Lp(a) concentration was determined using the double antibody enzyme-linked immunosorbent assay method. For inferential statistical analysis, one-factor analysis of variance tests and Student t test for independent observations were used according to each case, considered significant when P value was <0.05. No significant differences were observed when evaluating Lp(a) levels according to gender in all ages. Males showed no significant difference in Lp(a) levels between groups, but, in females, a significantly lower level (P < 0.03) in the group 5 to 9 years of age was found. When considering only age, significantly lower levels were observed (P < 0.03) in the 5- to 9-year-old group. When studying family risk factors of cardiovascular diseases, it was found that the group with family risk factors had a significantly higher Lp(a) concentration (P < 0.01) than those without family risk factors, observing that those who had four or more factors exhibited a significantly higher concentration than those with two to three risk factors (30.6 +/- 4.5 mg/dL versus 18.5 +/- 12.2 mg/dL, P < 0.009) and than those with one risk factor (30.6 +/- 4.5 mg/dL versus 21.6 +/- 1.4 mg/dL, P < 0.03). These results emphasize the clusters of family risk factors of cardiovascular disease with higher Lp(a) levels and also indicate that the evaluation of its concentration should be taken as an independent risk factor of atherosclerosis for the population in developmental ages.

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 μg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12 h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.

Alternative splicing of P2X6 receptors in developing mouse brain and during in vitro neuronal differentiation

We have previously demonstrated that intestinally infused bovine lactoferrin (bLF) is transported into the blood circulation via the lymphatic pathway, not via the portal circulation. Therefore, in the present study, we further investigated whether intragastrically infused enteric-formulated bLF (EF-bLF) was more efficiently absorbed than bLF from the intestine in adult rats. The rats were randomly divided into three groups: 30 and 300 mg kg(-1) non-enteric-formulated bLF (non-EF-bLF) groups and a 30 mg kg(-1) EF-bLF group. Thoracic lymph was collected from a thoracic lymph duct under general anaesthesia. Bovine lactoferrin was infused into the stomach or duodenal lumen via a needle for a period of over 1 min in a volume of 1 ml kg(-1). The bLF transported into the lymph was assayed quantitatively by double-antibody enzyme-linked immunosorbent assay (ELISA). Following the intragastric administration of bLF, the three groups showed almost the same lymph flow, but the bLF concentration in the lymph fluid in the EF-bLF group increased significantly and peaked 3 h after administration. With intraduodenal administration, the bLF concentration in the lymph fluid of the higher non-EF-bLF group was significantly higher than those of the other groups. The amount of absorbed bLF in the EF-bLF group was, however, about 10 times higher than that in the lower non-EF-bLF group, when it was administered intragastrically. These data show that enteric-formulated bLF is less susceptible to gastric pepsin and is more efficiently absorbed from the intestine than is non-enteric-formulated bLF.

Development of a sensitive enzyme immunoassay for anti‐Müllerian hormone and the evaluation of potential clinical applications in males and females

Recent studies have found anti-Müllerian hormone (AMH) to be a potentially important marker for the assessment of ovarian reserve and prediction of the success of in vitro fertilization (IVF) treatment. The objectives of this study were to develop a sensitive and specific assay for AMH and to evaluate the potential application of the assay. This assay will be then available to our collaborators in the UK and overseas. Samples obtained as part of another prospective cross-sectional study from infertility patients and another prospective longitudinal study from pregnant women were used in this study to measure AMH using a new double-antibody enzyme-linked immunosorbent assay (ELISA). AMH levels were evaluated in (i) serum and seminal fluid from males (normal and male factor infertility males), (ii) serum and follicular fluid from females (normal and female with unexplained infertility) and (iii) serum, amniotic fluid (AF) and coelomic fluid (CF) from pregnant women. AMH levels in the samples were measured by a newly developed ELISA. The assay had a detection limit of<0.078 ng/ml. High recoveries of spiked recombinant protein were observed from male and female sera and also from follicular, seminal, coelomic and amniotic fluids. The intra- and interassay coefficients of variation (CVs) were 3.6% and 4.0%, respectively. Serially diluted human samples gave dose-response curves parallel to the standard curve. Immunoreactivity was stable to sample storage at room temperature for several days and to multiple cycles of freezing and thawing. In seminal fluid, the AMH concentrations in a group of men with male factor infertility were insignificantly different from those in fertile men. By contrast, serum AMH concentrations were lower in the male factor infertility group than the normal group of patients. Women with unexplained infertility had similar concentrations of AMH in serum and follicular fluid compared to controls. Pregnant women had higher concentrations of AMH in the circulation in early pregnancy compared with nonpregnant women, suggesting a foeto-placental contribution and a possible biological role for this molecule in early pregnancy. We have developed a sensitive and specific assay for AMH. Serum AMH in men with male factor infertility is lower than in normal men. Levels of AMH in pregnancy are higher than normal menstrual cycle levels suggesting a foeto-placental contribution.

The determinants of dust mite allergen and its relationship to the prevalence of symptoms of asthma in the Asia-Pacific region.

The role that house dust mites play in the primary causation of asthma is controversial. Approximately thirty-six 10-yr-old children in each of 10 centres in the Asia-Pacific region participated. Researchers collected dust from mattresses and living room floors using standardized procedures. Der p1 and Der f1 were analysed using a double monoclonal antibody enzyme-linked immunosorbent assay. Geometric mean allergen levels were calculated for each centre. An ecological analysis was conducted to show the regression of the geometric mean allergen level, using the highest household level, against asthma symptom and severity prevalence data from the International Study of Asthma and Allergies in Childhood, Phase I. Among children aged 13-14 yr, the change in asthma symptom prevalence was associated with per unit change in Der p1 microg/g (1.08, 95% CI 0.10-2.06) and Der 1 microg/g (Der p1 + Der f1) (0.64, 95% CI 0.02-1.26). The change in having four or more attacks of asthma in the last 12 months was associated with per unit change in Der p 1 microg/g (0.29, 95% CI -0.02 to 0.60) and Der 1 microg/g (0.20, 95% CI 0.01-0.38). There was no effect for total Der p1 or Der f1 (total or microg/g). Among children aged 6-7 yr, neither allergen was related to symptoms or severity prevalence. While our findings suggest that Dermatophagoides pteronyssinus may have a role in the primary causation of asthma, the complexity of this association reinforces the need for prospective studies.

An association of autoantibody status and serum cytokine levels in type 1 diabetes.

At onset of type 1 diabetes, the islet autoantibody status of patients has been reported to predict progression of the disease. We therefore tested the hypothesis that the systemic immunoregulatory balance, as defined by levels of circulating cytokines and chemokines, is associated with islet autoantibody status. In 50 patients with recent-onset type 1 diabetes, antibodies to GAD and insulinoma-associated antigen 2 (IA-2) were analyzed by radioimmunoassay; cytoplasmic islet cell antibodies were determined by indirect immunofluorescence. Cytokine and chemokine concentrations were measured by rigidly evaluated double antibody enzyme-linked immunosorbent assay. Of four classically defined Th1/Th2 cytokines (gamma-interferon, interleukin [IL]-5, IL-10, IL-13), none showed an association with multiple autoantibody positivity. Of six mediators mainly produced by innate immunity cells, three were associated with multiple autoantibody status (IL-18 increased, MIF and MCP-1 decreased) and three were unaffected (IL-12, MIP-1beta, IP-10). GAD and/or IA-2 antibody titers negatively correlated with systemic concentrations of MIF, MIP-1beta, and IL-12. Combining the data of several cytokine and chemokine levels made it possible to predict islet antibody positivity in individual patients with 85% sensitivity and 94% specificity. These data suggest a close association of islet antibody status with systemic immunoregulation in type 1 diabetes.

Is inhibin B a potential marker of gonadotoxicity in prepubertal children treated for cancer?

Chemotherapy treatment of childhood cancer may impair gonadal function, which may be manifested only in adulthood as permanent sterility. Detection of gonadal dysfunction in prepubertal children has been hampered by the absence of a sensitive marker. Inhibin B is secreted by small antral follicles and Sertoli cells in females and males, respectively, and may be a marker of gonadal function in prepubertal children. The aim of this pilot study was to evaluate inhibin B in relation to sensitive measurements of gonadotrophins as markers of the early gonadotoxic effects of chemotherapy in prepubertal children treated for cancer. Twenty-five prepubertal children (9 females), median age 4.5 years (range 1.2-12.8 years) with cancer (16 solid tumours, nine acute lymphoblastic leukaemia, ALL) were studied longitudinally. Blood samples were collected before and during chemotherapy (solid tumours) or immediately following induction and first intensification (ALL). Post-treatment (1-6 months) samples were collected in 12 of the patients (5 females). Dimeric inhibin B was measured by double antibody enzyme-linked immunosorbent assay (ELISA). FSH and LH were measured by sensitive time-resolved immunofluorescence. Girls: Pretreatment inhibin B was slightly high in one girl but normal for age and sex in all others: median 16.1 (range 9.4-186.2) ng/l, median SD score +0.2 (-1.3 to +2.6). Inhibin B decreased to undetectable levels (< 8 ng/l) in 8/9 girls during treatment (P = 0.03), with no accompanying rise in FSH or LH. Post-treatment recovery of inhibin B was variable: median 16.1 (range < 8.0-44.2) ng/l, median SD score +0.1 (range < -2.4 to +1.8). Sustained undetectable inhibin B levels were observed in 2/5 girls with correspondingly elevated FSH concentrations (11.8 and 10.9 U/l). Boys: Inhibin B was normal for age and sex in all boys before treatment with no significant change during or after treatment (medians 93 ng/l, 85 ng/l and 94 ng/l, SD scores -0.3, -0.6 and -0.2, respectively). Inhibin B decreased to undetectable levels in one boy post-treatment with no accompanying increase in FSH or LH. In prepubertal girls with cancer, chemotherapy is associated with suppression of inhibin B, usually transient, which may indicate arrest of follicle development. Sustained suppression of inhibin B following treatment may be indicative of permanent ovarian damage. In prepubertal boys, chemotherapy had little immediate effect on Sertoli cell production of inhibin B, although one boy showed a delayed effect. Inhibin B, together with sensitive measurements of FSH, may be a potential marker of the gonadotoxic effects of chemotherapy in prepubertal children with cancer.

Serum MMP-3 and MMP-1 and progression of joint damage in early rheumatoid arthritis.

Expression and activation of matrix metalloproteinases such as MMP-3 (stromelysin-1) and MMP-1 (collagenase-1) are increased in patients with rheumatoid arthritis (RA). Previous negative reports of their value as predictors of joint damage may be due to the lack of a large longitudinal study of early RA patients. This study evaluated their use in assessing early untreated patients with RA and predicting subsequent joint damage. Ninety-eight patients with early untreated RA of less than 12 months duration and 20 normal controls had baseline serum samples tested with a double-antibody enzyme-linked immunosorbent assay for each of MMP-1 and MMP-3. The subsequent changes in Larsen score (DeltaLarsen) and Health Assessment Questionnaire (DeltaHAQ) over the first 12 months were recorded. Baseline serum levels of MMP-3 and MMP-1 correlated significantly with baseline C-reactive protein (CRP) (r=0.42 and 0.49, P<0.001), DeltaHAQ (r=0.32 and 0.30, P<0.01) and DeltaLarsen (r=0.23 and 0.32, P<0.05) respectively. Analysis of the group of patients with a normal CRP at presentation (n=21) showed correlation of the baseline MMP-3 and MMP-1 with the presence of erosive disease during the first 12 months (r=0.52 and 0.65 respectively, P<0.05). Logistic regression analysis, in the patients who were non-erosive at presentation, showed that the strongest correlation with progression in Larsen score was the baseline MMP-3 level (r=0.30, P=0.01). Baseline serum MMP-1 and MMP-3 levels correlate with disease activity and predict functional and radiographic outcome in early untreated RA. They may have a particular value in predicting the progression of erosive disease in patients who are not erosive at presentation.