The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH, <0.1% with porcine FSH and <0.01% with equine FSH. Using this EIA on different time series of plasma in cows, we have confirmed the presence of a FSH pre-ovulatory peak at estrus, of periodic FSH fluctuations accompanying the waves of terminal follicular development, and of FSH pulses, mainly asynchronous with LH ones, in the peri-ovulatory phase of the cycle. In a second objective, the EIA was used to assess the role of FSH in regulating the development of ovarian follicles up to the small antral stage in young calves. To answer this question, six calves were submitted to weekly blood sampling during their first 3 months of life, and FSH changes were studied concomitantly to those of anti-Müllerian hormone (AMH), a well-established endocrine marker of the ovarian population of small antral follicles in cows. In the ovaries of 3-month calves, the population of 3 to 5 mm follicles contained the highest intra-follicular AMH amounts, and the number of 3 to 5 mm follicles on ovaries was closely correlated with AMH concentrations in the plasma of calves at this age (rs = 0.94). Before 3 months of age, only two out of six calves showed a clear postnatal FSH peak in plasma, and no correlation was found between plasma FSH and AMH concentrations. These results indicate that female calves undergo different patterns of FSH secretion and that postnatal activation of follicular growth up to the small antral stage appears independent and not directly related to circulating FSH levels.

Imazaquin movement in a field site that had Karnak silty clay soil was evaluated during a 2-yr study to determine if leaching was a significant dissipation mode for imazaquin. Imazaquin in water samples collected from subsurface tile drainage was measured by immunoassay. Total imazaquin losses due to leaching in 1993 were 0.8% of the total applied and in 1994, practically zero. Imazaquin concentrations as high as 80 μg L−1 were observed in drain water when rainfall occurred shortly after herbicide application. Imazaquin concentrations were lower with each successive drain flow event. Higher concentrations of imazaquin were associated with higher drainage flow, shortly after application. However, 40 d after application, the levels of imazaquin were low (<7 μg L−1) even when high tile flow occurred. The time between application and the first rainfall is an important factor in imazaquin leaching. In the dry growing season of 1994, imazaquin concentration in drain water did not exceed 5 μg L−1.

The expression of a candidate auxin-responsive gene was evaluated for use in a diagnostic assay for plant growth regulator (PGR) herbicide injury in soybean leaves. Expression of GH3, a primary auxin-responsive gene, was evaluated in response to dicamba and clopyralid at the RNA and protein levels, and proteomic analysis evaluated global expression of proteins in response to dicamba. Expression of GH3 was also analyzed in response to heat, drought, salt stress, and infection by soybean mosaic virus (SMV) and bean pod mottle virus (BPMV) to determine the specificity of GH3 expression as a diagnostic marker for PGR herbicide injury. At the RNA level, GH3 was strongly induced by dicamba and clopyralid within 8 h after application. Expression peaked 1 to 3 d after treatment (DAT) in response to 10% and 1% of a labeled dose of dicamba and clopyralid, with higher expression levels detected at higher herbicide rates. At the protein level, GH3 expression was also strongly induced at 1, 2, and 3 DAT by 10% vs. 1% of a labeled dose of dicamba and clopyralid. Heat, drought, and salt stress and infection with SMV or BPMV had no effect on GH3 expression at either the RNA or protein level. Proteomic analysis identified three proteins that were up-regulated in response to dicamba. Two were induced for less than 7 DAT, and a third was identified as a stress-response enzyme (superoxide dismutase) that is likely not specific to PGR herbicide injury. Expression of GH3 was highly induced by PGR herbicides at the RNA and protein level and was not affected by environmental stresses or viral infection, indicating that GH3 expression has excellent potential for use in a diagnostic assay for PGR herbicide injury.

A two-year surface water reconnaissance of the Bogue Phalia and its tributaries was conducted in 1997 and 1998. Cyanazine and metolachlor in surface water samples were quantified using enzyme-linked immunosorbent assays (ELISA). Cyanazine and metolachlor were detected in 101 and 132 of 160 samples, respectively. Cyanazine concentrations ranged from 0.1 to 2.2 g L−1 and exceeded the U.S. Environmental Protection Agency (EPA) lifetime health advisory level (HAL) of 1 g L−1 in eight samples. However, concentrations never exceeded the HAL for shorter exposure times. Metolachlor concentrations never reached the lifetime HAL of 100 g L−1. Metolachlor concentrations ranged from 0.1 to 20.6 g L−1. Metolachlor was detected more frequently and found to be more persistent throughout the growing season than was cyanazine. Higher cyanazine and metolachlor concentrations were detected at sampling dates that coincided with herbicide applications. One of the Bogue Phalia's tributaries, Clear Creek, was found to be a point-source of cyanazine for the watershed.

Background: Elevated plasma homocysteine concentrations have been associated with both cognitive impairment and dementia. However, it is unclear whether some cognitive domains are more affected than others, or if this relationship is independent of B12 and folate levels, which can also affect cognition. We examined the relationship between plasma homocysteine and cognitive decline in an older hypertensive population.

Methods: 182 older people (mean age 80 years) with hypertension and without dementia, were studied at one center participating in the Study on COgnition and Prognosis in the Elderly (SCOPE). Annual cognitive assessments were performed using a computerized assessment battery and executive function tests, over a 3–5 year period (mean 44 months). Individual rates of decline on five cognitive domains were calculated for each patient. End of study plasma homocysteine, folate and B12 concentrations were measured. The relationship between homocysteine levels and cognitive decline was studied using multivariate regression models, and by comparing high versus low homocysteine quartile groups.

Results: Higher homocysteine showed an independent association with greater cognitive decline in three domains: speed of cognition (β = −27.33, p = 0.001), episodic memory (β = −1.25, p = 0.02) and executive function (β = −0.05, p = 0.04). The association with executive function was no longer significant after inclusion of folate in the regression model (β = −0.032, p = 0.22). Change in working memory and attention were not associated with plasma homocysteine, folate or B12. High homocysteine was associated with greater decline with a Cohen's d effect size of approximately 0.7 compared to low homocysteine.

Conclusions: In a population of older hypertensive patients, higher plasma homocysteine was associated with cognitive decline.

A solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomatic E. histolytica cyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 72·7% of cases of amoebic liver abscess, 18·1% of the cyst passers and 2·5% of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess.

An indirect ELISA and an immunoblot were standardized to detect anti-Toxoplasma gondii antibodies in sheep, and were compared with a commercial ELISA as reference. Sensitivity was 92·0 and 96·0%, and specificity 88·0 and 75·0%, respectively. Then the serum samples of 103 sheep on a ranch located in the Eastern region of Mexico were analysed. A frequency ranging from 77 to 84% was observed, with a heterogeneous pattern among the animals by immunoblotting. Ten months later 56 sheep were sampled and tested again. Six animals became negative while 1 case was negative the first time and strongly positive 10 months later. Considering this to be a new case we calculated an incidence rate of 2·1% (CI95% 0·6–4·8%). IgG avidity ELISA was performed on 36 positive samples, 33 being of high-avidity at both times; slight increases in 2 samples and conservation of low-avidity in 1 sheep were also observed. Higher prevalence rates of toxoplasmosis in a moist warm compared to a cold atmosphere was attributed to the long viability of T. gondii oocysts. This may explain the high frequency of T. gondii in this region, which apparently has favourable climatic conditions for the transmission of this protozoan, besides the presence of both domestic and wild cats.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50–60°C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56°C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6·7 deg C (D60°C=24·6 min) and 6·8 (D60°C=23·0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0·994) with those obtained by the colorimetric assay. A similar high correlation (r=0·998) was obtained when industrially thermized milks (62–67°C for 20–90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

An immunoassay to quantify αS1-casein (αS1-CN) in milk using an optical biosensor, based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-αS1-CN antibodies directed against each extremity of the molecule. This strategy permits only intact αS1-CN to be quantified and not its degradation products. The calibration curve was obtained using a reference milk powder with a known αS1-CN concentration. Analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. Detection limit was established at 0·87 μg/ml and the intra- and inter-assay variation coefficients were 2·86 and 5·31%, respectively. The method was applied to raw milk to quantify intact αS1-CN, with no pre-treatment of the sample. An initial analysis of 48 milk samples permitted αS1-CN concentrations ranging from 8·8 to 12·06 mg/ml to be obtained.

T lymphocytes (T cells) play critical roles in the regulation of immune responses, and are responsible for mediating many of the effector mechanisms of the immune system. For this reason, there has always been a need for assays to measure accurately the activity of populations of T cells, both in model (animal) systems and in humans. The expansion of the biotechnology industry has led to a dramatic increase in the number of novel immunotherapeutics that are being developed for the treatment of cancer, autoimmune disorders and infectious diseases. This increase in activity in the field of immunotherapy, coupled with the expense of clinical trials, has led to renewed interest in methods that accurately assess T-cell function, as researchers seek to maximise the amount of information that can be obtained from each clinical study. Assessing the quantitative and qualitative nature of a T-cell response, for example following vaccination or immunosuppressive therapy, can provide valuable information about the efficacy of a treatment, in place of a clinical endpoint. This article reviews some of the established methods that are used to monitor human T-cell activity, and describes some new approaches that are in development to increase the speed, sensitivity and relevance of such methods.

This paper attempts to evaluate the clinical usefulness of CYFRA 21-1 as a serum tumour marker in patients with head and neck squamous cell carcinoma (HNSCC).

The serum concentration of CYFRA 21-1 was measured utilizing a new electrochemiluminescent immunoassay (ECLIA) in 142 patients with HNSCC before and after treatment, 68 patients with benign tumours of the head and neck, and 50 healthy controls.

Serum levels of CYFRA 21-1 in patients with HNSCC were significantly higher than those of benign tumours and healthy controls (p < 0.001). The diagnostic sensitivity and specificity of CYFRA 21-1 for HNSCC were 62 per cent and 100 per cent, respectively. The positive rates of CYFRA 21-1 increased with progression of HNSCC, serum CYFRA 21-1 levels were related to the tumour stage expressed by primary tumour (T) and nodal status (N) (p < 0.001), but not related to patient age, gender, smoking and drinking habit, or histopathological grade (p > 0.05). Post-treatment levels of CYFRA 21-1 in HNSCC decreased significantly (p < 0.001). Among 38 patients with clinical or radiological evidence of a recurrence during follow-up, 78.9 per cent (30 of 38) showed an increase in CYFRA 21-1.

The analytical ECLIA performance for serum CYFRA 21-1 provides a new means of clinical assessment for HNSCC. The results of ECLIA suggest that the serum marker CYFRA 21-1 is valuable not only for diagnosis but also for close monitoring of patients with HNSCC.

A nephelometric immunoassay, with a detection range of 0·3 to 5 g IgG1/l, was developed for the determination of immunoglobulin in bovine milk. The assay exhibited no significant cross-reactivity with αS1-casein, αS2-casein, β-casein, κ-casein or β-lactoglobulin and 39% cross-reactivity with IgG2. The nephelometric assay was compared with ELISA and RID (24 h and 48 h incubations) assays using 105 duplicate milk samples covering IgG1 values ranging from 0·45 to 1·8 g1. The results obtained from all assays showed good agreement with the exception of those obtained by the RID assay (24 h incubation) which gave lower results in samples containing more than 1·2 g IgG1/l. It was concluded that the nephelometric assay is a reliable, rapid and convenient method suitable for the quantification of IgG1 in milk. The assay can be configured for routine high-throughput milk quality assurance for IgG1 in dairy laboratories.

Since they were first utilized, immunoassays have witnessed phenomenal growth in the range and scope of their applications. A vast array of different labels and assay strategies has been developed to meet the requirements of sensitivity, accuracy and convenience. The development of increasingly sensitive labels and detection equipment has seen a drastic improvement in the sensitivity of immunoassay systems, allowing an ever-increasing range of analytes to be measured accurately. At the same time, simple to use, inexpensive assay systems have been developed with the necessary reliability, accuracy and sensitivity to bring immunoassay technology to much more diverse areas such as home testing, near-patient monitoring, and large screening programmes in developing countries. Recent developments in molecular biology techniques have made possible the production of fusion antibody conjugates, which can lead to further improvements in sensitivity and cost of reagents, as well as possibly revolutionizing the production of monoclonal antibodies. However, dissatisfaction with various aspects of existing immunoassay technology will necessarily lead to the continued development of this already widely diverse subject.

Two specific Enzyme Linked lmmunosorbent Assays (ELISA) for vitellogenin were developed using two different antisera anti-plasma vitellogenin purified by SDS polyacrilamide gel electrophoresis and electroelution. Purified vitellogenins migrated as single bands under SDS conditions with an apparent molecular mass of 154 and 152 kDa for Chrysichthys nigrodigitatus and Heterobranchus longifilis respectively.The ELISA was performed on 96 well microtiter plates using specific antibodies raised on rabbits and a second antibody (Goat anti-rabbit) labeled with peroxidase. Parallel displacement curves were obtained between purified vitellogenin and female plasma as well as plasma from estradiol treated fish. Both antibodies cross-reacted with the heterologous vitellogenin but the curves were not parallel, indicating different antigenic structures of the two vitellogenin molecules from the two catfish species. The reliability and the specificity of these assays allowed us to quantify vitellogenin in plasma samples during vitellogenesis of the two species. The annual vitellogenin plasma levels presented a good correlation with mean oocyte diameter and reproduction period in Chrysichthys nigrodigitatus. In Heterobranchus longifilis the mean oocyte diameter is almost constant all year round, but vitellogenin levels presented significant variations with higherlevels corresponding mainly to the low gonado-somatic index values observed during the dry seasons.