Noncompetitive Enzyme-linked Immunosorbent Assay Research Articles

Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes

Perfluorochemicals (PFCs) are emerging persistent organic pollutants (POPs) and are widely present in the environment, wildlife and humans. Recently, reports have suggested that PFCs may have endocrine-disrupting activities. In the present study, we have developed a non-competitive enzyme-linked immunosorbent assay (ELISA) method to investigate estrogenic activities of selected PFCs using vitellogenin (VTG) induction in primary cultured hepatocytes of freshwater male tilapia ( Oreochromis niloticus). Cultured hepatocytes were exposed to various concentrations of perfluorooctanyl sulfonate (PFOS), pentadecafluorooctanoic acid (PFOA), 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH), 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH) and 1H, 1H, 2H, 2H-perfluoro-1-decanol (8:2 FTOH) for 48 h, while 17β-estradiol (E2) and 4-nonylphenol (4-NP) were used as positive controls. A dose-dependent induction of VTG was observed in E2-, 4-NP-, PFOS-, PFOA- and 6:2 FTOH-treated cells, whereas VTG levels remained unchanged in the 4:2 FTOH and 8:2 FTOH exposure groups at the concentrations tested. The estimated 48-h EC 50 values for E2, 4-NP, PFOS, PFOA and 6:2 FTOH were 4.7 × 10 −7, 7.1 × 10 −6, 1.5 × 10 −5, 2.9 × 10 −5 and 2.8 × 10 −5 M, respectively. In the time-course study, significant VTG induction took place at 24 h (E2), 6 h (4-NP), 48 h (PFOS), 48 h (PFOA), 72 h (4:2 FTOH), 12 h (6:2 FTOH), 72 h (8:2 FTOH), and increased further after 96 h of exposure. Co-exposure to binary mixtures of individual PFCs and E2 for 48 h significantly inhibited E2-induced hepatocellular VTG production in a dose-dependent manner except for 4:2 FTOH. The estimated 48-h IC 50 (concentration of a compound that elicits 50% inhibition of maximally E2-induced VTG) values for PFOS, PFOA, 6:2 FTOH and 8:2 FTOH were 3.1 × 10 −7, 5.1 × 10 −7, 1.1 × 10 −6 and 7.5 × 10 −7 M, respectively. In order to further investigate the estrogenic mechanism of PFCs, the hepatocytes were co-exposed to binary mixtures of individual chemicals (E2, 4-NP, PFOS, PFOA and 6:2 FTOH) and the known estrogen receptor inhibitor tamoxifen for 48 h; tamoxifen significantly inhibited the ability of these chemicals to stimulate vitellogenesis. The overall results demonstrated that PFOS, PFOA and FTOHs have estrogenic activities and that exposure to a combination of E2 and PFCs produced anti-estrogenic effects. The results of the estrogen receptor inhibition assay further suggested that the estrogenic effect of PFCs may be mediated by the estrogen receptor pathway in primary cultured tilapia hepatocytes.

Development of Anti-Isoproturon Polyclonal Antibody

Abstract A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropy1)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 10 5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L -1 and 1.0 x 10 5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC 50) was 0.07 mg mL -1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.

Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus

Yellow head virus (YHV) is an invertebrate nidovirus that can cause mass mortality of the cultured Penaeus monodon shrimp . A single-chain variable fragment (scFv) antibody directed against the gp116 envelop glycoprotein of YHV was constructed from hybridomas. Variable heavy (V H) and light (V L) chain genes were amplified from cDNA using antibody-specific primers, linked to generate a full-length gene via a standard peptide linker, ligated into the pET28a expression vector and transformed into E. coli. The expressed insoluble scFv antibody was solubilized, purified using immobilized metal affinity chromatography and rapid refolded; final yield 1–1.5 mg/l. Solid-phase non-competitive enzyme-linked immunosorbent assay (non-competitive ELISA) determined the affinity constant ( K A) to be 3.34 ± 0.38 × 10 8 l/mol. The sensitivity and specificity of scFv antibody was demonstrated by ELISA, dot blot and Western blot analysis. The detection limit determined by dot blot and indirect ELISA was 9 ng and 45 ng of purified YHV, respectively. Dot-blot assays revealed that the scFv antibody could detect YHV-infected shrimp at 24 h post-infection and did not cross-react with White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) proteins. The scFv antibody therefore might find application in rapid, simple and sensitive diagnostic tests to detect YHV in farmed shrimp.

Quantitative Detection of Poultry in Cooked Meat Products

ABSTRACT: There is a growing awareness of perceived harm from meat species adulteration, both intentional and accidental. The present study developed a monoclonal antibody (Mab)-based enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chicken and turkey meat adulterated in cooked (100 °C, 15 min) mammalian meat. The specificity of Mab 5D2 to different species (pork, beef, lamb, deer, horse, duck, chicken, and turkey) and tissues (serum, gizzard, heart, and liver) was studied by noncompetitive ELISA. The detection of cooked chicken in beef, and turkey in pork was accomplished by competitive and noncompetitive ELISAs. Both ELISAs were optimized to quantify cooked poultry in red meats. The new Mab-based ELISAs enabled the detection of cooked poultry in red meats at levels as low as 1% (v/v) or better. The correlation (r > 0.994) between chicken or turkey concentrations and ELISA signals permitted the quantification of poultry adulterants in cooked non-poultry meats.

An enzyme-linked immunosorbent assay for monitoring of Aspergillus ochraceus growth in coffee powder, chilli powder and poultry feed

The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate. An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10-160 microg fungal biomass per millilitre extract of coffee (R(2)=0.989), poultry feed (R(2)=0.987) and chilli (R(2)=0.989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9.8 and 11.8 mg g(-1)) followed by coffee beans (6.8 and 11.3 mg g(-1)) and chilli (5.1 and 6.3 mg g(-1)) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 microg g(-1)) followed by coffee beans (8 and 24 microg g(-1)) and chilli (0.2 and 0.45 microg g(-1)) at 20% and 30% moisture after 20 days of incubation. The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition. The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of >or=0.02% (w/w) and can be used for early detection of specific fungal infestation in food commodities.

Effects of peroxisome proliferator-activated receptor α/δ agonists on HDL-cholesterol in vervet monkeys

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARalpha and PPARdelta agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following "treatments": a) vehicle only (vehicle); b) the PPARdelta selective agonist GW501516 (GW); c) the PPARalpha/delta agonist T913659 (T659); and d) the PPARalpha agonist TriCor (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARalpha and PPARdelta agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARalpha and PPARdelta agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.

Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou

Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1–128 and 2–256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17β (E 2). The changes in serum levels of Chgs and Vg in male fish differed according to the E 2 dose. When fish were given a 5 mg/kg body weight (BW) of E 2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 μg/kg BW of E 2 injection. A similar trend was seen in the response time to exogenous E 2. Serum Chgs were induced from 8 h after E 2 injection and reached a peak of about 5 μg/ml at 24 h. Although Vg was not detected until 8 h after E 2 injection, its levels remained considerably low at around 0.03 μg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 μg/kg BW of estrogen: the long-term exposure of fish to E 2 showed that Chg H could be induced from a lower dose of E 2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E 2.

Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design

Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11–25) and VP3(102–121), in rabbits immunised with either Freund’s adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund’s protocol.

Monoclonal antibodies as a probe for the unfolding of porcine growth hormone

Monoclonal antibodies (mAbs) were generated against pituitary porcine growth hormone (pGH). Ten mAbs were selected for their specificity and affinity for pGH. These mAbs were of the immunoglobulin G (IgG) 1 κ subclass, with dissociation constants ( K d) between 7.42 and 0.26 nM, and recognised seven non-overlapping epitopes. We measured whether the mAbs detected alterations of the pGH three-dimensional structure by comparing the antibody reactivity to native pGH and to pGH experimentally unfolded by heating at 50 °C, 75 °C and 100 °C or by reduction and S-carboxymethylation. The antibody–antigen interactions were studied with two enzyme-linked immunosorbent assays (ELISA), based either on a direct binding or inhibition format. The results show that: 1) one mAb, mAb D12, is a conformation-sensitive antibody that recognises an epitope present only in the native pGH. Because the intact three-dimensional structure is essential for the expression of biological activity, mAb D12 could be used to detect altered pGH molecules in biological samples (blood, pituitary extracts or material produced with recombinant technology), and for the one-step purification of biologically active pGH by immunoaffinity chromatography; 2) one mAb, mAb I4, binds to a linear epitope that is not significantly modified in the denatured hormone. This mAb was able to detect the hormone in assays where protein conformation is usually strongly altered, i.e. immunoblotting and immunohistochemistry; 3) the performances of the other eight mAbs differed significantly in the competitive and non-competitive ELISA.

Idiotype–anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7- N-acetylglucosaminides in human urine with subfemtomole range sensitivity

We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7- N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype–antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a β-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled α-type antiidiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246±184 (S.D.) μg ( n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC ( n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 μg/mg Ucre with the average of 5.41±4.53 (S.D.) μg/mg Ucre.

An enzyme-linked immunosorbent assay for measuring anti-sheep red blood cells antibodies in lead-exposed toads

Introduction: Immune function assays to screen immunotoxic effects of xenobiotics has recently become of major interest. In the framework of our studies, we standardized methods to quantify the humoral response of an amphibian species ( Bufo arenarum, Amphibia, Anura) exposed to sublethal lead (as acetate). Methods: The levels of agglutinins to heterologous red blood cells (RBC) were measured in serum from adult B. arenarum. Since agglutinin titers were very low, a noncompetitive enzyme-linked immunosorbent assay (ELISA) method was carried out. As toad serum showed marked nonspecific binding, we developed a new ELISA on microtiter plates for the quantitative determination of the heterophile antibodies. The method was based on that described by Hirvonen et al. [Vox Sang. 69 (1995) 341], employing sheep red blood cells (SRBC) sensitized with amphibian antibodies that were transferred to microplates; later the measurement of bound immunoglobulins was performed. Different variables such as the amount of antigen, blocking agents, and other experimental conditions (fixing solution and commercial plates) were studied. Toads ( n=22) received a weekly subcutaneous injection of 50 mg/kg lead (acetate) for 6 weeks, and the control ones ( n=26) were injected with Na acetate at the same time. Results: The anti-sheep RBC antibodies titers of adult toads were obtained with the improved ELISA method, being the absorbance range 0.12 to 1.58 AU (1/200 diluted serum). Titers from lead-exposed toads were also determined, being the final titers (expressed as x̄±S.E.M.) higher (0.79±0.06 AU), than those of Day 0 (0.57±0.06) ( P<.01). Discussion: It was concluded that the ELISA technique we developed was useful for measuring the humoral immune response in this animal model and that in these preliminary studies, lead showed an immunostimulating action on the humoral immune system.

Cytochrome P4501A responses in two tropical fish species, riverine catfish ( Rita rita) and marine mudfish ( Apocryptes bato)

The present study focuses on the establishment of methods for biomarker studies in freshwater and marine fish species as a basis for monitoring the extent of contamination of fisheries resources in tropical waters. Riverine catfish ( Rita rita) and marine mudfish ( Apocryptes bato) were given a single intraperitoneal injection with two selected inducing compounds; β-naphthoflavone (BNF, 50 mg/kg) and a polychlorinated biphenyl mixture (Clophen A50, 20 mg/kg), and the heavy metal compound cadmium chloride (CdCl 2, 1 mg/kg). Effects on cytochrome P4501A (CYP1A) were determined in post-mitochondrial supernatants (PMS) of liver at days 3 and 10 after treatment. EROD (7-ethoxyresorufin O-deethylase) activity and CYP1A protein level by indirect non-competitive enzyme-linked immunosorbent assay (ELISA) and Western blotting using a monoclonal antibody against fish CYP1A, were measured. BNF and Clophen A50 resulted in induction upto 9.5- and 5-fold, respectively, of CYP1A protein compared to control, while CdCl 2 showed significant inhibition in these species. The present study examined the phase-I cytochrome P450 monooxygenase activity and response in these two tropical fish species for the first time.

Increased levels of advanced glycosylation end products in the kidney and liver from spontaneously diabetic Chinese hamsters determined by immunochemical assay

Increased levels of advanced glycosylation end products (AGEs) have been reported in tissues in association with diabetes mellitus. Thus, we measured tissue AGE levels and detected an accumulation of AGEs in the kidney and liver from spontaneously diabetic Chinese hamsters (CHAD) to determine the relationship between AGEs and diabetes mellitus. Diabetic CHAD aged 12 to 13 months were studied together with age-matched nondiabetic CHAD. We used an AGE-specific noncompetitive enzyme-linked immunosorbent assay (ELISA) with polyclonal anti-AGE-bovine serum albumin (BSA) antibody to measure tissue AGE levels. The samples extracted from the kidney and liver obtained from diabetic and nondiabetic CHAD reacted with anti-AGE-BSA antibody. When the absorbance of standard AGE-BSA (0.1 microg/mL) was expressed as 1 U, AGE levels in the kidney and liver from diabetic CHAD were significantly increased as compared with nondiabetic CHAD (kidney, 0.26 +/- 0.05 v 0.10 +/- 0.03 U/microg protein, P< .01; liver, 0.20 +/- 0.03 v 0.09 +/- 0.02 U/microg protein, P< .01). Positive AGE staining was observed in the renal cortex, especially in the tubules of diabetic CHAD, but little AGE staining was observed in the glomerulus by the immunohistochemical study. AGE staining was diffuse in the hepatocytes. These AGE levels were significantly correlated with fasting plasma glucose and glycated hemoglobin (P < .01, respectively). In conclusion, we have confirmed that AGE structures were expressed in the kidney and liver from CHAD, and these AGE levels were increased in diabetic CHAD. AGE staining was observed in the renal tubules and hepatocytes. Tissue AGE levels were positively correlated with glycemic control in CHAD.

Causal Relationship between Conformational Change and Inhibition of Domain Functions of Glycoxidative Fibronectin

Glycoxidative modification of various body proteins, including fibronectin (FN), has been shown to change their structural and functional properties, and be implicated in pathogenesis of diabetic complications. Little is known about the role of secondary structure of glycoxidative FN (gFN) in its domain functions. gFN was prepared by incubation with 25 and 200 mM glucose in 0.2 M sodium phosphate buffer at 37°C on a shaking plate under aerobic and sterile conditions for various time intervals up to 49 days, being defined as gFN25 and gFN200, respectively. Unmodified FN (uFN) was prepared by incubation in 0.2 M sodium phosphate buffer without any glucose at 4°C for 49 days. The extent of glycoxidative modification was examined using a noncompetitive enzyme-linked immunosorbent assay with an antibody against N-(carboxymethyl)lysine (CML), one of the major glycoxidation products. The binding activities of uFN and gFN to collagen, gelatin and heparin were determined by a solid phase enzyme immunoassay or heparin-affinity HPLC. Cell attachment was estimated by the extent of adhesion of FITC-labeled smooth muscle cells to uFN or gFN. Conformational change in gFN was detected by SDS-polyacrylamide gel electrophoresis and spectroscopy (circular dichroism). CML was detected in gFN25 and gFN200 after 49 and 21 days of incubation, respectively. Levels of CML were about six-fold higher in gFN200 than in gFN25 after 49 days. Both gFN25 and gFN200 showed a significant decrease in the ability of binding to collagen and gelatin after 7 days of incubation. The binding activity for heparin was significantly decreased in both gFN25 and gFN200 after one day. Cell attachment activity was reduced to 89% and 76% of the unmodified form in both gFN25 and gFN200 after 49 days, respectively. High molecular weight materials were found in gFN25 and gFN 200 after 21 and 7 days, respectively. CD spectrum showed that gFN25 had lost its native conformation after 3 days of incubation, depending upon the concentration and incubation interval of the applied glucose. These in vitro results suggest that the loss of native conformation may reduce the domain functions of gFN, including binding activity to macromolecular ligands and cell attachment, and may play a major role in the pathogenesis of diabetic complications.

Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein.

Rabbits were immunized with purified almond major protein (AMP), the primary storage protein in almonds. Rabbit anti-AMP polyclonal antibodies (PA) could detect AMP when as little as 1-10 ng/mL were used to coat microtiter plates in a noncompetitive enzyme linked-immunosorbent assay (ELISA). Competitive inhibition ELISA assays detected the AMP down to 300 ng/mL. PA recognized the AMP in protein extracts from all U.S. major marketing cultivars of almonds (Mission, Neplus, Peerless, Carmel, and Nonpareil) with specific reactivity of 52.6-75% as compared to that of the AMP alone. Immunoreactivity of protein extracts prepared from commercial samples of blanched almonds, roasted almonds, and almond paste was respectively reduced by 50.0%, 56.6%, and 68.4% (noncompetitive ELISA) when compared to the immunoreactivity of the AMP. Moist heat (121 degrees C, 15 min) pretreatment of the AMP reduced the PA reactivity by 87% (noncompetitive ELISA). Exposing AMP to pH extremes (12.5 and 1.5-2.5) caused a 53% and 57% reduction in PA reactivity, respectively (noncompetitive ELISA). PA showed some cross-reactivity with the cashew major globulin, and to a lesser extent, the Tepary and Great Northern bean major storage protein (7S or phaseolin). The presence of almonds in a commercial food was detected using PA in a competitive ELISA.

Relationship between Autofluorescence and Advanced Glycation End Products in Diabetic Lenses

Autofluorescence and advanced glycation end product (AGE) levels were measured in the lenses of 9 diabetic Chinese hamsters and 6 age-matched controls. Lens autofluorescence also was measured in 37 diabetic patients and 14 age-matched controls. Lens autofluorescence values were measured noninvasively with a lens measurement system using color filters with peak transmission at 365- and 434-nm wavelengths (excitation and emission, respectively) that are characteristic of AGE fluorescence. The peak lens autofluorescence level was used as the lens autofluorescence value, and the mean lens autofluorescence values from both eyes of each subject were used for statistical analysis. The AGE levels in one lens from each hamster were measured by noncompetitive enzyme-linked immunosorbent assay with a polyclonal anti-AGE antibody. We found a 2.2 times increase of the mean lens autofluorescence value of diabetic hamsters in comparison with that of controls (P<0.01). We also found a 1.5 times increase of the mean AGE level from the lenses of diabetic hamsters in comparison with that of controls (P<0.01). Moreover, a statistically significant positive correlation between the AGE level and autofluorescence value in the same lenses was observed in all hamsters (ρ=0.58,P<0.05). In human subjects, we found a 1.4 times increase of the mean lens autofluorescence value of diabetic patients in comparison with that of age-matched controls (P<0.01). Our results suggest that non invasive measurement of lens autofluorescence may be a guide to AGE levels in lenses.

Critical factors affecting the diagnostic reliability of enzyme-linked immunosorbent assay formats.

This paper aims to evaluate different formats of the enzyme-linked immunosorbent assays (ELISAs) for detection of virus-specific antibodies and focuses on factors that may influence the diagnostic reliability of such tests. Newly developed and well-established ELISAs for detection of infections of bovine herpesvirus 1 (BHV1), bovine respiratory syncytial virus (BRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV) and bovine viral diarrhoea virus (BVDV) are used as examples. Differences between competitive and non-competitive ELISAs are described, with special reference to the influence of the antigen, the conjugated antibody and the test sample on the test results. Attention is drawn to interference, which may result in false positive or false negative test results, with special emphasis on the 'bridging' phenomenon. The use of monoclonal antibodies and discriminatory tests are briefly discussed. Diagnostic reliability is described for tests that are used in monitoring or eradication programmes, emphasising the consequences of false negative and false positive test results. Finally, reducing assay-time and functional quality control for such tests are discussed.

Development of an ELISA for Chum Salmon ( Oncorhynchus keta) Growth Hormone

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon ( Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus.

A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation of p53 status.

AIM: To develop a highly sensitive and specific enzyme linked immunosorbent assay (ELISA) system for analysis of p53 protein in cancer lysates. METHODS: The anti-p53 monoclonal antibodies DO7, 1801, BP53.12,...

Antisperm antibody and Klinefelter syndrome: does autoimmunity play a role in the pathogenesis?

Serum samples from 18 patients with Klinefelter syndrome (age range 20-22 years) and from a control group of 18 age-matched healthy subjects were analyzed for antisperm antibodies by a noncompetitive enzyme-linked immunosorbent assay. The antisperm antibody values were found higher in 5 patients (27.7%) than the level of 150 mU/100 microliters which was considered the upper level of the normal range (p < 0.01).