Specific Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Increase of laminin 5 synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines and growth factors, and lysophospholipids

Laminin 5 is known to induce the adhesion, spreading and migration of human keratinocytes. In skin wound healing, laminin 5 deposition beneath migrating keratinocytes occurs early and is followed by the formation of hemidesmosomes and then basement membrane. To identify factors that regulate the synthesis and secretion of laminin 5 by human keratinocytes during acute wound healing. Laminin 5 synthesis by human keratinocytes was determined by a specific sandwich enzyme-linked immunosorbent assay. To determine the total amount of laminin 5 synthesized, laminin 5 deposited on culture dishes and inside cells was solubilized by detergent solution and determined separately from conditioned medium, and the total laminin 5 synthesis was calculated. A quantitative polymerase chain reaction method was used to measure the expression levels of laminin 5 genes, LAMA3, LAMB3 and LAMC2, which correspond to the alpha3, beta3 and gamma2 chains of laminin 5. We also examined the effects of lysophospholipids, proinflammatory cytokines and growth factors, which are components in acute wound fluids, on laminin 5 synthesis in keratinocytes. Human acute wound fluid at days 1, 2 and 3 stimulated laminin 5 synthesis in cultured human keratinocytes in a concentration-dependent manner, although findings are restricted to one case. Human serum also increased laminin 5 production by human keratinocytes as strongly as the wound fluid did, suggesting that the major active components in acute wound fluid may be derived from those in human serum. Lysophospholipids such as lysophosphatidic acid (LPA), lysophosphatidylcholines (LPCs) and sphingosine-1-phosphate (S1P) increased laminin 5 synthesis in a concentration-dependent manner. Among growth factors, epidermal growth factor, insulin-like growth factor-1, interferon-gamma and keratinocyte growth factor increased laminin 5 production in keratinocytes, while platelet-derived growth factor, hepatocyte growth factor and basic fibroblast growth factor were ineffective. Although interleukin-1alpha had no effect, transforming growth factor (TGF)-alpha, tumour necrosis factor (TNF)-alpha and TGF-beta1 also stimulated laminin 5 synthesis, and TGF-alpha and TGF-beta1 showed a synergistic effect. Neutralizing antibodies to TGF-alpha and TGF-beta1 markedly inhibited the enhanced laminin 5 synthesis by human serum, suggesting that TGF-alpha and TGF-beta1 are important components to increase laminin 5 in human serum. In line with the increase of laminin 5 synthesis, the expression levels of all three laminin 5 genes were also augmented by TGF-alpha and TGF-beta1. Laminin 5 synthesis in human keratinocytes was augmented by inflammatory cytokines and growth factors such as TGF-alpha, TGF-beta1 and TNF-alpha, and lysophospholipids such as S1P, LPA and LPCs, which are supposed to be present in acute wound fluid. The increased laminin 5 protein in the wound area presumably enhances wound repair by stimulating adhesion and migration of keratinocytes on the wound bed and by facilitating basement membrane formation at the dermal-epidermal junction.

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol.

There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested. R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods. We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol

Objectives There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in α-, γ- and ω-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. Methods Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail glutenextraction procedure for heat-processed foods was also tested. Results R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barleybased products, and to estimate the gluten content of hydrolysed foods. Conclusion We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.

Oxidative stress correlates with left ventricular volume after acute myocardial infarction.

It has been suggested that oxidative stress may play a role in the pathophysiology of heart failure. However, little is known about the clinical relationship between oxidative stress and left ventricular dilatation after acute myocardial infarction (AMI). We prospectively studied 28 consecutive patients, successfully treated with primary coronary angioplasty, after their first AMI. To evaluate oxidative stress, plasma oxidized low-density LDL levels (U/mL) were measured serially 1 day, 7 days, 14 days, 30 days, and 90 days after the onset of AMI using a specific sandwich enzyme-linked immunosorbent assay. Left ventriculography and coronary angiography were obtained in all patients 3 months after the AMI and infarct-related arteries were all patent. Peak plasma oxidized LDL levels were seen 7 days after AMI (after 1 day: 14.7+/-1.5, 7 days: 21.0+/-2.8, 14 days: 20.2+/-2.8, 30 days: 18.3+/-2.5, 90 days: 16.5+/-2.3 U/mL). Plasma oxidized LDL levels 7 days after AMI were significantly correlated with left ventricular end-diastolic volume (115+/-7 mL; r=0.54, P=0.0025) and end-systolic volume (58+/-5 mL; r=0.49, P=0.008) 3 months after the AMI. Moreover, they were also correlated with end-diastolic volume index (68+/-4 mL/m2, r=0.40, P<0.05). However, no correlation was seen between peak plasma oxidized LDL levels and ejection fraction. These findings suggest that oxidative stress may play an important role in the development and progression of left ventricular remodeling after AMI.

Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway.

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.

PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER

No AccessJournal of UrologyINVESTIGATIVE UROLOGY1 Dec 2000PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER CHRIS L. BERENDSEN, THEO P.J. MULDER, and WILBERT H.M. PETERS CHRIS L. BERENDSENCHRIS L. BERENDSEN More articles by this author , THEO P.J. MULDERTHEO P.J. MULDER More articles by this author , and WILBERT H.M. PETERSWILBERT H.M. PETERS More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)66982-0AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Transitional cell carcinomas of the human bladder and many gastrointestinal tumors often contain high amounts of the detoxification enzyme glutathione S-transferase π (GSTP1-1). Elevated levels of GSTP1-1 have been found in serum and plasma from patients with gastrointestinal, lung or head and neck cancer. GSTP1-1 and glutathione S-transferase α (GSTA1-1) have been reported to be increased in 10 of 15 patients (67%) with bladder cancer. We evaluate the role of GSTP1-1 and GSTA1-1 as plasma tumor markers in 50 patients with bladder cancer before and after treatment. Materials and Methods: Blood from patients with bladder cancer was sampled in ethylenediaminetetraacetic acid tubes. Plasma GSTA1-1 and GSTP1-1 were measured using the sensitive and specific sandwich enzyme-linked immunosorbent assay. Results: Respective plasma GSTA1-1 and GSTP1-1 levels were above the upper normal reference limit in 2 (4%) and 14 (28%) of the 50 patients with bladder cancer. No significant decrease in plasma GSTA1-1 or GSTP1-1 was noted in matched pairs of plasma samples collected before and after treatment. Conclusions: In contrast to earlier reports, only a limited number of patients with bladder cancer had increased plasma GSTA1-1 or GSTP1-1, which did not decrease after tumor resection. These findings argue against the use of GSTP1-1 or GSTA1-1 as plasma markers for bladder cancer. References 1 : The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol1995; 30: 445. Google Scholar 2 : Nomenclature for human glutathione transferases. Biochem J1992; 282: 305. Google Scholar 3 : Preparation, characterization and properties of glutathione S-transferases. In: . Edited by . New York: Wiley J and Sons1985: 297. Google Scholar 4 : The isoenzymes of glutathione S-transferase. In: Advances in Enzymology. Edited by . New York: Wiley J and Sons1985: 357. Google Scholar 5 : Glutathione S-transferases: biomedical applications. Adv Clin Chem1993; 30: 282. Google Scholar 6 : Glutathione transferases and cancer. Crit Rev Biochem Mol Biol1992; 27: 337. Google Scholar 7 : Glutathione S-transferase activity and subunit composition in transitional cell cancer and mucosa of the human bladder. Urology1997; 49: 644. Google Scholar 8 : Enhanced glutathione S-transferase activity and glutathione content in human bladder cancer. Follow up study: influence of smoking. J Urol1993; 149: 1452. Google Scholar 9 : Glutathione S-transferase activity in human superficial transitional cell carcinoma of the bladder. Cancer1990; 9: 2064. Google Scholar 10 : Enzyme immunoassay for human serum glutathione S-transferase pi using monoclonal and polyclonal antibodies. Cancer J1989; 2: 225. Google Scholar 11 : Serum glutathione S-transferase-pi as a tumor marker for gastrointestinal malignancies. Cancer1989; 63: 317. Google Scholar 12 : Elevation of the placental glutathione S-transferase form (GST-pi) in tumor tissues and the level in sera of patients with cancer. Cancer Res1989; 49: 5225. Google Scholar 13 : Serum glutathione S-transferase-pi level as a tumor marker for non-small cell lung cancer. Cancer1994; 73: 1377. Google Scholar 14 : Radioimmunoassay for plasma glutathione S-transferase-pi and its clinical application in gastrointestinal cancer. Cancer1995; 76: 1363. Google Scholar 15 : Significance of glutathione S-transferase-pi as a tumor marker in patients with oral cancer. Cancer1992; 70: 2381. Google Scholar 16 : Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines. Biochim Biophys Acta1994; 1225: 223. Google Scholar 17 : Sandwich ELISA for glutathione S-transferase Alpha 1-1: plasma concentrations in controls and in patients with gastrointestinal disorders. Clin Chem1996; 42: 416. Google Scholar 18 : Measurement of glutathione S-transferase P1–1 in plasma; pitfalls and significance for screening and follow-up of patients with gastrointestinal cancer. Cancer1997; 80: 873. Google Scholar 19 : Hepatocellular integrity in liver donors and recipients indicated by glutathione transferase alpha. Transplantation1996; 61: 904. Google Scholar 20 : Glutathione S-transferase alpha as a marker for hepatocellular damage in pre-eclampsia and HELLP syndrome. Lancet1995; 345: 1571. Google Scholar From the Department of Urology, Atrium Medical Center Heerlen, Heerlen and Department of Gastroenterology, University Hospital St. Radboud, Nijmegen, The Netherlands© 2000 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 164Issue 6December 2000Page: 2126-2128 Advertisement Copyright & Permissions© 2000 by American Urological Association, Inc.Keywordsglutathione transferasetumor markers, biologicalenzyme-linked immunosorbent assaybladder neoplasmsMetricsAuthor Information CHRIS L. BERENDSEN More articles by this author THEO P.J. MULDER More articles by this author WILBERT H.M. PETERS More articles by this author Expand All Advertisement PDF downloadLoading ...

Effects of pro-inflammatory cytokines on apolipoprotein E secretion by a human astrocytoma cell line (CCF-STTG1).

Apolipoprotein (apo) E has been implicated in Alzheimer's disease; however, little is known about the regulation of its secretion in astrocytes. To investigate the effects of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on apoE secretion by CCF-STTG1 cells, a sensitive and specific double sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) was developed. Using a monoclonal anti-human apoE antibody as the capture antibody, this assay was carried out with commercially available reagents. The assay had a sensitivity of 0.013 ng per well, within-run and between-run variation coefficients of 6.0 and 8.6 per cent respectively. There was no cross-reactions between antibodies used and apoAI, apoAII, apoB, apoCI, apoCII and apoCIII. Low apoE concentrations were assessed using a serum-free HepG2 culture medium as secondary calibrator, containing 59 microg l(-1) of apoE. In serum-free medium, CCF-STTG1 cells secreted apoE, the accumulation of which in the cell medium increased linearly with time (27 microg per 48 h). After 48 h of incubation, apoE secretion was inhibited by TNF-alpha but not affected by IL-1beta and IFN-gamma. However, the effect of regulatory factors may depend upon culture conditions since in the presence of 10 per cent fetal calf serum, IFN-gamma significantly inhibited apoE secretion. Thus, apoE secretion by CCF-STTG1 cells is inhibited by specific pro-inflammatory cytokines. This new apoE ELISA presents the great advantage of using commercially available reagents which permit inter-laboratory comparability of results, involves relatively low cost and is adaptable for the measurement of low levels of apoE.

Determination of interleukin-1 receptor antagonist, interleukin-10, and transforming growth factor-β1 in synovial fluid aspirates of patients with temporomandibular disorders

Purpose: This study was undertaken to examine the presence of interleukin-1 receptor antagonist (IL-1ra), IL-10, and transforming growth factor-β1 (TGF-βl) in the synovial fluid (SF) lavage specimens of patients with temporomandibular disorders (TMDs). Patients and Methods: Synovial fluid lavage specimens were obtained from 14 temporomandibular joints (TMJs) of 12 patients with TMJ internal derangement (ID) and 17 TMJs of 15 patients with TMJ osteoarthritis (OA). Seven synovial fluid lavage samples of TMJs of four asymptomatic donors served as normal controls. The concentrations of IL-1ra, IL-10, and TGF-β1 were detected with sensitive and specific sandwich enzyme-linked immunosorbent assay (sandwich-ELISA). Results: IL-1ra, IL-10, and TGF-β1 in all the normal controls were undetectable. IL-lra concentrations were 175.78 ± 52.43 pg/mL in the patients with TMJ ID and 187.85 ± 59.51 pg/mL in those with TMJ OA. IL-10 was undetectable in all the TMJ ID and OA samples. The concentration of TGF-βl in TMJ ID patients (47.93 ± 88.25 pg/mL) was significantly less than in patients with TMJ OA (143.61 ± 108.00 pg/mL) ( P < .01). Conclusion: The results suggest that deficiencies of IL-1ra, IL-10, and TGF-β1 probably play an important role in the cause and pathogenesis of TMJ ID and OA.

Upregulated expression of cardiac endothelin-1 participates in myocardial cell growth in Bio14.6 Syrian cardiomyopathic hamsters

ObjectivesThe purpose of this study is to investigate the role of endogenous endothelin-1 (ET-1) in myocardial growth in Bio 14.6 Syrian cardiomyopathic hamsters (Bio).BackgroundWhile ET-1, as a growth-promoting peptide, has been implicated in the development of secondary cardiac hypertrophy, the role of endogenous ET-1 in cardiac growth in primary myocardial disease is unknown.MethodsWe measured left ventricular ET-1 levels by a specific sandwich enzyme-linked immunosorbent assay. Furthermore, we examined the chronic effect of T0201, an ET type A receptor-specific antagonist.ResultsThe ET-1 levels in the left ventricles were 1.8-fold higher (p < 0.0005) at 20 weeks and 6.4-fold higher (p < 0.0001) at 35 weeks in the Bio compared to age-matched control F1B hamsters (F1B). The Bio ET-1 levels in the lungs exhibited an only 1.3-fold elevation at 35 weeks. Immunohistochemistry demonstrated the localization of ET-1 mainly in the cardiac myocytes. The treatment with T0201 significantly reduced the heart weight/body weight ratio in the Bio, but did not affect the heart weight/body weight ratio in the F1B. Histologically, T0201 reduced the myocyte diameter of Bio to a level similar with that of F1B. However, T0201 did not affect the extent of fibrosis in Bio or F1B.ConclusionsThe ET-1 level in the heart of cardiomyopathic hamsters increases in stage-dependent and organ-specific manners. Though myocyte degeneration and subsequent replacement fibrosis do not require an ET-1 pathway, the accelerated synthesis of ET-1 in the heart may contribute to the pathological growth of remaining myocytes in this animal model.

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner's fiber.

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner's fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.

Partial Characterization of the Epitope on Excretory-Secretory Products of Fasciola hepatica Recognized by Monoclonal Antibody ES78

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of african swine fever virus antigens and antibodies

An improved solid-phase enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies was developed to detect an African swine fever virus protein (VP73) in pig samples. The use of monoclonal antibodies against VP73 allowed a sensitive and specific sandwich ELISA. This assay detected a limiting antigen concentration of 0.05 μg ml of VP73, lower than the detection limit of 0.6 μg ml obtained by using polyclonal antibodies by the same ELISA. The whole virus particle was detected with this method to a limit of 2.3 × 102 PFU ml . At the same time, an indirect ELISIA was developed to detect ASFV antibodies. The results also indicate that this method may be a useful technique for epidemiological surveys.

The Alternatively Spliced Kunitz Protease Inhibitor Domain Alters Amyloid β Protein Precursor Processing and Amyloid β Protein Production in Cultured Cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.

Soluble interleukin-6 receptor levels in pleural effusions

Generation of soluble cytokine receptors is a general phenomenon, and the roles of several such receptors have been investigated in clinical settings. Unlike other soluble cytokine receptors, soluble interleukin-6 receptor (sIL-6R) can act as an agonist and thus is implicated as an important modulator in the acute-phase reaction of prolonged inflammation. The purpose of the present study was to determine the roles of pleural sIL-6R in both differential diagnosis of pleural diseases and in the induction of acute-phase protein. Specific sandwich enzyme-linked immunosorbent assays were used to determine sIL-6R and IL-6 in 19 tuberculous, 48 malignant and 10 transudative effusions. Although IL-6 levels in pleural effusions were strikingly different, no significant differences in pleural sIL-6R levels were found between the groups. Pleural levels of IL-6 were invariably much higher, whereas those of sIL-6R were invariably lower than serum levels. Furthermore, IL-6, but not sIL-6R, levels in effusions correlated significantly with serum C-reactive protein levels. These results suggest that: (1) pleural levels of sIL-6R are not increased even in strong inflammation such as tuberculous pleurisy, nor significantly different among pleural diseases; and (2) the local levels of sIL-6R are not as important as expected for the induction of acute-phase proteins in patients with pleural diseases.

A Sandwich Enzyme-linked Immunosorbent Assay for .DELTA.3, .DELTA.2-Enoyl-CoA Isomerase in Rat-Tissue Homogenates.

We established two monoclonal antibodies (MAbs, A6-1-E4, 1gM; κ and BC-D1, IgG1, λ), that specifically recognize different epitopes of rat Δ3, Δ2-enoyl-CoA isomerase (ECI). We then developed a specific and reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using a capture monoclonal antibody and an indicator polyclonal antibody to evaluate ECI. The amounts of ECI were linearly determined in the ranges from 15 to 250 ng/well by this method. This assay can detect the amount of ECI in tissue homogenates without subfractionation. The ECI levels were 0.13 ng/μg homogenate in normal rat liver and 1.50 ng/μg homogenate in clofibrate-treated rat liver.

Reduction of tumor necrosis factor-α bioactivity by a human ovarian epithelial cancer cell line in vitro

OBJECTIVE: Our purpose was to determine the ability of an ovarian epithelial carcinoma cell line, Caov-3, to alter the bioactivity of exogenously added tumor necrosis factor-α. STUDY DESIGN: Caov-3 cells were cultured for up to 6 days in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The control and tumor necrosis factor-α-treated cells were analyzed for proliferation, distribution throughout the cell cycle by flow cytometry, their ability to release bioactive tumor necrosis factor-α by L929 bioassay, and their ability to release immunoreactive tumor necrosis factor-α by a specific double sandwich enzyme-linked immunosorbent assay. RESULTS: Tumor necrosis factor-α induced a dose- and time-dependent inhibition of cell proliferation accompanied by accumulation of cells in late S and G 2/M phases of the cell cycle. Tumor necrois factor-α bioactivity was undetectable in the media of control Caov-3 cell cultures, but these cells exhibited TNF-α messenger ribonucleic acid. After culture of the cells for 2 days in the presence of various doses of TNF-α (0.1, 1.0, 10, or 100 ng/ml), a significant decline ( p < 0.01) in bioactivity was observed in all groups with the exception of 100 ng of TNF-α. Further declines in bioactivity were observed 2 and 4 days later. Addition of TNF-α to Caov-3 cells did not affect its immunoactivity, and Western blots of media revealed major bands of immunoactivity at −17 kd, the expected molecular size of TNF-α. CONCLUSION: These results indicate that Caov-3 ovarian carcinoma cells reduce the bioactivity of TNF-α, an important growth regulation, by a novel yet unknown mechanism to escape the modulatory effects of the normal immune response during cancer cell growth.

Specific and sensitive quantitation of transforming growth factor β3 by sandwich enzyme-linked immunosorbent assay

Transforming growth factors beta (TGF-β) consist of a highly homologous family of 25 kDa dimers involved in a diverse array of biological functions. Progress in understanding the biology of the third isoform (TGF-β3) of this family has been limited by the absence of a quantitative assay for TGF-β3. Here we report the development of a sensitive and specific sandwich enzyme-linked immunosorbent assay (SELISA), which is based on an anti-TGF-β mouse monoclonal IgG as the capture antibody and chicken anti-recombinant-hTGF-β3 IgY as the secondary antibody. This assay can quantitate TGF-β3 in complex biological fluids, with a detection limit of 2 pg and no cross-reactivity or inteference with as high as 1000-fold molar excesses of either TGF-βs 1, 2 or 1.2. This TGF-β3 SELISA is the first reported assay for the direct and sensitive quantitation of TGF-β3 in complex biological fluids.

A 22 kDa human growth hormone specific enzyme-linked immunosorbent assay.

A specific sandwich enzyme-linked immunosorbent assay (ELISA) was developed for human growth hormone (hGH) with molecular weight of 22 kDa (22k hGH). To prepare the 22k hGH specific antibody, a peptide (P-15) corresponding to residues 32-46 in 22k hGH was synthesized and used to immunize rabbits, because a 20k hGH variant was known to lack this peptide sequence. The assay method was developed using a combination of an anti P-15 polyclonal antibody (YK-2) coated on the microtiter well as a catching antibody and another polyclonal antibody against 22k hGH as a peroxidase-conjugated detecting antibody. The assay sensitivity was 1 ng/ml for 22k hGH, and cross-reactivity with other pituitary protein hormones as well as the 20k hGH variant was less than 0.01%, respectively. Furthermore, the 22k hGH content was measured precisely even in the presence of 20k hGH in this ELISA system.

Cytokines in the peritoneal fluid of patients with endometriosis

OBJECTIVE: To determine which of the various cytokines interleukin-5 (IL-5), interleukin-6 (IL-6) and interleukin-1 (IL-1) are important in endometriosis. METHODS: Peritoneal fluid (PF) samples were taken from 55 women at the time of either laparotomy or laparoscopy, and were examined for the levels of IL-5, IL-6 and IL-1 using each cytokine specific sandwich enzyme-linked immunosorbent assay. Thirty-two patients had pelvic endometriosis, eight post-pelvic inflammatory disease, four advanced cancer, three adenomyosis, three benign ovarian tumor, and other diseases. Statistical analysis was performed with the Mann Whitney test, chi-square test or Fisher's test. RESULTS: Both IL-5 and IL-6 levels in PF specimens with endometriosis tended to be higher than normal, while the same specimens were mostly interleukin-1β (IL-1β) negative. Of great interest was the negative correlation between log (IL-5) and IL-6 (Fisher's test, P < 0.04). CONCLUSION: These findings support the concept that IL-5 and IL-6 but not IL-1 are associated with endometriosis.

Changes in serum immunoglobulin M (IgM) concentrations during early development of chum salmon ( Oncorhynchus keta) as determined by sensitive elisa technique

1. 1. A specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum immunoglobulin M (IgM) of chum salmon Oncorhynchus keta. 2. 2. In this assay, 5 μl serum was enough to measure the concentration of IgM and the minimum detectable concentration of serum IgM was about 5 ng/ml. 3. 3. Coefficients of variation within and between assays ranged from 2.90 to 9.61%. 4. 4. IgM concentrations remained at low level (< 300 ng/ml) until 40 days after hatching and then increased rapidly at the period of emergence (48 days after hatching).