Enzyme-linked Immunosorbent Assay-based Assay Research Articles

Antibodies against glutamic acid decarboxylase in intravenous immunoglobulin preparations can affect the diagnosis of type 1 diabetes mellitus.

Intravenous immunoglobulins (IVIgs) contain various autoantibodies, including those against glutamic acid decarboxylase (GADAb), a valuable biomarker of type 1 diabetes mellitus. Passive transfer of GADAb from IVIgs to patients poses a risk of misdiagnosis, and information on the specific titres of GADAb and their impact on diagnostic accuracy remains limited. This study aimed to provide further insights into the origin of GADAb detected in patient serum following IVIg infusion. GADAb titres in IVIg products from Japan and the United States were measured using enzyme-linked immunosorbent assay-based assays. For reliable quantification, GADAb titres in pooled plasma were quantified and compared with those in the IVIg products. The determined titres were used to estimate the likelihood of passively detecting acquired GADAb in individuals receiving IVIgs. GADAbs were prevalent in IVIg products; however, the titres varied significantly among different lots and products. Importantly, IVIg-derived GADAb was estimated to remain detectable in patient serum for up to 100 days following a dosage of 2000 mg/kg. Clinicians should consider that IVIg preparations may contain GADAb, which can lead to false-positive results in serological assays. Careful interpretation of the assay results is key to the definitive diagnosis of type 1 diabetes mellitus.

Usefulness of high-risk HPV early oncoprotein (E6 and E7) serological markers in the detection of cervical cancer: A systematic review and meta-analysis.

We reviewed the literature on the importance of selected anti-high-risk human papillomavirus (HR-HPV)antibodies (namely, 16/18 and early oncoproteinsE6 and E7) as potential serological markers for early detection of individuals at high risk of cervical cancer. We searched for studies in PubMed and Embase databases published from 2010 to 2020 on antibodies against HR-HPV E6 and E7 early proteins and cervical cancer. Pooled sensitivity and specificity for HPV16 and HPV18 antibodies were calculated using a bivariate hierarchical random-effects model. A total of 69 articles were identified; we included three studies with 1550 participants. For the three HPV16/18 E6 and E7 antibody tests, enzyme-linked immunosorbent assay-based assays had a sensitivity of 18% for detecting CIN2+ (95% confidence interval [CI]: 15-21) and a specificity of 96% (95% CI: 92-98), for slot-blot, sensitivity was 28.9% (95% CI: 23.3-35.1) and specificity was 72% (95% CI: 66.6-77.0) for detecting CIN2+, and for multiplex HPV serology assay based on a glutathione S-transferase, sensitivity was 16% (95% CI: 8.45-28.6) and specificity was 98% (95% CI: 97-99) for detecting invasive cervical cancer. HR-HPV16/18E6 and E7 serological markers showed high specificity, but sensitivity was suboptimal for the detection of cervical cancer in either population screening settings or as point-of-care screening tests.

Liver Cancer-Specific Serine Protease Inhibitor Kazal Is a Potentially Novel Biomarker for the Early Detection of Hepatocellular Carcinoma.

INTRODUCTION:Liver cancer–secreted serine protease inhibitor Kazal (LC-SPIK) is a protein that is specifically elevated in cases of hepatocellular carcinoma (HCC). We assessed the performance of LC-SPIK in detecting HCC, including its early stages, in patients with cirrhosis, hepatitis B virus (HBV), and hepatitis C virus (HCV).METHODS:We enrolled 488 patients, including 164 HCC patients (81 early HCC) and 324 controls in a blinded, prospective, case–control study. Serum LC-SPIK levels were determined by an enzyme-linked immunosorbent assay-based assay. The performance of serum LC-SPIK and α-fetoprotein (AFP), including area under the curve (AUC), sensitivity, and specificity, are compared. The performance of LC-SPIK was evaluated in an independent validation cohort with 102 patients.RESULTS:In distinguishing all HCC patients from those with cirrhosis and chronic HBV/HCV, LC-SPIK had an AUC of 0.87, with 80% sensitivity and 90% specificity using a cutoff of 21.5 ng/mL. This is significantly higher than AFP, which had an AUC of 0.70 and 52% sensitivity and 86% specificity using a standard cutoff value of 20.0 ng/mL. For early-stage HCC (Barcelona Clinic Liver Cancer stage 0 and A), LC-SPIK had an AUC of 0.85, with 72% sensitivity and 90% specificity, compared with AFP, which had an AUC of 0.61, with 42% sensitivity and 86% specificity. In addition, LC-SPIK accurately detected the presence of HCC in more than 70% of HCC patients with false-negative AFP results.DISCUSSION:The study provided strong evidence that LC-SPIK detects HCC, including early-stage HCC, with high sensitivity and specificity, and might be useful for surveillance in cirrhotic and chronic HBV/HCV patients, who are at an elevated risk of developing HCC.

Effect of immunization routes and protective efficacy of Brucella antigens delivered via Salmonella vector vaccine.

An anti-Brucella vaccine candidate comprised of purified Brucella lipopolysaccharide (LPS) and a cocktail of four Salmonella Typhimurium (ST)-Brucella vectors was reported previously. Each vector constitutively expressed highly conserved Brucella antigens (rB), viz., lumazine synthase (BLS), proline racemase subunit A, outer membrane protein-19 (Omp19), and Cu-Zn superoxide dismutase (SOD). The present study determined a relative level of protection conferred by each single strain. Upon virulent challenge, the challenge strain was recovered most abundantly in non-immunized control mice, with the ST-Omp19-, ST-BLS-, LPS-, and ST-SOD-immunized mice showing much less burden. Indirect enzyme-linked immunosorbent assay-based assay also confirmed the induction of antigen-specific immunoglobulin G for each antigen delivered. In a route-wise comparison of the combined vaccine candidate, intraperitoneal (IP), intramuscular (IM), and subcutaneous immunizations revealed an indication of highly efficient routes of protection. Splenocytes of mice immunized via IM and IP routes showed significant relative expression of IL-17 upon antigenic pulsing. Taken together, each of the Brucella antigens delivered by ST successfully induced an antigen-specific immune response, and it was also evident that an individual antigen strain can confer a considerable degree of protection. More effective protection was observed when the candidate was inoculated via IP and IM routes.

High levels of soluble thrombomodulin may be a marker of arterial disease and peripheral ischemia in Egyptian patients with diabetes mellitus

Background Thrombomodulin is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. It was found that soluble forms of thrombomodulin (sTM) exist in plasma and is considered a marker of endothelial dysfunction. Materials and methods The study included 160 individuals: 40 healthy controls and 120 diabetic patients (40 patients with type 1 diabetes and 80 patients with type 2 diabetes). sTM concentrations were measured by an enzyme-linked immunosorbent assay-based assay employing monoclonal antithrombomodulin antibodies. Results The diabetic patients had highly significantly increased plasma sTM concentrations compared with the control group (P Conclusion We recommend including sTM in the follow-up panel of diabetic patients because of its potential ability to predict the risk for ischemic manifestations.

Differential DNA Methylation Alterations in Radiation-Sensitive and -Resistant Cells

An understanding of cellular processes that determine the response to ionizing radiation (IR) exposure is essential to improve radiotherapy and to assess risks to human health after accidental radiation exposure. Exposure to IR induces a multitude of biological effects. Recent studies have indicated the involvement of epigenetic events in regulating the responses of irradiated cells. DNA methylation, where the cytosine bases in CpG dimers are converted to 5-methyl cytosine, is an epigenetic event that has been shown to regulate a variety of biological processes. We investigated the DNA methylation changes in irradiated TK6 and WTK1 human cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an enzyme-linked immunosorbent assay-based assay showed hypomethylation in both type of cells. Using an arbitrarily primed polymerase chain reaction (AP-PCR) approach, we observed time-dependent dynamic changes in the regional genomic DNA methylation patterns in both cell lines. The AP-PCR DNA methylation profiles were different between TK6 and WTK1 cells, indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells after irradiation but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells after radiation treatment. TET1, involved in the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns, and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns change at different genomic regions and are dependent on time after irradiation and the genetic background of the cell.

MicroRNA Signature in Waldenstrom Macroglobulinemia

Background. MicroRNAs (miRNAs) constitute a class of small, non-coding, 18–24 nucleotide RNAs, that act as negative regulators of gene expression. By repressing several target mRNAs, mature miRNAs play a pivotal role in regulating development, cell differentiation, apoptosis, and cell proliferation. Moreover, miRNAs have been described to play roles in both solid tumors and hematologic malignancies, however the role of miRNAs in WM has not been yet elucidated.Methods. We performed miRNA expression profiling of bone marrow-derived CD19+ WM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-155 knockdown WM cells. Effect of miRNA-155 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding enzyme-linked immunosorbent assay-based assay. Gene expression profile analysis has been performed on both miRNA-155 knockdown- and control probe-transfected WM cells in order to identify potential miRNA-155 targeted genes. Finally correlation between miRNA signature and prognosis has been evaluated.Results. We identified a WM specific miRNA signature characterized by increased expression of miRNA-363*/-206/-494/-155/-184/-542-3p; and decreased expression of miRNA-9* (ANOVA;P< 0.01). Our data showed that miRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-kB signaling pathways. Potential miRNA-155 target genes were identified using gene expression profiling and included genes involved in cell cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM (P<0.01). We further demonstrated that therapeutic agents commonly used in WM (rituximab, perifosine, bortezomib) alter the levels of the major miRNAs identified, by inducing down-modulation of five increased miRNAs (all but miR-206) and upmodulation of patient-downexpressed miR-9* microRNA.Conclusion. These data indicate that miRNAs play a pivotal role in the biology of WM; represent important prognostic markers; and provide the basis for the development of new miRNA-based targeted therapies in this disease.

Use of Early Passage Fetal Intestinal Epithelial Cells in Semi–High-Throughput Screening Assays: An Approach to Identify New Innate Immune System Adjuvants

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.

Characterization of Sugar Binding by Osteoclast Inhibitory Lectin

Osteoclast inhibitory lectin (OCIL) is a membrane-bound C-type lectin that blocks osteoclast differentiation and, via binding to its cognate receptor NKRP1D, inhibits natural killer cell-mediated cytotoxicity. OCIL is a member of the natural killer cell receptor C-type lectin group that includes CD69 and NKRP1D. We investigated carbohydrate binding of soluble recombinant human and mouse OCIL in enzyme-linked immunosorbent assay-based assays. OCIL bound immobilized high molecular weight sulfated glycosaminoglycans, including fucoidan, lambda-carrageenan, and dextran sulfate, but not unsulfated dextran or sialated hyaluronic acid. Carbohydrate binding was Ca(2+)-independent. Binding of immobilized low molecular weight glycosaminoglycans, including chondroitin sulfate (A, B, and C forms) and heparin, was not observed. However, the soluble forms of these low molecular weight glycosaminoglycans competed for OCIL binding of immobilized fucoidan (as did soluble fucoidan, dextran sulfate, and lambda-carrageenan), indicating that OCIL does recognize these carbohydrates. Inhibition constants for chondroitin sulfate A and heparin binding were 380 and 5 nm, respectively. Immobilized and soluble monosaccharides did not bind OCIL. The presence of saturating levels of fucoidan, dextran sulfate, and lambda-carrageenan did not affect OCIL inhibition of osteoclast formation. The fucoidan-binding lectins Ulex europaeus agglutinin I and Anguilla anguilla agglutinin did not block osteoclast formation or affect the inhibitory action of OCIL. Although the osteoclast inhibitory action of OCIL is independent of sugar recognition, we have found that OCIL, a lectin widely distributed, but notably localized in bone, skin, and other connective tissues, binds a range of physiologically important glycosaminoglycans, and this property may modulate OCIL actions upon other cells.

Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum.

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.

New insights into evaluating effective T-cell responses to HIV.

Towards addressing questions related to HIV pathogenesis and vaccine design we are fortunate to have the availability of the SIV-infected rhesus macaque model. The strengths of this model which include a rapid rate of progression to AIDS and knowledge of the dose route and strain of the infecting virus complement studies in HIV-infected patients in which the reagents host genetics and access to samples are more extensive and better defined. Unfortunately there is currently still too little known about the antiviral immune responses in either system to directly and accurately compare their similarities and differences and to draw any definitive conclusions. Therefore the data and views presented herein will simply reflect what has recently been discovered in both humans and non-human primate studies. (excerpt)

Identification of a Binding Site on the Type II Activin Receptor for Activin and Inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.

Binding of Haemophilus ducreyi to extracellular matrix proteins.

We developed an enzyme-linked immunosorbent assay-based assay to assess Haemophilus ducreyi binding to extracellular matrix (ECM) proteins. H. ducreyi 35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in ftpA or losB bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for H. ducreyi to bind to ECM proteins.

Carbohydrate-dependent binding of the cell-free hemagglutinin of Vibrio cholerae to glycoprotein and glycolipid.

The carbohydrate-binding specificity of the cell-free hemagglutinin (HA) of Vibrio cholerae (K.K. Banerjee, A.N. Ghose, K. Datta-Roy, S.C. Pal, and A.C. Ghose, Infect. Immun.58:3698-3705, 1990) was studied by using glycoconjugates with defined sugar sequences. The HA was not inhibited by simple sugars including glucobiose, galabiose, and their N-acetylated derivatives. The hemagglutination of rabbit erythrocytes by the HA was inhibited moderately by fetuin, calf thyroglobulin, and human alpha 1-acid glycoprotein, all of which contain multiple asparagine-linked complex-type oligosaccharide units alone or in combination with serine/threonine-linked oligosaccharide units. The inhibitory potencies of the glycoproteins increased approximately 10-fold following removal of the terminal sialic acid and were completely destroyed by exhausative proteolysis. The HA agglutinated phosphatidylcholine liposomes containing GM1-ganglioside or its asialo-derivative in the presence of Ca2+ ions. The association constants of the complexes of the HA with asialofetuin, asialothyroglobulin, GM1-ganglioside, and asialo-GM1-ganglioside were determined by an enzyme-linked immunosorbent assay-based assay and found to be 1.7 x 10(7) M-1, 1.5 x 10(7) M-1, 1.8 x 10(7) M-1, and 2.4 x 10(7) M-1, respectively. Studies using chemically modified glycoproteins and plant lectins with defined sugar specificity revealed that the HA recognized the terminal beta 1-galactosyl moiety of these glycoconjugates. There was no evidence for the presence of an extended carbohydrate-binding domain in the HA molecule or a preference of the HA for a complex, branched oligosaccharide structure. Similar to the mechanisms proposed for the binding of cholera toxin and Shiga toxin to glycolipids and neoglycoproteins, the strong interaction of V. cholerae cell-free HA with glycoconjugates appeared to be a consequence of multiple weak binding to terminal beta1-galactosyl moieties of the glycoproteins or glycolipids.

Regulation of the expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in kidney.

Previous studies (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373; Galili, U., Shohet, S. B., Korbrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762) have established that there is a unique evolutionary distribution of glycoconjugates carrying the Gal alpha 1-3Gal beta 1-4GlcNAc epitope. These glycoconjugates are expressed by cells from New World monkeys and non-primate mammals, but not by cells from humans, Old World monkeys, or apes. The lack of expression of this epitope in the latter species appears to result from the suppression of gene expression for the enzyme UDP-galactose:nLc4Cer alpha 1-3-galactosyltransferase (alpha 1-3GalT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Although many non-primate species are known to express this carbohydrate epitope, the nature (i.e. glycoprotein or glycosphingolipid) of the glycoconjugate carrying this epitope is only known for a few tissues in a few animal species. Furthermore, it is not known whether all animal species express this epitope in the same tissues. We have investigated these questions by analyzing the glycosphingolipids in kidney from several non-primate animal species. Immunostained thin layer chromatograms of glycosphingolipids from sheep, pig, rabbit, cow, and rat kidney with the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipid-specific monoclonal antibody, Gal-13, demonstrated that kidney from all of these species except rat contained Gal alpha 1-3Gal beta 1-4GlcNAc neutral glycosphingolipids. A lack of expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in rat may be due to the lack of expression of the enzyme (alpha 1-3GalT) which catalyzes the formation of the Gal alpha 1-3Gal nonreducing terminal sequence of these compounds or to the lack of expression of glycosyltransferases which are necessary for the synthesis of the neolacto core structure of these compounds. These possibilities were evaluated in two ways. First, the three enzymes (UDP-N-acetylglucosamine:LacCer beta 1-3-N-acetyl-glucosaminyltransferase, UDP-galactose:Lc3Cer beta 1-4-galactosyltransferase, and alpha 1-3GalT) involved in the synthesis of the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids were assayed using an enzyme-linked immunosorbent assay-based assay system and carbohydrate sequence-specific monoclonal antibodies. Second, TLC immunostaining was done to determine if the glycosphingolipid precursors (i.e. Lc3Cer and nLc4Cer) are expressed in rat kidney. Interestingly, rat kidney had a relatively high level of alpha 1-3GalT activity compared with the other animals tested.(ABSTRACT TRUNCATED AT 400 WORDS)