scholarly journals The identification of epitopic sites in human α1-proteinase inhibitor

1987 ◽  
Vol 246 (1) ◽  
pp. 25-36 ◽  
Author(s):  
X J Zhu ◽  
S S Kang ◽  
K Hargrove ◽  
D Shochat ◽  
M Jarrells ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Proteinase Inhibitor ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Proteolytic Digestion ◽  
Group Iii ◽  
Group I ◽  
Hybridoma Technology ◽  
Cnbr Cleavage

Human α 1-proteinase inhibitor (α 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized α 1-PI and mouse polyclonal antibodies against native α 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between α 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native α 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized α 1-PI as the antigen. The ability of the monoclonal antibodies to bind native α 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding α 1-PI and only fragment I. Antibodies in groups II and III bound α 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between α 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in α 1-PI. Specific monoclonal antibodies to three of these sites were obtained.

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

1985 ◽  
Vol 31 (10) ◽  
pp. 1654-1658 ◽  
Author(s):  
S Marcovina ◽  
D France ◽  
R A Phillips ◽  
S J Mao
Keyword(s):  
Monoclonal Antibodies ◽  
Apolipoprotein B ◽  
Polyclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Radial Immunodiffusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Density ◽  
Apo B ◽  
Blotting Technique

Abstract We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

Urinary Claudin-1 Level as a Marker of Podocyte Injury in Patients with Proteinuria

QJM ◽  
2020 ◽  
Vol 113 (Supplement_1) ◽  
Author(s):  
M E Nasser ◽  
S M Shawky ◽  
E A M Sanad
Keyword(s):  
Wide Spectrum ◽  
Cell Junction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Podocyte Injury ◽  
Group Iii ◽  
Group I ◽  
Significant Difference ◽  
Diaphragm Cell ◽  
Control Study

Abstract Background The biology of claudins is a rapidly evolving field, and many intriguing questions remain unanswered. Although it had been assumed that the reason there are ≥24 isoforms of claudin is that each one has distinct permeability properties. The nephron displays a wide spectrum of claudins, whose distribution varies in each tubular segment. In diabetic nephropathy and glomerulonephritis the gene expression of claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit diaphragm (cell-cell junction made by the glomerular podocytes) and the appearance of TJ-like structures between the foot processes causing further podocytes injury and proteinuria. Aim of the work to assess urinary claudin -1 level as a marker of podocyte injury in patients with proteinuria. Patients and Methods it is a case control study which was conducted upon 90 subjects who were divided into three groups: group I included 30 patients with type II DM, group II included 30 patients with glomerulonephritis and group III had 30 healthy subjects as controls. Urinary claudin-1 level was measured by Enzyme linked Immunosorbent Assay (ELISA) Results In this study, we found that urinary claudin-1 level was significantly higher in diabetics group and GN group than in control group. In comparison between GN group and diabetic group, we found that urinary claudin-1 level was higher in GN group than in diabetics group but with no statistically significant difference between the two groups. Conclusion urinary claudin-1 level was significantly higher in diabetics and GN group and has positive correlation with uACR. So it can be used as marker of podocytes injury and proteinuria

scholarly journals Structure of the murine rab3A gene: correlation of genomic organization with antibody epitopes

1993 ◽  
Vol 293 (1) ◽  
pp. 157-163 ◽  
Author(s):  
M Baumert ◽  
G Fischer von Mollard ◽  
R Jahn ◽  
T C Südhof
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Protein Domains ◽  
Amino Acid Sequences ◽  
Sequence Motifs ◽  
Rab Protein ◽  
Gtp Binding ◽  
Gene Structures ◽  
Antibody Epitopes

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.

scholarly journals Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

1996 ◽  
Vol 8 (1) ◽  
pp. 68-75 ◽  
Author(s):  
H. E. Jensen ◽  
B. Aalbaek ◽  
P. Lind ◽  
H. V. Krogh ◽  
P. L. Frandsen
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunohistochemical Techniques ◽  
Immunohistochemical Diagnosis ◽  
Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Detection of Abrin in Food Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies

2008 ◽  
Vol 71 (9) ◽  
pp. 1868-1874 ◽  
Author(s):  
ERIC A. E. GARBER ◽  
JENNIFER L. WALKER ◽  
THOMAS W. O'BRIEN
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Dairy Products ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ribosome Inactivating Protein ◽  
Limits Of Detection ◽  
Abrus Precatorius ◽  
A Priority

Abrin is a toxic ribosome-inactivating protein present in beans of Abrus precatorius, also known as rosary peas. The possibility that abrin could be used to adulterate food has made the development of assays for the detection of abrin a priority. Rabbit-derived polyclonal antibodies and mouse monoclonal antibodies were prepared against a mixture of abrin isozymes. The specificity and cross-reactivity of the antibodies were evaluated against a challenge library of 40 grains, nuts, legumes, and foods. An enzyme-linked immunosorbent assay (ELISA) and an electrochemiluminescence (ECL)–based assay were assembled and optimized. Polyclonal (capture) and polyclonal (detection) ELISAs, polyclonal and monoclonal ELISAs, and polyclonal and monoclonal ECL assays had limits of detection (LODs) of 0.1 to 0.5 ng/ml for abrin in buffer. The LOD for abrin dissolved into juices, dairy products, soda, chocolate drink, and condiments and analyzed with the ECL assay ranged from 0.1 to 0.5 ng/ml in the analytical sample. In contrast, the LODs for the ELISAs ranged from 0.5 to 10 ng/ml in the analytical sample.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Serotyping Ureaplasma urealyticum Strains Using Monoclonal Antibodies

2001 ◽  
Vol 8 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Fedoua Echahidi ◽  
Gaëtan Muyldermans ◽  
Sabine Lauwers ◽  
Anne Naessens
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Ureaplasma Urealyticum ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cross Reactions ◽  
Antigenic Preparation ◽  
Negative Controls ◽  
Reference Strains

ABSTRACT Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains ofU. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

scholarly journals Neurovirulence of Type 1 Polioviruses Isolated from Sewage in Japan

2002 ◽  
Vol 68 (1) ◽  
pp. 138-142 ◽  
Author(s):  
Hitoshi Horie ◽  
Hiromu Yoshida ◽  
Kumiko Matsuura ◽  
Miwako Miyazawa ◽  
Yoshihiro Ota ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Restriction Enzyme ◽  
Mutant Analysis ◽  
Group Iii ◽  
Group I ◽  
Poliomyelitis Vaccine ◽  
Vaccine Type ◽  
Enzyme Cleavage ◽  
Human Sera

ABSTRACT Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.

scholarly journals H13 Subtype Avian Influenza Viruses Are Crossing Host Species Barrier From Wild Waterfowl to Land Fowl

2021 ◽  
Author(s):  
Weiyang SUN ◽  
Menglin ZHAO ◽  
Zhijun YU ◽  
Yuanguo LI ◽  
Xinghai ZHANG ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Chicken Embryo Fibroblast ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Post Inoculation ◽  
Species Barrier ◽  
Group Iii ◽  
Mammalian Cell Lines ◽  
Group I ◽  
Wild Waterfowl

Abstract The avian influenza virus H13 subtype circulates primarily in waterfowl. To explore the ability of the H13 virus to cross the host barriers, we genetically analysed two H13 isolates from wild birds in China and evaluated the infectivity of these subtypes in chickens. Genetic and molecular analyses showed differences in the lineages and amino acid sequences between the two subtypes; A/mallard/Dalian/DZ-137/2013 (H13N6) belonged to Group I, while A/Eurasian Curlew/Liaoning/ZH-385/2014 (H13N8) belonged to Group III. The nucleotide sequence results showed high homology (approximately 96.9%-100%) to sequences in GenBank. Neither H13 isolate replicated in adult chickens or 20-day-old chicks; however, the H13N8 strain replicated in 1- and 10-day-old chicks. Viruses were recovered from the nasal turbinate, tracheal, lung and colon tissues of chicks at 1, 3 and 5 days post-inoculation. The H13N6 isolates replicated inefficiently in 1-day-old chicks and did not replicate in 10-day-old chicks. Serological surveillance results showed that domestic chickens had a 4.6%-10.4% (15/328-34/328) positive antibody titre to the H13 virus. H13N6 and H13N8 isolates replicated in mammalian cell lines, including 293T, Madin-Darby canine kidney and chicken embryo fibroblast cells. Our results suggest that the AIV H13 subtype may cross the host barrier from wild waterfowl to land fowl.

scholarly journals Does thyroid dysfunction influence inflammatory mediators in experimental periodontitis?

2021 ◽  
Vol 55 (3) ◽  
pp. 131-141
Author(s):  
Vitaliy Shcherba ◽  
Inna Krynytska ◽  
Mariya Marushchak ◽  
Mykhaylo Korda
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Mediators ◽  
Thyroid Dysfunction ◽  
Solid Phase ◽  
White Male ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Group Iii ◽  
Group I ◽  
Tnf Α

Abstract Objective. The aim of the present study was to investigate the presence of inflammatory mediators in rats with only periodontitis and periodontitis in a setting of hyper- and hypo-thyroidism and to analyze the correlative linkages between inflammatory mediators and thyroid hormones. Methods. White male 12–14 weeks old inbred rats (n=48) weighing 180–200 g were employed in the experiment. They were randomly divided into the following groups: Group I – control group, Group II – group with a model of periodontitis, Group III – group with a periodontitis in a setting of hyperthyroidism, and Group IV – group with periodontitis in a setting of hypothyroidism. The presence of tumor-necrosis factor-α (TNF-α) and interleukins IL-1β and IL-10 in the periodontal homogenate supernatant was studied by a solid-phase enzyme-linked immunosorbent assay. Results. It was shown that experimental lipopolysaccharide (LPS)-induced periodontitis is accompanied by hyperproduction of pro-inflammatory cytokines (TNF-α, IL-1β) and reduction of anti-inflammatory cytokines (IL-10), whereas TNF-α underwent to maximum changes. Thyroid dysfunction exacerbates cytokine imbalance and severity of inflammation in experimental LPS-induced periodontitis, especially pronounced at hyperthyroidism, as evidenced by the predominance of TNF-α and IL-1β levels in the periodontal homogenate supernatant by 38.5% (р<0.01) and 75.6% (p<0.001), respectively, hyperthyroid over the euthyroid, and by 20.1% (p<0.05) and 24.1% (p<0.05), respectively, over the hypothyroid rats. Conclusions. Thyroid dysfunction, especially hyperthyroidism, may play an important role in the pro-inflammatory response in periodontitis. Hyperproduction of inflammatory mediators in thyroid dysfunction can induce a noticeable damage in the whole apparatus of the periodontium, thereby causing progression of periodontitis.

scholarly journals An Evaluation and Correlation of Leptin in Gingival Crevicular Fluid and Serum in Health, Gingivitis and Periodontitis

2012 ◽  
Vol 1 (2) ◽  
pp. 93-97
Author(s):  
Vinay Vadvadgi ◽  
Neeta Padmawar
Keyword(s):  
Periodontal Disease ◽  
Gingival Crevicular Fluid ◽  
Gingival Tissue ◽  
Enzyme Linked Immunosorbent Assay ◽  
Group Iii ◽  
Group I ◽  
The Mean ◽  
Group Ii ◽  
Crevicular Fluid ◽  
Mean Concentration

ABSTRACT Background and objective Plasma leptin is associated in patients with inflammatory diseases. A high concentration of leptin is associated with healthy gingival tissue. The purpose of this study was to assess the concentration of human leptin in gingival crevicular fluid (GCF) and serum within healthy and diseased gingiva, further to explore the possibility of using the levels of leptin in GCF and serum as a biochemical marker of periodontal disease progression. Materials and methods Ninety subjects were selected with age (30-39 years) and sex (15 males and 15 females) matched, to eliminate age and sex as confounders. The subjects were divided into three groups consisting of 30 subjects in each group based on the clinical and radiological parameters; healthy (group I), gingivitis (group II), periodontitis (group III), from whom the GCF samples were collected with Periopaper GCF collection strips (Proflow, Amityville, NY, USA) for 30 seconds and blood samples with 20-gauge needle syringe respectively. Leptin concentration was determined from individual GCF and serum samples by enzyme-linked immunosorbent assay (ELISA). Results The highest mean leptin concentration in GCF was observed in group I (2,664.30 pg/ml ± 324.73) and least mean leptin concentration was obtained in group III (1,309.43 pg/ml ± 202.45). The mean concentration of group II (1,639.43 pg/ml ± 344.46) was intermediate between the highest and lowest values. In contrast, the highest mean leptin concentration in serum was obtained for group III (12,086.57 pg/ml ± 1,698.23) and least mean leptin concentration was obtained for group I (8,715.09 pg/ml ± 1,649.19). The mean concentration of the group II (10,694.01 pg/ml ± 1,777.72) were intermediate between the highest and lowest values. Conclusion The results indicated a statistically significant decrease in the GCF leptin concentration and increase in serum leptin concentration as the periodontal disease progressed. How to cite this article Vadvadgi VH, Saini R, Padmawar N. An Evaluation and Correlation of Leptin in Gingival Crevicular Fluid and Serum in Health, Gingivitis and Periodontitis. Int J Experiment Dent Sci 2012;1(2):93-97.

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