scholarly journals Identification and Characterization of Immunogenic Proteins of Mycoplasma genitalium

2006 ◽  
Vol 13 (8) ◽  
pp. 913-922 ◽  
Author(s):  
Helle Friis Svenstrup ◽  
Jørgen Skov Jensen ◽  
Kris Gevaert ◽  
Svend Birkelund ◽  
Gunna Christiansen
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycoplasma Genitalium ◽  
Cross Reactivity ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Immunogenic Proteins ◽  
Recombinant Fragments

ABSTRACT Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.

scholarly journals Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

2001 ◽  
Vol 8 (2) ◽  
pp. 454-459 ◽  
Author(s):  
Mitsushi Tsujimura ◽  
Chuzo Ishida ◽  
Yasuko Sagara ◽  
Takashi Miyazaki ◽  
Koichi Murakami ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Samples ◽  
Aerococcus Viridans ◽  
Sds Page

ABSTRACT Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide fromAerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose,N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection

Parasitology ◽  
2020 ◽  
Vol 147 (6) ◽  
pp. 715-720
Author(s):  
Dayane Costa de Souza ◽  
Lucas Silva de Faria ◽  
José Eduardo Neto de Sousa ◽  
Camila de Almeida Lopes ◽  
Vanessa da Silva Ribeiro ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Egg Yolk ◽  
Diagnostic Value ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hookworm Infection ◽  
Serum Samples

AbstractDefinitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

scholarly journals Production of polyclonal antibody against the recombinant PirBvp protein of Vibrio parahaemolyticus

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ngoc-Diem Duong ◽  
Khai-Hoan Nguyen-Phuoc ◽  
Kim-Yen Thi Do ◽  
Nguyet-Thu Thi Nguyen ◽  
Thuoc Linh Tran ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Detection Threshold ◽  
Cross Reactivity ◽  
Rabbit Serum ◽  
Lateral Flow Strip ◽  
Specificity And Sensitivity

Abstract Background Acute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirAvp and PirBvp encoded in pVA1 plasmid. The polyclonal antibodies against PirBvp protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND. Results In this study, PirBvp gene was amplified, cloned, and expressed in E. coli. The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirBvp (rPirBvp) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirBvp, and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot. Conclusion This study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.

scholarly journals Detection of Soy Proteins in Processed Foods: Literature Overview and New Experimental Work

2004 ◽  
Vol 87 (6) ◽  
pp. 1398-1407 ◽  
Author(s):  
Stef J Koppelman ◽  
Catriona M M Lakemond ◽  
Riek Vlooswijk ◽  
Susan L Hefle
Keyword(s):  
Soy Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Soy Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Foods ◽  
Food Ingredients ◽  
Advantages And Disadvantages ◽  
Limit Of Quantitation

Abstract Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 μg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 μg/mL, corresponding to 0.4 μg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.

scholarly journals A Preliminary Study on Cross-Reactivity of Heat-Treated Quail and Hen’s Egg White Proteins in Young Children

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2172
Author(s):  
Jeongmin Lee ◽  
Purevsan Gantulga ◽  
Changhoon Lee ◽  
Kyunguk Jeong ◽  
Eunjoo Lee ◽  
...  
Keyword(s):  
Young Children ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Cross Reactivity ◽  
Inhibition Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hen’S Egg ◽  
Sds Page ◽  
Elisa Inhibition

We investigated the effects of different types of heat treatments on hen’s egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.

scholarly journals Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate

2019 ◽  
Vol 12 (4) ◽  
pp. 477-482
Author(s):  
Melani Wahyu Adiningsih ◽  
Retno Damajanti Soejoedono ◽  
Rahmat Setya Adji ◽  
Dwi Desmiyeni Putri ◽  
Trioso Purnawarman ◽  
...  
Keyword(s):  
Wild Boar ◽  
Antibody Production ◽  
Specific Antibody ◽  
Sus Scrofa ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Meat Extract ◽  
The Third

Aim: Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate. Materials and Methods: Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund's complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund's incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. Results: Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster. Conclusion: The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Identification of a laminin-like substance on the surface of high-malignant murine fibrosarcoma cells

1984 ◽  
Vol 65 (1) ◽  
pp. 139-151
Author(s):  
J.P. McCoy ◽  
R.V. Lloyd ◽  
M.S. Wicha ◽  
J. Varani
Keyword(s):  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoperoxidase Staining ◽  
Normal Rabbit Serum ◽  
Malignant Cells ◽  
Fibrosarcoma Cells ◽  
Murine Fibrosarcoma

High- and low-malignant murine fibrosarcoma cells were stained with anti-laminin antibodies using immunoperoxidase techniques and examined by electron microscopy. With the high-malignant cells, specific staining was observed along the cell surface. Use of normal rabbit serum in place of the rabbit anti-laminin or pretreatment of the anti-laminin with soluble laminin completely eliminated this staining. No immunoperoxidase staining was observed with the low-malignant cells. In additional studies, membrane fractions were prepared from the high- and low-malignant cells and used to immunize rabbits. The animals immunized with the membrane fractions from the high-malignant cells produced antibodies that reacted by enzyme-linked immunosorbent assay (ELISA) with murine laminin obtained from the EHS sarcoma. The animals immunized with membrane fractions from the low-malignant cells did not. These studies provide strong evidence that the high-malignant cells (but not the low) express on their cell surface a substance that is immunologically cross-reactive with laminin. In addition, the high-malignant cells (but not the low) secreted a material into the cell culture fluid that could be specifically immunoprecipitated with antilaminin antibodies. The immunoprecipitated material co-migrated with purified laminin when examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis under reducing conditions. The existence of this substance associated with the surface of the high-malignant cells and its absence from that of the low-malignant cells may explain the previously noted difference between these cells in their ability to attach to type IV collagen. This difference may also contribute to the dissimilarity between these cells in their metastatic potential.

Antigenic and immunogenic activity of flagella and fimbriae preparations from uropathogenic Proteus mirabilis

1991 ◽  
Vol 37 (4) ◽  
pp. 325-328 ◽  
Author(s):  
C. Legnani-Fajardo ◽  
P. Zunino ◽  
G. Algorta ◽  
H. F. Laborde
Keyword(s):  
Proteus Mirabilis ◽  
Mechanical Treatment ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sodium Deoxycholate ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ammonium Sulfate Precipitation ◽  
Urea Treatment

The antigenic and immunogenic activities of fimbriae and flagella from three uropathogenic strains of Proteus mirabilis were compared. Flagella were obtained by mechanical treatment and fimbriae were isolated from cells by heat shock, ammonium sulfate precipitation, sodium deoxycholate and urea treatment, and gel filtration. Both preparations inoculated to mice demonstrated high antigenicity. Titers up to 1: 80 000 were determined by an enzyme-linked immunosorbent assay either against the homologous or heterologous strains. When immunized mice were challenged with homologous or heterologous hematogenous infecting doses, a good cross protection was achieved only when fimbriae were used as antigens. Cross-reactivity found between the three fimbriae antisera, and the presence of common proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of fimbriae, should validate the study of these proteins to determine the existence of a shared adhesin. Key words: fimbriae, flagella, Proteus mirabilis, and antigenicity.

scholarly journals Dogs as Sentinels for Flavivirus Exposure in Urban, Peri-Urban and Rural Hanoi, Vietnam

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 507
Author(s):  
Long Pham-Thanh ◽  
Thang Nguyen-Tien ◽  
Ulf Magnusson ◽  
Vuong Bui-Nghia ◽  
Anh Bui-Ngoc ◽  
...  
Keyword(s):  
Mosquito Control ◽  
Risk Mitigation ◽  
Significant Risk ◽  
Cross Sectional Study ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Cross Sectional ◽  
Major Health ◽  
Household Level

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

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