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

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.

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Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

Indian Journal of Fisheries ◽

10.21077/ijf.2019.67.2.84594-08 ◽

2020 ◽

Vol 67 (2) ◽

Author(s):

Snatashree Mohanty ◽

M. Makesh ◽

K. V. Rajendran ◽

P. P. Suresh Babu ◽

Deepika Anand ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Reactivity ◽

Catla Catla ◽

Cirrhinus Mrigala ◽

Indian Major Carps ◽

Sds Page ◽

Igg1 Isotype ◽

Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

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Variation of egg proteins between bird varieties by using SDS-PAGE

Al-Anbar Journal of Veterinary Sciences ◽

10.37940/ajvs.2019.12.1.8 ◽

2019 ◽

Vol 12 (1) ◽

pp. 68-73

Author(s):

Questan Amin ◽

Hemn Zhahir ◽

Ahmed Shaker

Keyword(s):

Sodium Dodecyl Sulfate ◽

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Egg Yolk ◽

Egg White ◽

Yolk Proteins ◽

Egg White Proteins ◽

Sds Page

Proteins are essential constituents of all organisms; both egg white proteins and egg yolk are source of protein. The aim of this study was conducted to perform preliminary studies to analyses and compare egg white proteins and yolk proteins from different avian species (guineafowl, dwarf hens, local hen, Shami, turkey, duck, geese, partridge and quail) via or with SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis ). 18 Fresh eggs of different poultry species (guineafowl, dwarf hens, local hen, Shami, turkey, duck, geese, partridge and quail) were collected from various farms in the Sulaimani province. Data on egg proteins were analyzed using Statistical Xlstate used for dendrogram construction and PCA. The main egg white proteins were Ovomicin, Ovotransferrin, Ovalbumin, Flavoprotein, α- chymotrypsinogen, and Trypsin inhibitor. The main lipoproteins were Apovitellenin VI, Apovitellenin Vb, Apovitellenin V, Apovitellenin IIIa, Apovitellenin III, Apovitellin 7, B-Livetin, Apovitellenin IIa, Apovitellenin II, and Apovitellenin I. All these lipoproteins were observed in the nine birds species. The egg white proteins and yolk lipoproteins for nine species were examined. It can be concluded the large differences were found in a mount of egg white proteins and yolk lipoproteins of the nine species of birds.

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Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219878407 ◽

2019 ◽

Vol 25 (3) ◽

pp. 310-319

Author(s):

Yuan Dong ◽

Hanjin Hou ◽

An Chen ◽

Wei Ma ◽

Moli Yin ◽

...

Keyword(s):

Monoclonal Antibody ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Reactivity ◽

Enzyme Hydrolysis ◽

Clinical Samples ◽

Sds Page ◽

Thrombotic Diseases ◽

D Dimer

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

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Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽

10.1182/blood.v65.2.496.496 ◽

1985 ◽

Vol 65 (2) ◽

pp. 496-500 ◽

Cited By ~ 16

Author(s):

M Wolf ◽

C Boyer ◽

A Tripodi ◽

D Meyer ◽

MJ Larrieu ◽

...

Keyword(s):

Antithrombin Iii ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Reactivity ◽

Two Dimensional ◽

Western Blots ◽

Sds Page ◽

New Variant ◽

At Iii ◽

Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

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Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00058-06 ◽

2006 ◽

Vol 13 (10) ◽

pp. 1155-1161 ◽

Cited By ~ 22

Author(s):

Donghee Cho ◽

Michael T. Collins

Keyword(s):

Solid Phase ◽

Expression Profiles ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Size Exclusion ◽

Enzyme Linked Immunosorbent Assay ◽

Infected Cattle ◽

Mycobacterium Paratuberculosis ◽

Serologic Tests ◽

Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

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Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽

10.1182/blood.v79.1.161.161 ◽

1992 ◽

Vol 79 (1) ◽

pp. 161-168 ◽

Cited By ~ 23

Author(s):

Y Tomiyama ◽

R Kekomaki ◽

J McFarland ◽

TJ Kunicki

Keyword(s):

Immune Thrombocytopenia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Surface Protein ◽

Normal Subjects ◽

Enzyme Linked Immunosorbent Assay ◽

Two Dimensional ◽

Platelet Protein ◽

Sds Page ◽

Significant Difference

Abstract We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

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Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽

10.1182/blood.v73.5.1202.1202 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1202-1206 ◽

Cited By ~ 7

Author(s):

MG Bolyard ◽

ST Lord

Keyword(s):

Escherichia Coli ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Reactivity ◽

Dependent Manner ◽

Beta Chain ◽

Gamma Chain ◽

Human Fibrinogen ◽

E Coli ◽

Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

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MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

Infection and Immunity ◽

10.1128/iai.66.1.289-296.1998 ◽

1998 ◽

Vol 66 (1) ◽

pp. 289-296 ◽

Cited By ~ 33

Author(s):

Morten Harboe ◽

Harald G. Wiker ◽

Gunni Ulvund ◽

Bent Lund-Pedersen ◽

Åse Bengård Andersen ◽

...

Keyword(s):

Protein Localization ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Culture Fluid ◽

Secreted Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Sds Page ◽

Shock Proteins ◽

Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

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Highly-Purified Natural Rubber by Saponification of Latex: Analysis of Residual Proteins in Saponified Natural Rubber

Rubber Chemistry and Technology ◽

10.5254/1.3548192 ◽

2008 ◽

Vol 81 (1) ◽

pp. 121-137 ◽

Cited By ~ 8

Author(s):

Jintana Yunyongwattanakorn ◽

Yasuyuki Tanaka ◽

Jitladda Sakdapipanich ◽

Voratep Wongsasutthikul

Keyword(s):

Natural Rubber ◽

Sodium Hydroxide ◽

Room Temperature ◽

Sodium Hydroxide Solution ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Aqueous Sodium Hydroxide ◽

Enzyme Linked Immunosorbent Assay ◽

Nitrogenous Compounds ◽

Sds Page

Abstract Highly purified natural rubber (NR) was prepared by saponification of fresh latex (FL-latex) and preserved high-ammonia latex (HA-latex) in the presence of surfactant to reduce the residual proteins in resulting solid NR. Saponification of latex diluted to 30% DRC was carried out with 1–7% (w/v) sodium hydroxide at room temperature for 1–7 hr at 70 °C and coagulated with formic acid. The nitrogen content of NR obtained by coagulation of the saponified latex markedly decreased to less than 0.014% by centrifugation of the saponified latex or soaking the coagulum in aqueous sodium hydroxide solution. The nitrogenous compounds from saponified NR (SAP-NR) were extracted with sodium dodecyl sulphate (SDS) aqueous solution and subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis to check the molecular weight of extracts. The extract from SAP-NR and deproteinized NR by protease (DPNR) for comparison was subjected to the analysis of allergic protein by FIT Kit method, based on Enzyme-Linked Immunosorbent Assay (ELISA) method. No extractable protein was observed in SAP-NR, whereas DPNR contained 1.5 μg/ml proteins. The results from SDS-PAGE analysis and FIT Kit test demonstrated that NR free from allergic proteins is obtainable by saponification of FL-latex with 1.5% NaOH at 70 °C for 1 hr or at room temperature for 24 hr.

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.2.454-459.2001 ◽

2001 ◽

Vol 8 (2) ◽

pp. 454-459 ◽

Cited By ~ 37

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.

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