scholarly journals Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays

Micromachines ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 614 ◽  
Author(s):  
Jenny Elomaa ◽  
Laura Gallegos ◽  
Frank A. Gomez
Keyword(s):  
Alkaline Phosphatase ◽  
Antibody Concentration ◽  
Microfluidic Chips ◽  
Igg Antibody ◽  
Capillary Action ◽  
Glucose Assay ◽  
Rabbit Igg ◽  
Mouse Immunoglobulin ◽  
Artificial Urine ◽  
Blue Intensity

This paper describes the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. In this study, biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose are quantitatively detected. In the ELISA systems, the antibody is spotted on the cord at the detection site and a series of washes, followed by streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody and colorimetric substrate, completing the experiment. The devices are subsequently scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity and antibody concentration. For the first ELISA, a linear range of detection was observed at lower concentrations (2.50 × 10−4–1.75 × 10−3 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 × 10−4). For the second ELISA, the L50 was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution containing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the detection sites realizing a yellow-brown color indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed to show the correlation between yellow inverse intensity and glucose. Glucose in artificial urine showed good correlation using the devices.

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

scholarly journals Immune complexes containing factor V in a patient with an acquired neutralizing antibody

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 810-818 ◽  
Author(s):  
HC Chiu ◽  
AK Rao ◽  
C Beckett ◽  
RW Colman
Keyword(s):  
Immunosorbent Assay ◽  
Immune Complexes ◽  
Neutralizing Antibody ◽  
Circulating Immune Complexes ◽  
Heat Inactivation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration ◽  
Factor V ◽  
Igg Antibody ◽  
Plasma Factor

Abstract An 82-year-old woman presented with extensive hematomas and melena associated with markedly decreased plasma factor V coagulant activity (FV:C). Using a competitive enzyme-linked immunosorbent assay developed in our laboratory, we made serial measurements of factor V antigen (FV:Ag) in plasma and found it to be normal or elevated. The patient's plasma was demonstrated to contain an IgG antibody that could neutralize FV:C in normal plasma. The antibody was of restricted heterogeneity (IgG1, IgG2,kappa). Circulating immune complexes containing antibody to factor V and FV:Ag were demonstrated directly in the plasma by immunoelectrophoresis with polyclonal monospecific antibody and with a monoclonal antibody using an enzyme-linked immunosorbent assay. Presence of neutralizing antibody could be demonstrated in vitro even at times when FV:C was within normal limits by heat inactivation of FV:C. Treatment with plasma and platelet transfusions as well as plasmapheresis induced definite but transient elevation of FV:C. Steroid therapy lowered the neutralizing antibody concentration and produced a rapid and persistent elevation of FV:C during two separate hospitalizations. This report describes a patient in whom levels of FV:Ag have been serially measured, and the presence of circulating immune complexes consisting of factor V and a neutralizing antibody have been directly demonstrated.

scholarly journals Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.

1986 ◽  
Vol 34 (4) ◽  
pp. 423-428 ◽  
Author(s):  
E Claassen ◽  
D M Boorsma ◽  
N Kors ◽  
N Van Rooijen
Keyword(s):  
Alkaline Phosphatase ◽  
Staining Method ◽  
Direct Detection ◽  
Direct Method ◽  
Simultaneous Detection ◽  
Single Section ◽  
Mouse Immunoglobulin ◽  
Enzyme Conjugate ◽  
One Step ◽  
Triple Staining

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.

scholarly journals The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 69-75 ◽  
Author(s):  
K Sasaki ◽  
S Moriyama ◽  
Y Tanaka ◽  
H Sumi ◽  
N Toki ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Intestinal Tract ◽  
Gel Filtration ◽  
Intestinal Transport ◽  
Plasminogen Activators ◽  
Protein Fractions ◽  
Igg Antibody ◽  
Chromatography Method ◽  
Dog Model ◽  
Rabbit Igg

Abstract In an attempt to elucidate the mechanism of fibrinolytic enhancement by orally administered urokinase, studies on the intestinal transport of urokinase were carried out, using 125I-labeled human high mol wt urokinase, administered intraduodenally in the experimental dog model with a saphenous vein thrombus. Using the plasma sample obtained from blood 45 minutes after intraduodenal administration of the urokinase, protein fractions were isolated by a sequential two-step affinity chromatography method, first with [N alpha-(epsilon-aminocaproyl)-DL- homoarginine hexylester]-Sepharose followed by a specific anti-human low mol wt urokinase rabbit IgG-Sepharose (adsorbed-eluted and unadsorbed). Each of the isolated protein fractions was further purified by gel filtration on Sephacryl S-300. The proteins isolated by the two-step affinity chromatography method were transported human urokinase with radioactivity in the adsorbed-eluted fraction, and newly synthesized and/or released dog plasminogen activators, probably urokinase-type and tissue-activator type, without radioactivity. In an antibody quenching assay, dog urokinase and the immuno-affinity unadsorbed fraction were not neutralized, but the immuno-affinity adsorbed-eluted fraction was completely neutralized by the specific anti-urokinase IgG antibody. Proteins isolated from control plasma (after administration of saline) by the two-step affinity chromatography method in the unadsorbed fraction had negligible amounts of activator activity. In these studies, we were able to show that synthesis of plasminogen activators was stimulated, with the activators being released, from either the liver or the vascular endothelium. Also we showed that urokinase is transported across the intestinal tract in the dog model.

Electron and Immunoelectron Microscopical Studies on the Extracellular Protease (Keratinase) of Dermatophytes

1982 ◽  
Vol 40 ◽  
pp. 158-159 ◽  
Author(s):  
Y. Sei ◽  
I. Takiuchi ◽  
M. Masutani
Keyword(s):  
Electron Microscopy ◽  
Human Skin ◽  
Human Hair ◽  
Phosphate Buffer ◽  
Proteolytic Enzyme ◽  
Microsporum Canis ◽  
Horny Layer ◽  
Double Immunodiffusion ◽  
Igg Antibody ◽  
Rabbit Igg

Dermatophytes, known to contain the proteolytic enzyme keratinase can grow in keratinized structures. Here we describe the digestion of the horny layer and the first localization of the enzyme by immunoelectronmicroscopy. Microsporum canis isolated from an outpatient was cultured in a medium of: human hair, 2.9g; glucose, 0.5g; MgSO4-7H2O, 0.06 g; thiamine 0.01g; pyridoxine 0.01 g; and inositol 0.05 g in one liter of 0.028 M phosphate buffer (pH 7.8). The enzyme was isolated from the medium and purified by chromatography. Human skin was incubated with the purified protease (2mg/ml) for 12 hrs at 37° C, and then processed for electron microscopy. Controls were incubated without enzyme. Using rabbit IgG antibody against the purified enzyme the IgG-Fab'-peroxidase complex was synthesized according to the method of Nakane and Kawaoi. That complex gave a single band in double immunodiffusion against purified protease and in immunoelectrophoresis at pH 4.0 and 9.3. Human hairs with fungi were removed from the keratin medium on the 12th day, fixed with periodate-lysin-paraformaldehyde for 24 hrs at 4°C. They were again washed in PBS, preincubated in 3,3'-diaminobenzidine (DAB) for 30 min at room temp., put in a Graham and Karnovsky medium containing DAB and H2 O2 as a substrate for 5 min at room temp., postfixed in 1% OSO4 in PBS for 1 hr at 4°C., and embedded in Epon.

scholarly journals Grb10 characterization in bovine cumulus oocyte complexes from different follicle sizes

2015 ◽  
Vol 45 (5) ◽  
pp. 898-904
Author(s):  
Paulo Roberto Antunes da Rosa ◽  
Rodrigo Camponogara Bohrer ◽  
Charles Alencar Ludke ◽  
Matheus Pedroti De Cesaro ◽  
Gabriel Ribas Pereira ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Protein Localization ◽  
Primary Antibody ◽  
Fluorescence Signal ◽  
Immunofluorescence Analysis ◽  
Pcr Assay ◽  
Igg Antibody ◽  
Alexa Fluor ◽  
Nuclear Maturation ◽  
Rabbit Igg

The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs) from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and >8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and >8mm; P>0.05), had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P>0.05). After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and >8mm) to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100). Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500). Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.

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