scholarly journals An Improved Protocol of Biotinylated Tyramine-based Immunohistochemistry Minimizing Nonspecific Background Staining

2003 ◽  
Vol 51 (1) ◽  
pp. 129-132 ◽  
Author(s):  
Seok Hyung Kim ◽  
Young Kee Shin ◽  
Kyung Mee Lee ◽  
Jung Sun Lee ◽  
Ji Hye Yun ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Blocking Agent ◽  
Immunohistochemical Method ◽  
Tween 20 ◽  
Distilled Water ◽  
Blocking Agents ◽  
Weak Staining ◽  
Optimized Protocol ◽  
Background Staining

An immunohistochemical method using biotinyl tyramine was recently introduced to amplify weak staining signals. Despite its high sensitivity, however, tyramine-based immunostaining has been limited by its increased background staining. In this study, to develop an improved protocol of biotinyl tyramine-based immunohistochemistry minimizing the background staining, we determined which staining steps lead to the nonspecific reaction and the most appropriate blocking agents for background-provoking steps. Trypton casein peptone and distilled water with Tween-20 were shown to be most effective as a blocking agent and a rinsing solution, respectively. In conclusion, we developed an optimized protocol for biotinyl tyramine-based immunohistochemistry with minimal background staining.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection

2021 ◽  
Vol 24 ◽  
Author(s):  
Nur Hanina Izzati Khairol Mokhtar ◽  
Ainulkhir Hussin ◽  
Aidil Abdul Hamid ◽  
Shahrul Hisham Zainal Ariffin ◽  
Muhammad Ashraf Shahidan
Keyword(s):  
Sialic Acid ◽  
Specific Binding ◽  
Specific Antigen ◽  
Blocking Agent ◽  
Microtiter Plate ◽  
Tween 20 ◽  
Nonspecific Binding ◽  
Binding Assays ◽  
High Background ◽  
Sialic Acid Linkage

Aims: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on prostate-specific antigen (PSA). Background: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based enzyme-linked lectin sorbent assays (ELLSAs), as they can mask specific binding signals and cause false-positive results. Methods: In this study, we constructed an ELLSA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELLSA. Results: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELLSA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. Conclusion: The ELLSA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

scholarly journals Immunohistochemical detection of soya protein – optimisation and verification of the method

2009 ◽  
Vol 27 (No. 1) ◽  
pp. 11-19 ◽  
Author(s):  
M. Pospiech ◽  
B. Tremlová ◽  
E. Renčová ◽  
Z. Randulová
Keyword(s):  
Indirect Elisa ◽  
Soya Protein ◽  
Immunohistochemical Method ◽  
Immunohistochemical Detection ◽  
Elisa Method ◽  
Background Staining ◽  
Soya Proteins ◽  
Abc Method ◽  
Soya Isolate ◽  
Avidin Biotin Complex

A functional immunohistochemical method for soya proteins detection was developed. The procedure is based on the avidin-biotin complex (ABC) method that attains sufficient sensitivity. The method was verified by the analysis of the model samples of different forms of soya additives containing various concentrations of soya isolate. The detection limit of soya present in the model samples was 0.5%. Different possibilities of the background staining were tested. The best results were obtained with the background staining according to Calleja. The results were confirmed by the accredited indirect ELISA method. The method allows the identification of various forms of soya proteins such as isolates, texturates, concentrates, and flour.

scholarly journals False Positivity in a Cyto-ELISA for Anti-Endothelial Cell Antibodies Caused by Heterophile Antibodies to Bovine Serum Proteins

2000 ◽  
Vol 46 (2) ◽  
pp. 273-278 ◽  
Author(s):  
Ronan Revelen ◽  
Anne Bordron ◽  
Maryvonne Dueymes ◽  
Pierre Youinou ◽  
Josiane Arvieux
Keyword(s):  
Endothelial Cell ◽  
Bovine Serum ◽  
Serum Proteins ◽  
Solid Phase ◽  
Calf Serum ◽  
Blocking Agent ◽  
Screening Tests ◽  
Tween 20 ◽  
Serum Samples ◽  
Antibody Absorption

Abstract Background: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. Methods: AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum (FCS) proteins were demonstrated and characterized in an ELISA—the interference assay—that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immunoblotting. Results: In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. Conclusions: Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer.

Fractionation of the motor unit: response to tetanus

1961 ◽  
Vol 200 (4) ◽  
pp. 689-693
Author(s):  
Simeon Locke
Keyword(s):  
Action Potential ◽  
Motor Unit ◽  
Nerve Terminal ◽  
Differential Effect ◽  
Blocking Agent ◽  
Single Motor Unit ◽  
Single Motor ◽  
Blocking Agents ◽  
Before And After ◽  
Post Tetanic Potentiation

The effect of a tetanus on the motor unit of the gastrocnemius of the rat has been studied before and after administration of blocking agents. Post-tetanic potentiation of action potential of the single motor unit occurs following depression of response by curare or decamethonium. Increased amplitude of unit potential results from partial resynchronization of subunit potential contributions which had been desynchronized by the differential effect of the blocking agent on subunit latency. Decline of unit potential subsequent to post-tetanic potentiation results from desynchronization of component contributions as had been observed with initial administration of blocking agent. The occurrence of these events in a single motor unit indicates that they take place at the nerve terminal or subterminal portion of the unit.

Increased Sensitivity to Depolarizing and Nondepolarizing Neuromuscular Blocking Agents in Young Rat Hemidiaphragms

2001 ◽  
Vol 95 (2) ◽  
pp. 478-484 ◽  
Author(s):  
L. Philippe Fortier ◽  
Richard Robitaille ◽  
François Donati
Keyword(s):  
Neuromuscular Junctions ◽  
Relative Sensitivity ◽  
Optimal Dose ◽  
Blocking Agent ◽  
Neuromuscular Blocking ◽  
Neuromuscular Blocking Agents ◽  
Response Curves ◽  
Potency Ratio ◽  
Blocking Agents ◽  
Increased Sensitivity

Background Newborn neuromuscular junctions are more sensitive to d-tubocurarine than more mature preparations. It is unclear whether the same modifications occur with newer nondepolarizing agents and depolarizing agent succinylcholine. The purpose of this study was to determine the relative sensitivity of newborn neuromuscular junctions to succinylcholine and five nondepolarizing agents. Methods The phrenic nerve-hemidiaphragm preparation from 60 rats was used, 30 aged 9-12 days (newborn) and 30 aged 27-33 days (adult). Five rats from each group were exposed to one of six neuromuscular blocking agents (d-tubocurarine, cisatracurium, atracurium, vecuronium, rocuronium, and succinylcholine). Indirectly elicited twitch tension was measured during control conditions in the absence of blocking agent, followed by four concentrations of one of the six agents. Concentration-response curves were constructed and the EC50 (concentration required to produce 50% depression of twitch tension) was obtained. Potency ratios (EC50adult/EC50newborn) were derived for each agent. Results Newborn preparations were significantly (P < 0.001) more sensitive than their adult counterparts for all six agents tested. For nondepolarizing agents, the potency ratio was in the 6-12 range. The EC50adult/EC50newborn were as follows, in decreasing potency order: d-tubocurarine, 1.68/0.23 microM; cisatracurium, 2.73/0.47 microM; vecuronium, 5.47/0.59 microM; rocuronium, 9.7/0.78 microM; and atracurium, 12.3/1.9 microM. Succinylcholine was three times as potent in newborn rats, with an EC50adult/EC50newborn of 21.3/7.3 microM. The ratio for succinylcholine was significantly less than for all nondepolarizing drugs (P < 0.02). Conclusion The newborn neuromuscular junction of the rat shows an increased sensitivity to all neuromuscular blocking agents tested, including succinylcholine. However, the potency ratio was greater for nondepolarizing than depolarizing drugs. The optimal dose of these agents for certain situations such as cesarean section and anesthesia in neonates should be reassessed.

Background staining in immunoblot assays reduction of signal caused by cross-reactivity with blocking agents

1993 ◽  
Vol 158 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Wendy Y. Craig ◽  
Sue E. Poulin ◽  
Marilyn F. Collins ◽  
Thomas B. Ledue ◽  
Robert F. Ritchie
Keyword(s):  
Cross Reactivity ◽  
Blocking Agents ◽  
Background Staining

Effect of thiol blocking agents on the binding of T3 and T4 in rat liver nuclear extract

1981 ◽  
Vol 98 (1) ◽  
pp. 68-72 ◽  
Author(s):  
J. Knopp ◽  
J. Brtko
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Nuclear Extract ◽  
Blocking Agent ◽  
Binding Affinities ◽  
Structural Configuration ◽  
Blocking Agents ◽  
Nuclear Extracts ◽  
Liver Nuclei ◽  
Apparent Association

Abstract. Liver nuclei bind thyroid hormones (T3 and T4) with different binding affinities. In our studies it was estimated that the apparent association constant (Ka) in the liver nuclei for T3 was 8.8 × 1010 l mol−1 and for T4 was 4.1 × 109 l mol−1. We have found that the binding sites for T4 are also saturated by an excess of unlabelled T4 and that saturation was dependent on the purification step of liver binding proteins. In liver nuclear extracts with 0.4 mol l−1 KCl the specific binding of T3 and T4 was blocked with a typical -SH blocking agent N-ethylmaleinimide (NEM) and by new types of -SH blocking agents such as p-bromphenylisothiocyanate (p-BPI) and 2,3 dicyano-1,4-dithio-9,10 antrachinone (Delan). NEM and p-BPI increased the non-specific binding of T4 and completely abolished specific binding. All these agents blocked the specific binding of T3. These results demonstrate that T3 and T4 binding sites in the liver nuclei may not be altogether identical and that the different effects of -SH blocking agents on the binding of T3 and T4 is probably associated with the structural configuration of these drugs.

Generalized Convulsions in a Patient Receiving Ultrashort-Acting Beta-Blocker Infusion

1988 ◽  
Vol 22 (6) ◽  
pp. 484-485 ◽  
Author(s):  
Gopal Das ◽  
Janine C. Ferris
Keyword(s):  
Heart Disease ◽  
Beta Blocker ◽  
Blocking Agent ◽  
Literature Survey ◽  
Receptor Blocking ◽  
Blocking Agents ◽  
Beta Receptor ◽  
Generalized Seizures ◽  
Beta Blocking ◽  
Therapeutic Doses

Beta-receptor blocking agents are commonly used to treat patients with heart disease, and generalized seizures due to therapy with these agents are rare. All reported cases of seizures due to beta blocking agents have occurred only in those subjects who ingested large doses of the drugs. We observed generalized convulsions in a patient who was receiving therapeutic doses of an ultrashort-acting beta-blocking agent (esmolol hydrochloride) intravenously. A literature survey and possible mechanisms by which these agents induce seizures are presented.

scholarly journals Comparison of Different Double Immunostaining Protocols for Paraffin Embedded Liver Tissue

1999 ◽  
Vol 18 (4) ◽  
pp. 227-233 ◽  
Author(s):  
Alexander Schütz ◽  
Andrea Tannapfel ◽  
Christian Wittekind
Keyword(s):  
Alkaline Phosphatase ◽  
Liver Tissue ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Time Cost ◽  
Double Immunostaining ◽  
Intracellular Protein ◽  
Background Staining ◽  
Fresh Frozen ◽  
Sensitivity Specificity

Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue‐specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non‐specific background staining in pathological liver specimens. We compared peroxidase–anti‐peroxidase, alkaline phosphatase–anti‐alkaline phosphatase (PAP/APAP), labelled‐avidin–biotin (LAB/LAB) and digoxigenin–anti‐digoxigenin (dig–a‐dig/PAP) techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP‐technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig–a‐dig/PAP protocol. In contrast to the dig–a‐dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.

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