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 An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.

1987 ◽  
Vol 35 (11) ◽  
pp. 1199-1204 ◽  
Author(s):  
C M van der Loos ◽  
P K Das ◽  
H J Houthoff
Keyword(s):  
Monoclonal Antibodies ◽  
Staining Method ◽  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Reaction Products ◽  
Tissue Sections ◽  
Beta Galactosidase ◽  
Immunohistological Analysis ◽  
Triple Staining ◽  
Avidin Biotin Complex

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

2012 ◽  
Vol 178 (3-4) ◽  
pp. 357-365 ◽  
Author(s):  
Wenqiang Lai ◽  
Junyang Zhuang ◽  
Juan Tang ◽  
Guonan Chen ◽  
Dianping Tang
Keyword(s):  
Simultaneous Detection ◽  
One Step ◽  
Electrochemical Immunosensing

One-step automated alkaline phosphatase analysis

1966 ◽  
Vol 14 (2) ◽  
pp. 263-269 ◽  
Author(s):  
Darlene Comfort ◽  
D.J. Campbell
Keyword(s):  
Alkaline Phosphatase ◽  
One Step

scholarly journals A simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections

Microscopy ◽  
2018 ◽  
Vol 67 (6) ◽  
pp. 331-344 ◽  
Author(s):  
Shunichi Morikawa ◽  
Azusa Sato ◽  
Taichi Ezaki
Keyword(s):  
Staining Method ◽  
Tissue Sections ◽  
One Step

A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites

2018 ◽  
Vol 118 (1) ◽  
pp. 191-201 ◽  
Author(s):  
Arif Ciloglu ◽  
Vincenzo A. Ellis ◽  
Rasa Bernotienė ◽  
Gediminas Valkiūnas ◽  
Staffan Bensch
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Haemosporidian Parasites ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
One Step

scholarly journals One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins.

1991 ◽  
Vol 39 (12) ◽  
pp. 1719-1723 ◽  
Author(s):  
T Krenács ◽  
H Uda ◽  
S Tanaka
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
B Lymphocytes ◽  
Enzyme Reaction ◽  
Molecular Complexes ◽  
Simultaneous Application ◽  
One Step ◽  
Reticular Cells ◽  
Secondary Antibodies ◽  
Double Immunolabeling

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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