scholarly journals Immunohistochemical detection of wheat protein in model samples

2010 ◽  
Vol 28 (No. 6) ◽  
pp. 516-519 ◽  
Author(s):  
Z. Řezáčová-Lukášková ◽  
B. Tremlová ◽  
M. Pospiech ◽  
E. Renčová ◽  
Z. Randulová
Keyword(s):  
Polyclonal Antibodies ◽  
Protein Detection ◽  
Immunohistochemical Method ◽  
Complex Method ◽  
Pork Meat ◽  
Immunohistochemical Detection ◽  
Wheat Protein ◽  
Elisa Method ◽  
Immunohistochemical Examination ◽  
Gluten Content

The study focused on the optimisation of immunohistochemical examination for gluten content detection in model samples (pork meat with wheat semi-smooth flour, pork meat with wheat protein edible vital). The best results were achieved with immunohistochemical method based on ABC (avidin–biotin complex) method utilising polyclonal antibodies diluted 1:1000. The results demonstrate that for pure wheat protein detection, the utilisation of immunohistochemical detection, which can detect as little as 0.1% of the added wheat protein, is more advantageous, while the commonly used ELISA method reliably proves this additive approximately from 0.4% upwards.

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 Quantitative immunohistochemical method for detection of wheat protein in model sausage

2014 ◽  
Vol 83 (10) ◽  
pp. S71-S76 ◽  
Author(s):  
Zuzana Řezáčová Lukášková ◽  
Matěj Pospiech ◽  
Bohuslava Tremlová ◽  
Eva Renčová ◽  
Michaela Petrášová
Keyword(s):  
Coeliac Disease ◽  
Immunohistochemical Method ◽  
Quantitative Detection ◽  
Wheat Protein ◽  
Elisa Method ◽  
Quantitative Results ◽  
Immunohistochemical Methods ◽  
Reliable Methods

Since gluten can induce coeliac symptoms in hypersensitive consumers with coeliac disease, it is necessary to label foodstuffs containing it. In order to label foodstuffs, it is essential to find reliable methods to accurately determine the amount of wheat protein in food. The objective of this study was to compare the quantitative detection of wheat protein in model sausages by ELISA and immunohistochemical methods. Immunohistochemistry was combined with stereology to achieve quantitative results. High correlation between addition of wheat protein and compared methods was confirmed. For ELISA method the determined values were r = 0.98, P < 0.01; for stereologythe determined values were r = 0.94, P < 0.01. Although ELISA is an accredited method, it was not reliable, unlike immunohistochemical methods (stereology SD = 3.1).

CD4+CD45RA+ cells (suppressor-inducer T cells) in thyroid tissue from patients with Graves' disease

1991 ◽  
Vol 125 (6) ◽  
pp. 687-693 ◽  
Author(s):  
A. Kawakami ◽  
K. Eguchi ◽  
M. Matsunaga ◽  
H. Tezuka ◽  
Y. Ueki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
Cell Surface ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Thyroid Tissue ◽  
Immunohistochemical Method ◽  
Graves Disease ◽  
Complex Method ◽  
Cell Surface Antigens

Abstract. Previously, we used dual immunofluorescent analysis and showed that the thyroid gland from patients with Graves' disease had a reduced number of CD4+CD45RA+ cells, but an increased number of complementary CD4+CDw29+ cells. An immunohistochemical study, however, produced opposite results; interstitial lymphocytes predominantly expressed the CD45RA+ rather than the CDw29+ phenotype. Because the difference in findings may be due to differences in the techniques used, we did the following experiments: Mononuclear cells were treated with various amounts of collagenase (50-1000 mg/l) which had no effect on the cell surface antigens CD3, CD4 and CD45RA. A dual immunofluoresent study showed that the numbers of CD4+CD45RA+ and CD8+CD45RA+ cell population among CD45RA+ cell population were markedly decreased in the thyroid tissue, and that the CD45RA antigen on the intrathyroidal mononuclear cells was mainly expressed on the CD20+ cells. As the thyroid section had been fixed with acetone before immunohistochemical staining, CD45RA− cells were treated with acetone and stained with anti-CD45RA monoclonal antibody using an avidin-biotin peroxidase complex method. The results of this experiment suggest that there are cell surface molecules which react with anti-CD45RA monoclonal antibody after treatment with acetone in CD45RA− cells. The above findings confirm our previous results which showed that the thyroid glands of patients with Graves' disease have decreased numbers of suppressor-inducer T cells. Also, several problems exist in the detection of CD45RA+ cells when using an immunohistochemical method.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Serotyping Ureaplasma urealyticum Strains Using Monoclonal Antibodies

2001 ◽  
Vol 8 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Fedoua Echahidi ◽  
Gaëtan Muyldermans ◽  
Sabine Lauwers ◽  
Anne Naessens
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Ureaplasma Urealyticum ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cross Reactions ◽  
Antigenic Preparation ◽  
Negative Controls ◽  
Reference Strains

ABSTRACT Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains ofU. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

scholarly journals Immunohistochemical detection of human brown adipose tissue uncoupling protein in an autopsy series.

1993 ◽  
Vol 41 (5) ◽  
pp. 759-764 ◽  
Author(s):  
M L Kortelainen ◽  
G Pelletier ◽  
D Ricquier ◽  
L J Bukowiecki
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Immunohistochemical Method ◽  
Histopathological Diagnosis ◽  
Positive Staining ◽  
Post Mortem ◽  
Immunohistochemical Detection ◽  
Intense Staining ◽  
Brown Adipose

We used an immunohistochemical method for the inner mitochondrial membrane uncoupling protein (UCP) to examine whether human brown adipose tissue UCP could be detected by an anti-rat UCP antibody. Samples of human brown adipose tissue were obtained at medicolegal autopsies. Fat tissue was excised from around the common carotid arteries and in the subscapular region and from around the thoracic aorta. The subjects were either known alcohol consumers, in which thermogenically active brown adipose tissue (BAT) is often found, or victims of sudden infant death syndrome (SID). UCP was detected in all the cases examined, even when the post-mortem time from death to autopsy reached several days, but the intensity of the immunostaining was variable. Intense staining was observed in three cases with a post-mortem time under 24 hr, but in the SID cases a strong positive staining was seen even with a post-mortem delay of 4 days. These results show that an anti-rat UCP antibody can be used for immunohistochemical detection of UCP in human brown adipose tissue and that it provides a useful method for distinguishing between white and brown fat in paraffin-embedded samples. It can be used to detect UCP in the BAT of obese and diabetic individuals and probably also in the histopathological diagnosis of brown adipose tissue lipoma, known as hibernoma.

scholarly journals A method for detecting IL-6 in serum of patients with uremia

2021 ◽  
Vol 292 ◽  
pp. 03081
Author(s):  
Qingbo Bi ◽  
Shih-Mo Yang
Keyword(s):  
Fluorescence Intensity ◽  
Gold Standard ◽  
Sandwich Structure ◽  
Magnetic Beads ◽  
Protein Detection ◽  
Solid Support ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Fluorescent Substrate ◽  
Digital Elisa

As the gold standard of protein detection, enzyme-linked immunosorbent assay (ELISA) is widely used in medical treatment and biology. Here, we report a digital ELISA method that combines fluorescence-coded magnetic beads with micropore arrays to effectively improve the accuracy of the detection. Fluorescence coded magnetic beads were used as solid support of ELISA, which were modified to specifically capture IL-6 in serum, and then combined with galactosidase to form a sandwich structure. These beads are then mixed with a fluorescent substrate and passed into a microfluidic chip. Under the action of gravity, the beads are trapped and isolated by an array of micropores in the chip. Combined with image recognition technology, the fluorescence intensity of micropores containing enzymes will increase rapidly. By mining image information, the IL-6 content in uremia patients can be detected with high precision.

scholarly journals Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

2022 ◽  
Author(s):  
Rasel A. Al-Amin ◽  
Phathutshedzo M. Muthelo ◽  
Eldar Abdurakhmanov ◽  
Cecile Vincke ◽  
Serge Muyldermans ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Protein Detection ◽  
Specific Protein ◽  
Protein Biomarkers ◽  
High Quality ◽  
Affinity Reagents ◽  
Protein Assay ◽  
Enzyme Coupling ◽  
Target Binding ◽  
Dna Strand

High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

Immunohistochemical method of simultaneous detection of neurons and astrocytes in the rat brain

2018 ◽  
Vol 18 (3) ◽  
pp. 46-51
Author(s):  
Yu A Kalinina ◽  
D A Sufieva
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Light Microscopy ◽  
Rat Brain ◽  
Simultaneous Detection ◽  
Immunohistochemical Method ◽  
Nuclear Antigen ◽  
Immunohistochemical Detection ◽  
Pathological Conditions ◽  
Staining Protocol

This study aimed to develop a method of simultaneous immunohistochemical detection of neurons and astrocytes using peroxidase labeling with subsequent analysis by light microscopy. A pair of antigens, each of which specifically identifies either neurons or astrocytes, was chosen (as markers neuronal nuclear antigen and glial fibrillary acidic protein were used, respectively). This method not only identifies the studied cells, but it also assesses their functional state under normal and pathological conditions. The proposed staining protocol allows significant simplification and optimization to simultaneously detect two antigens and to improve the quality of the slices obtained.

scholarly journals Immunohistochemical detection of FGF-23 protein in tumors that cause oncogenic osteomalacia

2003 ◽  
pp. 269-276 ◽  
Author(s):  
T Larsson ◽  
R Zahradnik ◽  
J Lavigne ◽  
O Ljunggren ◽  
H Juppner ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Fibroblast Growth Factor 23 ◽  
Immunohistochemical Analysis ◽  
Current Evidence ◽  
Immunohistochemical Detection ◽  
Mesenchymal Tumors ◽  
Fgf 23

OBJECTIVE: Oncogenic hypophosphatemic osteomalacia (OOM) is a rare disease characterized by hypophosphatemia, inappropriately low levels of circulating 1,25-dihydroxyvitamin D(3) and osteomalacia. The disease is most commonly caused by benign mesenchymal tumors that produce, among several other factors, fibroblast growth factor-23 (FGF-23). Current evidence thus suggests that this protein has an important role in the regulation of phosphate homeostasis. By producing polyclonal antibodies against human FGF-23 protein we wanted to determine the localization of FGF-23 protein in OOM tumors that express FGF-23 mRNA. DESIGN AND METHODS: Three polyclonal antibodies were raised in rabbits against three different peptides with sequences derived from human FGF-23: [Cys-70]FGF-23(51-69)amide, [Tyr-223]FGF-23(206-222)amide and [Tyr-224]FGF-23(225-244)amide. One of the resulting antisera was subsequently used for immunohistochemistry on sections from five different tumors causing OOM. FGF-23 mRNA expression was confirmed with in situ hybridization. RESULTS: After affinity purification, two of three antisera detected recombinant human FGF-23 by Western blot analysis. Through immunohistochemical analysis using the anti-[Tyr-224]FGF-23(225-244)amide antibody and through in situ hybridization using full-length antisense FGF-23 cRNA as a probe, we showed that abundant amounts of FGF-23 protein and mRNA are present in certain tumor cells of five different OOM tumors. CONCLUSIONS: We conclude that OOM tumors express FGF-23 protein and that the immunohistochemical detection of FGF-23 in OOM tumors is feasible and may help in establishing the diagnosis of tumor-induced hypophosphatemia through analysis of biopsies or surgical specimens.

scholarly journals A multiepitope fusion protein-based p-ELISA method for diagnosing bovine and goat brucellosis

2021 ◽  
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Negative Predictive Value ◽  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Simultaneous Detection ◽  
Protein Detection ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Roc Curve Analysis

In recent years, the incidence of brucellosis has increased annually, which has caused tremendous economic losses in agriculture and husbandry in various countries. Therefore, developing rapid, sensitive and specific diagnostic techniques for brucellosis has become critical brucellosis research. Bioinformatics technology was used to predict the B cell epitopes of the main antigen proteins of Brucella, and the validity of each epitope was verified by indirect enzyme-linked immunosorbent assay (iELISA). The verified epitopes were connected in series to construct a multiepitope fusion protein, goat, bovine brucellosis sera, and rabbit sera were collected to verify the antigenicity and specificity of this protein. Then, the fusion protein was used as a diagnostic antigen to construct paper-based ELISA (p-ELISA) technology. A total of 22 effective epitopes were predicted, and a fusion protein was successfully constructed, which showed good antigenicity and specificity. The constructed p-ELISA method was used for the simultaneous detection of bovine and goat brucellosis. ROC curve analysis showed that the sensitivity and specificity of protein detection in goat serum were 98.85% and 98.51%, respectively. The positive and the negative predictive value was 99.29% and 98.15%, respectively. When assessing bovine serum, the sensitivity and specificity were 97.85% and 96.61%, respectively. The positive and the negative predictive value was 98.28% and 97.33%, respectively. This study combined bioinformatics, fusion protein development and p-ELISA technologies to establish a sensitive and specific rapid diagnosis technology for brucellosis that can be used to assess the serum of bovine, goats and other livestock.

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