Concentration Of Primary Antibody Research Articles

OPTIMIZATION AND VALIDATION OF FOXP3, BRAFV600E AND MMR IMMUNOHISTOCHEMISTRY STAINING PROTOCOLS

To ensure consistent and repeatable results, t Here, we described could enhance the IHC signals of the targeted antibodies. Mismatch Repair (MMR) proteins such as MutL homolog 1 (MLH1), Mut S homolog 2 (MSH2), Mut S homolog 6 (MSH6), and postmeiotic segregation 2 (PMS2), including B-rapidly accelerated fibrosarcoma with V600E mutation (BRAFV600E) protein and forkhead box P3 (FOXP3) protein, are important for colorectal cancer diagnosis. The IHC staining was performed using different archival tissue controls and commercial antibodies. MLH1 and PMS2 showed higher staining intensity after an overnight incubation at a higher concentration of primary antibody (1:50) compared to BRAFV600E (1:100). The enhanced MSH2 signal was likewise generated at a one-hour incubation period with an equivalent antibody concentration. However, even with a shorter incubation duration of one hour, MSH6 and FOXP3 generated good IHC signals when incubated with primary antibody at a lower antibody concentration of 1:100 and 1:300, respectively. The addition of a primary antibody linker improved the IHC signals for all targeted proteins. In conclusion, when using archival tissues, modifying the aforementioned IHC staining variables produces optimal staining for the MMR, BRAFV600E, and FOXP3 proteins.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.Graphical

Development of a Multiplex HIV/TB Diagnostic Assay Based on the Microarray Technology.

Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen-antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.

Development of a Competitive Enzyme Immunoassay Technique for the Detection of Peanut Traces in Gluten-free Products

The aim of this work was to develop a competititve enzymeimmunoassay technique, to detect the presence of traces of peanut in gluten-free products. Specific rabbit polyclonal antiserum against peanut was used as primary antibody. The optimal antigen concentration to be immobilized on the plate and the concentration of primary antibody to be used in competition was determined. The calibration curve was fitted using increasing concentrations of an extract of peanut product. The peanut product was extracted with Tris-HCl buffer 0.0625M with 3% sodium dodecylsulfate (SDS) and 2% sulphite (S) 0,1 M. All validation parameters studied were appropriate. Commercial samples of gluten-free products were analysed with this enzyme immunoassays and a commercial ELISA kit. Significant differences were observed in the quantitative results obtained with both methods; nevertheless the developed enzyme immunoassay could be used as screening method.

Immunodetection of Furcraea Necrotic Streak Virus-FNSV in fique plants (Furcraea macrophylla Baker) using a polyclonal antibody IgY produced in chicken egg yolk

The necrotic streak of the fique (Furcraea spp.) or “Macana” disease is considered the most limiting disease for this crop in Colombia, whose causal agent is the Furcraea Necrotic Streak Virus - FNSV (RNA+). Currently, there are no strategies to control the disease, being necessary to develop methods for detection of this pathogen in the planting material before being taken to the field. In this study, polyclonal antibodies produced in egg yolk (IgY) were produced and assesses for detection FNSV. Two immunoenzymatic methodologies were standardized: dot blot immunobinding assay (DBIA) and enzyme-linked immunosorbent assay (ELISA), determining their specificity and sensitivity. The detection limit by DBIA corresponded to 8 μg/mL of purified virus suspension using 10 μg/mL of primary antibody. In the ELISA test, the primary antibody concentration of 3 μg/mL (1:800 dilution) detected the antigen at concentrations between 10 and 70 μg/mL. The polyclonal antibody anti-FNSV IgY allowed the detection of FNSV in samples of purified virus and extracts of roots and leaves of fique plants with symptoms of “Macana” disease and did not produce any signal with the control samples. Results showed the potential of using egg yolk IgY in immunological tests for the detection of FNSV in fique plants.

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections.

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:

Abstract PS1-04: Utility of rapid- immunohistochemistry using an alternating current electric field for intraoperative diagnosis of sentinel lymph nodes in breast cancer

Abstract Axillary lymph node status and pathological diagnosis of sentinel lymph nodes (SLNs) is an important factor that influences management of postoperative therapy. Recent reports indicate that one-step nucleic acid amplification (OSNA) and hematoxylin and eosin (HE)-stained frozen sections are effective for intraoperative diagnosis of SLNs. In the present study, we report a rapid-immunohistochemical staining (R-IHC) method that enables intraoperative detection of SLN metastases within 16 min using an anti-cytokeratin antibody. With this R-IHC system, we apply a high-voltage, low-frequency AC electric field to lymph node sections while they are incubating with the antibodies. The antibodies are mixed within microdroplets and the opportunity for contact between the antibody and antigen is increased. This greatly reduces the time required for the antigen-antibody reaction. This is the report on SLN diagnosis using R-IHC in patients with breast cancer. We prospectively examined 632 dissected SLNs from 260 breast cancer patients who underwent surgery at our institute between July 2014 and March 2020. The dissected SLNs were sectioned and conventionally stained with HE or immunohistochemically labeled with anti-cytokeratin antibody using R-IHC procedures. Intraoperative R-IHC analyses were completed within 16 min, after which diagnoses were made by two pathologists. The total time required for intraoperative diagnosis was about 20 min. In this study, R-IHC detected four metastatic SLNs that were undetected using conventional HE staining (10/52, 19.2%). Compared with subsequent permanent diagnosis, R-IHC offered 98.0% sensitivity, 100% specificity and 99.8% accuracy while those values for intraoperative HE were 80.4%, 100%, 98.4%, respectively (isolated tumor cells were counted as negative). These findings indicate R-IHC is a clinically applicable technique that enables precise, and quick intraoperative detection of micro- and macrometastases in breast cancer. Moreover, R-IHC is cost-effective method: it’s cost is less than a quarter of OSNA and previous report showed that the concentration of primary antibody could be reduced by more than 90% by using R-IHC because AC mixing activated antigen-antibody reaction. Another benefit is its ability to shorten the time needed for microscopic diagnosis itself. When using R-IHC, pathologists are able to readily find metastatic lesions, even within low-power fields (e.g. 40x). This reduces not only the time needed for intraoperative diagnosis, but also the effort necessary to find unclear lesions like small cancer cells (even though it is macrometastasis), micrometastasis, and artifacts of frozen section. This study was conducted at a single institute. Further investigation in a multi-institutional collaborative prospective study will be needed to confirm the utility of this method. But for now, R-IHC appears to be a cost-effective and clinically applicable method for diagnosis of breast cancer and SLN metastasis. Citation Format: Kaori Terata, Hiroshi Nanjo, Eriko Takahashi, Ryuta Nakamura, Yoichi Akagami, Ayuko Yamaguchi, Misako Yatsuyanagi, Chiaki Kudo, Yoshihiro Minamiya. Utility of rapid- immunohistochemistry using an alternating current electric field for intraoperative diagnosis of sentinel lymph nodes in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS1-04.

Using Immunohistochemistry Without Linkers to Determine the Optimum Concentrations of Primary Antibodies for Immunofluorescence Staining of Formalin-fixed Paraffin-embedded Tissue Sections.

The use of immunofluorescence (IF) technique to detect and evaluate expression levels and localization of cellular proteins and other antigens of interest through the antibodies in their cellular or tissue context has become a standard approach among researchers. Optimizing primary antibody concentrations/dilutions is an essential step in the fluorescent antibody staining protocol. The steps in IF staining are similar to those of the immunohistochemistry (IHC) technique. The use of IHC technique to determine the optimal working dilutions of primary antibodies for IF staining of formalin-fixed paraffin-embedded (FFPE) tissues sections can minimize time wasting and cumbersome approach of using direct IF single labeling using variable dilutions of both primary and secondary antibodies. We used IHC staining technique to determine the working dilutions of the respective primary antibodies by staining 3-µm sections of recommended positive FFPE tissue sections using 3 different dilutions of the primary antibodies and an isotype control (used at the highest concentration). Digital images of sections stained were reviewed in ImageScope by a Consultant Pathologist for positivity, intensity, and histologic distribution. We adopted the IHC predetermined optimal dilutions of primary antibodies to CD4, CD8, CD16, CD21, CD56, CD68, CD163, FOXP3, and PD1 to carry out IF staining of FFPE tissue sections. This approach has helped to remove the complexities associated with grappling with 2 unknown to optimize for both the primary and secondary antibodies using IF technique.

Abstract A030: Systematic optimization of multiplex immunofluorescence of bone marrow eliminates false positives in the detection of disseminated tumor cells

Abstract Approximately 30% of prostate cancer (PCa) patients develop biochemical recurrence after radical prostatectomy or radiation therapy; 40% of these patients develop metastasis. Importantly, 100% of men who die of PCa have cancer in the bone. Our goal is to determine whether we may identify disseminated tumors cells (DTCs) in the bone marrow (BM) of PCa patients. This will allow us to study and characterize the biology of BM-DTCs, allowing us to develop innovative strategies to target these cells before they develop overt lethal metastatic lesions. However, the identification of BM-DTCs has been hampered by strong background staining of non-tumor BM cells in immunofluorescence (IF) assays. This prevalent false positive staining calls into question the true identity of what had previously been classified as DTCs. The purpose of this study was to rigorously and systematically optimize our multiplex IF staining protocol so as to eliminate the background signal, thereby increasing confidence that positively stained cells are truly BM-DTCs. The IF protocol we employ involves applying BM (spiked with PCa cells as a positive control) onto positively charged agar-coated slides following a red blood cell lysis step. We then apply heat to promote proper seeding of the cells onto the slide, followed by fixation and permeabilization. We then use indirect IF with primary and fluorescently labeled secondary antibodies before staining the nucleus and mounting a coverslip. We primarily stained pan-cytokeratin (CK) to label cancer cells, CD45 to label white blood cells (WBCs), and DAPI to label the nuclei. This base protocol resulted in false positive staining of many WBCs in the fluorescent channel reserved for CK-positivity. In addition, we observed ubiquitous autofluorescent signal throughout the BM, making it difficult to detect true positives. We then went through the protocol step by step to determine the source of the background signal. We assessed fixation time, permeabilization, antigen retrieval, blocking reagents, primary antibody concentration, secondary antibody concentration, nuclear stain, wash reagents, and reagents to eliminate autofluorescence. Each step provided us with information about background signal. After these optimizations, our current novel protocol has eliminated autofluorescence and false positive signal. We now consistently observe high signal-to-noise ratio for CK-positivity in the tumor cells and no CK-positivity in the WBC population. We have also performed this protocol on a small number of PCa patient BM and blood samples and saw no background signal. These data lead us to conclude that we have, for the first time, a multiplex IF protocol that can be used to stain patient BM tissue to identify BM-DTCs. Future studies will determine the true number of BM-DTCs present at different time points during PCa progression, as well as study the biology of these rare cells in relation to development of metastasis and patient prognosis. Citation Format: Haley Axelrod, Kenneth J. Pienta, Kenneth C. Valkenburg. Systematic optimization of multiplex immunofluorescence of bone marrow eliminates false positives in the detection of disseminated tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A030.

High sensitive ELISA for detection of atrazion

To set up ELISA for detection of atrazine with high precision. The reaction condition of indirect-ELISA was optimized, including atrazine-ovalbumin(AT-OVA) concentration and primary antibody concentration, organic solvent, goat anti-rat immunoglobin G-horseradish peroxidase(IgG-HRP) concentration. The actual samples were detected by the ELISA method established in our laboratory. Then the ELISA method was compared with the HPLC. The specification curve of indirect-ELISA was set up after optimization. The relation coefficient R2=0.9958. The limit of detection (LOD) was 1.972 ng/ml. The percent recovery of the actual samples was in range of 80%~120%. The ELISA detection sensitivity was higher than the HPLC in the range of 0 ng/ml~6 ng/ml atrazine. The ELISA to detect atrazine has good specificity and high precision. And it can be applied in testing real atrazine samples replacing of the large-scale instrument.

Optimization and characterization of a biosensor assembly for detection of Salmonella Typhimurium

The performance of biosensors depends directly on the strategies adopted during their development. In this paper, a fast and sensitive biosensor for Salmonella Typhimurium detection was assembled by using optimization studies in separate stages. The pre-treatment assays, biomolecular immobilization (primary antibody and protein A concentrations), and analytical response (hydroquinone and hydrogen peroxide concentrations) were optimized via voltammetric methods. In the biosensor assembly, a gold surface was modified via the self-assembled monolayer technique (SAM) using cysteamine thiol and protein A for immobilization of anti-Salmonella antibody. The analytical response of the biosensor was obtained through the use of a secondary antibody labeled with a peroxidase enzyme, and the signal was evaluated by applying the chronoamperometry technique. The biosensor was characterized by infrared spectroscopy and cyclic voltammetry. Optimization of protein A and primary antibody concentrations enabled higher analytical signals of 7.5 and 75 mg mL−1, respectively, to be achieved. The hydroquinone and H2O2 concentrations selected were 3 and 300 mM, respectively. The biosensor developed attained a very low detection limit of 10 CFU mL−1 and a fast response with a final detection time of 125 min. These results indicate that this biosensor is very promising for the food safety and emergency response applications.

OR45 Proof of principle: Bead-based ABO antibody assessment

Aim ABO antibody (ABO-Ab) titration is performed for ABO-incompatible (ABOi) transplantation management and is traditionally assessed by hemagglutination. However, hemagglutination cannot readily differentiate Ab isotype, nor distinguish between Abs to ABO-subtype antigens, which are expressed differently on organs vs erythrocytes. Our goal was to explore Luminex as a potential platform for the detection of IgG and IgM antibodies to ABO subtype antigens. Methods Phase I: Human blood group antigen subtype A-II was synthesized and conjugated to bovine serum albumin (BSA). BSA-A-II conjugates were coupled to Luminex beads. Coupling was assessed using phycoerythrin (PE)-labeled mouse monoclonal IgG and IgM anti-human ABO-A. Increasing BSA-A-II antigen concentrations were tested to optimize MFI. A Luminex-200 was used for bead acquisition. Phase II: Antigens ABO A-III and A-IV were similarly tested (alone and multiplexed) using additional Luminex beads. Phase III: Dilutions of PE-labeled anti-human IgG conjugate were tested against dilutions of anti-ABO-A plasma (blood group B donor) in a checkerboard titration. BSA-A-II coupled beads were used. Results Bead coupling was optimal at 5ug of BSA-A-II antigen. The coupling of BSA-A-II, -III and -IV was successful as demonstrated by a linear increase in MFI with increasing concentration of primary antibody (Figure). Beads were successfully labeled with monoclonal ABO-Ab when multiplexed. The assay appears most sensitive with the lowest background when patient plasma is diluted 1/25. Conclusions HLA laboratories are uniquely situated to assess ABO antibodies by Luminex as expertise and instrumentation already exist. This novel assay has the potential to be adopted by clinical HLA laboratories for rapid, specific, and sensitive assessment of IgM/IgG ABO subtype-specific ABO antibodies in ABOi transplantation. We continue to optimize the assay, include additional controls, and assess additional ABO subtypes. We are also testing flow-based bead platforms. Download : Download high-res image (185KB) Download : Download full-size image

Optimized CLARITY technique detects reduced parvalbumin density in a genetic model of schizophrenia

BackgroundNovel tissue clearing technologies have, for the first time, made it possible to study intact tissue samples. This approach provides a tool for further clarifying findings from animal models of schizophrenia by studying parvalbumin-positive (PV+) interneuron density from a 3D perspective. New methodThis study has developed an optimised CLARITY protocol, including an improved electrophoretic tissue clearing (ETC) chamber, an evaluation of antibody diffusion into cleared tissue slices, and a computational method for detecting PV+ interneurons in 3D. ResultsA reduced PV+ interneuron density was found in both prelimbic and motor cortex regions of the Df(h15q13)/+ mice, while no changes were observed in the Df(h22q11)/+ mice. Comparison with existing methodThe developed ETC chamber enables tissue clearing of variable tissue sizes while minimizing the resistance. It was found that a high concentration of primary and secondary antibodies were necessary for sufficient antibody staining of PV+ interneurons. Additionally, the developed computational method showed improved detection rates of interneurons compared to non-processed image stacks. ConclusionOur optimization of the CLARITY technology and automated 3D counting of cells were found to be useful for quantification of PV+ interneuron density. The results may provide insight into understanding the pathophysiology underlying the phenotype observed in Df(h15q13)/+ mice.

Proof of the quantitative potential of immunofluorescence by mass spectrometry

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2=0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

Development of a QCM-D biosensor for Ochratoxin A detection in red wine

Ochratoxin A (OTA), a highly toxic compound, is one of the most widely spread mycotoxins that contaminates a large variety of agricultural commodities. Due to its presence in the food chain, it imposes a hazard on both human and animal health. Therefore, there is a need for precise, fast and simple methods for toxin quantification. Herein, a novel sensor based on a quartz crystal microbalance with dissipation monitoring (QCM-D) and antibodies for specific analyte recognition was developed for rapid and sensitive detection of OTA in red wine.The combination of indirect competitive assay with QCM-D gives a straightforward device, which can simultaneously measure frequency (Δf) and dissipation (ΔD) changes resulting in detailed information about the mass attached to the sensor surface as well as conformational changes, viscoelastic properties and the hydration state of the film.Small molecules (such as OTA) suffer from poor LOD due to the high concentration of primary antibody needed to generate adequate signal. In the present study, amplification of the QCM-D signal was obtained by applying secondary antibodies conjugated with gold nanoparticles (AuNPs).Thanks to this, a linear detection range of 0.2–40ngmL−1 has been achieved with an excellent LOD of 0.16ngmL−1, which is one order of magnitude lower than LOD specified by European Union legislation concerning the limit of OTA in food.Moreover, a matrix effect (caused by the occurrence of polyphenols in wine) and associated non-specific interactions with the sensor surface was completely eliminated by a simple pre-treatment of the wine with the addition of 3% poly(vinylpyrrolidone) (PVP).

Development of a Competitive Enzyme Immunoassay Technique for the Detection of Soy Traces in Meat Products

The aim of this work was to develop a competitive enzyme immunoassay technique, to detect the presence of traces of soy in meat products. Specific rabbit polyclonal antiserum against soy protein was used as primary antibody. The optimal antigen concentration to be immobilized on the plate and the concentration of primary antibody to be used in competition was determined. The calibration curve was fitted using increasing concentrations of an extract of soy product. The soy product was extracted with Tris-HCl buffer 0.0625M with 3% sodium dodecylsulfate and 2% mercaptoethanol. The working range used in the enzyme immunoassay to detect soy was 9-280ppm SP with adequate linearity (R2: 0.9880). All validation parameters studied were appropriate. Commercial samples of meat products were analyzed with this enzyme immunoassays and a commercial ELISA kit. Significant differences were observed in the quantitative results obtained with both methods; nevertheless the developed enzyme immunoassay could be used as screening method.

High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP.

A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.

A flow-cytometry-based method for detecting simultaneously five allergens in a complex food matrix.

To avoid carry-over contamination with allergens, food manufacturers implement quality control strategies relying primarily on detection of allergenic proteins by ELISA. Although sensitive and specific, this method allowed detection of only one allergen per analysis and effective control policies were thus based on multiplying the number of tests done in order to cover the whole range of allergens. We present in this work an immunoassay for the simultaneous detection of milk, egg, peanut, mustard and crustaceans in cookies samples. The method was based on a combination of flow cytometry with competitive ELISA where microbeads were used as sorbent surface. The test was able to detect the presence of the five allergens with median inhibitory concentrations (IC50) ranging from 2.5 to 15mg/kg according to the allergen to be detected. The lowest concentrations of contaminants inducing a significant difference of signal between non-contaminated controls and test samples were 2mg/kg of peanut, 5mg/kg of crustaceans, 5mg/kg of milk, 5mg/kg of mustard and 10mg/kg of egg. Assay sensitivity was influenced by the concentration of primary antibodies added to the sample extract for the competition and by the concentration of allergenic proteins bound to the surface of the microbeads.

The Importance of Titrating Antibodies for Immunocytochemical Methods.

When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. © 2016 by John Wiley & Sons, Inc.

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.