A Comparison of Line Blots, Enzyme-linked Immunosorbent, and RNA-immunoprecipitation Assays of Antisynthetase Antibodies in Serum Samples from 44 Patients
Objective To determine the differences between anti-aminoacyl tRNA synthetase (ARS) antibodies among line blots, enzyme-linked immunosorbent assay (ELISA) anti-ARS tests, and RNA-immunoprecipitation (IP) assays. Methods Sera from patients with confirmed or suspected antisynthetase syndrome (ASS) that were positive for either the anti-ARS test or the line-blot assay were used to perform an RNA-IP assay and ELISA to detect individual anti-ARS antibodies. Results Among the 44 patients, 10 were positive only in line-blot assays, 6 were positive only in the anti-ARS test, and 28 were positive in both assays. We compared the accuracy of these assays against the gold standard RNA-IP assay.
The κ coefficient was 0.23 in the line-blot assay, but this increased to 0.75 when the cut-off was increased from 1+ to 2+. The κ coefficient was 0.73 in the anti-ARS test. The κ coefficient was 0.85 for positivity in both assays. Patients with ASS that was positive in an RNA-IP assay more frequently had mechanic’s hand (62.1% vs. 20%: p=0.031), myositis (51.7 vs. 10%: p=0.028) and more ASS symptoms than those who were positive only in line-blot assays (3.48 vs. 2.2: p=0.019). Conclusions Clinicians need to understand the features of each assay and determine diagnoses by also considering clinical presentations. Diagnoses should not be judged based only on the results of line-blot assays due to the risk of a misdiagnosis from false positives.
A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.
Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub.
The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.
HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions.
The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (Kd), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the Kd of antigen-antibody interactions in solution.
The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the Kd values of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent Kd values. This HiBiT-qIP assay showed a considerable variation in the Kd values among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.
Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus.
BACKGROUND
Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies.
RESULTS
Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result.
CONCLUSIONS
The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.
Immunoprecipitation (IP) Kit |
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| K286-25 | Biovision | each | 502.8 EUR |
Immunoprecipitation Kit (OKRA00051) |
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| OKRA00051 | Aviva Systems Biology | 1 kit | 1248 EUR |
Immunoprecipitation IP Lysis Buffer |
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| 20831004-1 | Bio-WORLD | 50 mL | 37.06 EUR |
Immunoprecipitation IP Lysis Buffer |
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| 20831004-2 | Bio-WORLD | 100 mL | 60.06 EUR |
Immunoprecipitation IP Lysis Buffer |
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| 20831004-3 | Bio-WORLD | 500 mL | 90.69 EUR |
OKRA00051-1U - Immunoprecipitation Kit |
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| OKRA00051-1U | Aviva Systems Biology | 1kit | 790 EUR |
OKRA00051-1KIT - Immunoprecipitation Kit |
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| OKRA00051-1KIT | Aviva Systems Biology | 1Kit | 719 EUR |
Topoisomerase II Immunoprecipitation Kit |
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| TG1035 | TopoGen | >50 analyses | 657.6 EUR |
Control for Immunoprecipitation IgG beads |
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| CIG01-beads | Cytoskeleton | 10 assays | 115.44 EUR |
HiPer® Immunoprecipitation Teaching Kit |
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| HTI016-5PR | EWC Diagnostics | 1 unit | 75.78 EUR |
Immunoprecipitation Kit: Protein A-Agarose |
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| BS688 | Bio Basic | 20Rxn, 20prep | 242.7 EUR |
Chromatin Immunoprecipitation (ChIP) Assay Kit |
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| TBS8050 | Tribioscience | 25 tests | 560 EUR |
Immunoprecipitation Kit: Protein A-Sepharose |
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| BS690 | Bio Basic | 20Rxn, 20prep | 237.48 EUR |
Immunoprecipitation Starter Pack 2 x 2mL - EACH |
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| 17600235 | Scientific Laboratory Supplies | EACH | 851.85 EUR |
EpiQuik Chromatin Immunoprecipitation (ChIP) Kit |
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| P-2002 | EpiGentek |
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EpiQuik Methylated DNA Immunoprecipitation Kit |
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| P-2019 | EpiGentek |
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Control for SUM01 Immunoprecipitation IgG beads |
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| CIG03-beads | Cytoskeleton | 10 assays | 115.44 EUR |
HDAC3 Immunoprecipitation (IP) & Activity Assay Kit |
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| K344-25 | Biovision | each | 901.2 EUR |
HDAC2 Immunoprecipitation (IP) & Activity Assay Kit |
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| K341-25 | Biovision | each | 1045.2 EUR |
HDAC1 Immunoprecipitation (IP) & Activity Assay Kit |
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| K342-25 | Biovision | each | 1038 EUR |
Immunoprecipitation Kit: Protein A/G PLUS-Agarose |
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| BS689 | Bio Basic | 20Rxn, 20prep | 237.48 EUR |
Control for Acetylation Immunoprecipitation IgG beads |
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| CIG02-beads | Cytoskeleton | 10 assays | 115.44 EUR |
Validation and application of a novel APC antibody in western blotting, immunoprecipitation, and immunohistochemistry.
Adenomatous polyposis coli (APC) is a large protein with multiple binding partners, suggesting diverse functions besides its well-known role in the destruction of β-catenin. To elucidate these complex functions, it is crucial to evaluate the precise subcellular distribution of APC within a cell and tissue. However, most of the commercially available anti-APC antibodies can only be used for limited applications, resulting in the use of independently generated antibodies.
This has led to various discrepancies between studies as a common antibody has not been established. In this study, we generated an antibody against the c-terminal domain of human APC, designated APC-C antibody, and evaluated its specificity and application in various immunological methods. Our data indicate that this novel APC-C antibody is a specific and versatile antibody that can be used in western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. Widespread use of this APC antibody will help enhance our understanding of APC’s function in both normal and cancer cell biology.