Bioactive flax cyclic peptides (orbitides and linusorbs) were site-specifically ligated through methionine with bovine serum albumin (BSA) to produce immunogenic compounds. In this study, modified flaxseed immunosuppressant orbitides (linusorbs or LOs) containing hydroxyl (OH) groups were synthesized for use as haptens.
These compounds were extensively characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, high-performance liquid chromatography-tandem mass spectrometry, and Fourier transform infrared spectroscopy.
The haptens were conjugated to BSA, and the extent of hapten incorporation was determined by matrix-assisted laser desorption and ionization, liquid chromatography-electrospray ionization-mass spectrometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The BSA hapten complexes were used to elicit polyclonal antibody (pAbs) production in rabbits.
A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed that used orbitide-specific pAbs and horseradish peroxidase (HRP) conjugates.
The LO assay detection limit was approximately 0.01 μg/mL (ppm), and thus, ELISA can be used for the detection of LOs in tissue and plant samples.
The pAbs can be used to detect and quantify LOs in flax and flaxseed samples, to verify the presence of LOs in flaxseed containing foods, and for the detection of LOs in tissue samples, wastes, and body fluids of animals fed flaxseed.
Determination of a new antibacterial peptide S-thanatin in rat plasma by an indirected-ELISA.
In this study, antimicrobial peptide S-thanatin (Ts) was chemically synthesized and linked to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) by carbodiimide reagent.
Rabbits were immunized with Ts-KLH and polyclonal antibody against Ts was purified by fractional precipitation of ammonium sulfate, coupled with anion-exchange chromatography.
The purified antibody specifically binding to Ts residues but not BSA molecules was observed by Western-Blot analysis.
Ts-BSA was selected as immobilized antigen and reacted with the residual antibody after the excess of anti-Ts antibody was combined with Ts in the sample. The binding antibody was recognized by HRP–conjugated secondary antibody. Finally, the horseradish peroxidase in the complex could catalyze the TMB substrate, resulting in color development.
The method was evaluated by analysis of linearity, precision and accuracy and successfully applied in the determination of Ts in rat plasma. The data of the pharmacokinetic parameters were also obtained. The proposed ELISA has a great value in routine analysis of Ts for its therapeutic monitoring and pharmacokinetic studies.
Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis.
In this paper, the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin.
Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection.
In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.
Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively.
The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively.
After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration.
The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for the quantitative determination of toxins in seafood.
The preparation of polyclonal antibody against chlordimeform and establishment of detection by indirect competitive ELISA
Objective: Chlordimeform is a chemical pesticide that is highly carcinogenic and toxic. The purpose of this study was to establish an enzyme-linked immunosorbent assay (ELISA) method for the detection of chlordimeform in aquaculture and fish farming.
Methods: Chlordimeform was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) as carrier proteins.
A chlordimeform-BSA conjugate was used as an immunogen, and chlordimeform-OVA was used as a coating antigen.
Chlordimeform-BSA was used to immunize rabbits, and a polyclonal antibody was prepared. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established to detect chlordimeform.
Results: The working range of the established IC-ELISA method for chlordimeform detection was 1-20 ng/mL. The IC50 was 3.126 ng/mL, and the lower limit of detection (LOD) of chlordimeform was 0.637 ng/mL. The recovery of chlordimeform from spiked water samples ranged from 81% to 107%.
Conclusion: An anti-chlordimeform polyclonal antibody was successfully developed, and a novel IC-ELISA was established to detect chlordimeform in aquaculture.
Keywords: Chlordimeform; IC-ELISA; artificial antigen; polyclonal antibody.
A paper-based ELISA for rapid sensitive determination of anaphylaxis-related MRGPRX2 in human peripheral blood
Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis.
Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions.
For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively.
We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody.
Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively.
This strategy improved the quantity, activity, and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood.
Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA while offering similar sensitivity, specificity, and accuracy.
Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.
Generation of Antibodies Targeting Cleavable Cross-Linkers
Chemical cross-linking has become a powerful tool for the analysis of protein structures and interactions by mass spectrometry.
A particular strength of this approach is the ability to investigate native states in vivo, investigating intact organelles, cells, or tissues.
For such applications, the cleavable cross-linkers disuccinimidyl sulfoxide (DSSO) and disuccinimidyl butyric urea (DSBU) are gaining increasing popularity, as they allow for the analysis of complex mixtures.
It is inherently difficult to follow the reaction of cross-linkers with proteins in intact biological structures, stalling the optimization of in vivo cross-linking experiments.
We generated polyclonal antibodies targeting DSSO- and DSBU-modified proteins, by injection of cross-linked bovine serum albumin (BSA) in rabbits. We show that the cross-linker-modified BSA successfully triggered an immune response and that DSSO- and DSBU-specific antibodies were generated by the animals.
Rabbit Serum Albumin Polyclonal Antibody, HRP Conjugated |
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| A57593 | EpiGentek |
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Rabbit anti-dog Serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1495509-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-dog Serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1495509-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-dog Serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1495509-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715383-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715383-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715383-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Rat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715414-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Rat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715414-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Rat Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715414-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Bovine serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1497211-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Bovine serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1497211-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Bovine serum albumin polyclonal Antibody, HRP conjugated |
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| MBS1497211-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715561-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715561-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715561-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Pigeon Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715094-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Pigeon Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715094-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Pigeon Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715094-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Human Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715283-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Human Serum Albumin polyclonal Antibody, HRP conjugated |
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| MBS715283-01mg | MyBiosource | 0.1mg | 270 EUR |
Using affinity-purified antibodies specific for the individual cross-linkers, we demonstrate their application to the detection of cross-linker-modified proteins in Western blot and immunocytochemistry experiments of intact and permeabilized cells.
Furthermore, we show their ability to immunoprecipitate DSSO/DSBU-modified proteins and provide evidence for their affinity toward water-quenched dead-links. These antibodies provide a valuable tool for the investigation of proteins modified with the cross-linkers DSSO and DSBU.