A sensitive and specific indirect competitive fluorescence immunoassays (FIA) has been developed for the quantitative determination of dicyclohexyl phthalate (DCHP) using an antigen-coated plate format.
The polyclonal antibodies raised against dicyclohexyl 4-amino phthalate conjugated to bovine serum albumin (BSA) by the amino diazotization linkage method.
Antiserum with a sufficiently high titer was generated in rabbits and fluorescein isothiocyanate (FITC) was used as sensitive labels to construct the fluorescence immunoassay (FIA) for measurement of targeted compounds. Under optimized FIA condition, the quantitative working range was from 0.1 to 200 μg L−1 with a limit of detection of 0.05 μg L−1.
Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%.
Four kinds of water samples (tap water, lake water, river water and leachate) had been detected in this assay, the recovery was 91.3–107.8%.
The proposed fluorescence immunoassay turned out to be a powerful tool for monitoring of dicyclohexyl phthalate in water samples at trace level.
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 dibutyric 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.
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.
Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples
Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions.
H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases.
Therefore, development of a simple and selective H2S measurement method would be advantageous.
This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples.
We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM).
We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively.
The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM.
By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.
Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis
Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte.
In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonalrabbitserum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits.
A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants.
Rabbit Serum Albumin Polyclonal Antibody, FITC Conjugated |
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| A57594 | EpiGentek |
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Rabbit anti-Rat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715050-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Rat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715050-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Rat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715050-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Human Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS1495489-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Human Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS1495489-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Human Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS1495489-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Mouse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715435-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Mouse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715435-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Mouse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715435-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715417-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715417-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Goat Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715417-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715193-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715193-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Horse Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715193-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Sheep Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715235-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Sheep Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715235-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Sheep Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715235-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Chicken Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715101-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Chicken Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715101-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Chicken Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715101-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Canine Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715478-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Canine Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715478-01mg | MyBiosource | 0.1mg | 270 EUR |
Rabbit anti-Canine Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715478-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Rabbit anti-Porcine Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715332-005mg | MyBiosource | 0.05mg | 190 EUR |
Rabbit anti-Porcine Serum Albumin polyclonal Antibody, FITC conjugated |
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| MBS715332-01mg | MyBiosource | 0.1mg | 270 EUR |
When spiked samples (commercial POA alone) were analyzed, the half-maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL).
However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable.
We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.