Production of an egg yolk antibody against Parietaria judaica 2 allergen
Riccardo AlessandroAlessia GalloMarilisa BarrancaStefania PrincipeSimona TavernaGiovanni DuroGiovanni CassataMichel BecchiSimona FontanaGiacomo De Leo
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Abstract:
Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.Keywords:
Polyclonal antibodies
Yolk
Yolk from the ova of mature hens (ova yolk) was evaluated chemically, physically, and functionally. The ova yolk contained the same proportions of moisture, solids, protein, fat, ash, and cholesterol as conventional egg yolk material. Average yield from birds with recoverable ova was 48.00 g. Mean ovum size was 12.68 g. Mean yield of ova yolk from intact ova was 89.05%. The emulsifying capacity of ova yolk was similar to that of conventional egg yolk. We were unable to demonstrate differences in the chemical, physical, or functional properties of ova yolk due to size of the ovum.
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Egg albumen
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Polyclonal anti-idiotypic antibodies to AIV were prepared from rabbits vaccinated with purified chicken anti-AIV antibodies, and isolated by aldehydated chicken red blood cells to bind unspecific chicken antibodies and to absorb rabbit-anti-chicken antibodies. The acquired antibodies were capable of agglutinating chicken red blood cells and inhibiting AIV in hemagglutinating weekly, and competed original AIV in binding chicken anti-AIV antibodies.
Polyclonal antibodies
Isolation
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Juge-Aubry CE, Liang H, Lang J, Barlow JW, Burger AG. Synthesis and characterization of anti-idiotypic anti-thyroxine antibodies. Eur J Endocrinol 1994;130:107–12. ISSN 0804–4643 We injected rabbits with purified monoclonal murine immunoglobulin (IgG1) or polyclonal anti-thyroxine antibodies (anti-T 4 ) and polyclonal anti-triiodothyroacetic acid (anti-Triac) antibodies to stimulate the production of anti-idiotypic antibodies. Purified immunoglobulins from all five rabbits immunized with monoclonal primary antibodies were able to inhibit the interaction between [ 125 I]T 4 and the primary antibody. The preimmune sera were inactive. This effect was not due to endogenous T 4 contamination or contamination with the injected primary antibody. Half-maximal inhibition of binding of primary antibody with anti-idiotype was between 1.6 and 30 μg of total immunoglobulins. Addition of normal mouse IgG1 did not alter the inhibitory effect of the anti-idiotypic antibody. suggesting that this effect is specific. These anti-idiotypic antibodies reacted differently with different polyclonal antibodies, reflecting the heterogeneous nature of polyclonal antibody populations. Polyclonal antibodies were less effective in stimulating anti-idiotypic antibody production. One polyclonal anti-T 4 and one anti-Triac antibody produced weak anti-idiotypic antibody that had to be used at a concentration of > 600 μg of total immunoglobulins to be inhibitory. Both inhibited the binding of T 4 to the monoclonal anti-T 4 antibody. However, they were ineffective in inhibiting the function of their own antigen, the polyclonal anti-T 4 or anti-Triac antibody. We tested the most potent anti-idiotypic antibodies for their ability to compete with T 4 for other T 4 -binding proteins. Specific inhibition of T 4 binding to thyroid-binding globulin was observed with half-maximal effect at approximately 450 μg of total IgG. The antibody was negative when tested against Transthyretin, rat liver deiodinase type I, triiodothyronine cell uptake and liver cytoplasmic triiodothyronine binding. In conclusion, the technique described herein allows production of anti-idiotypic anti-T 4 , which can be useful in the characterization of the range of iodothyronine-binding sites involved in thyroid hormone action. AG Burger, Unité de la Thyroïde, Hôpital Cantonal Universitaire, 1211 Genève 4, Switzerland
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TRIAC
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OBJECTIVE To prepare high-quanlity polyclonal antibody against chitinase,α-mannosidase and GST. METHODS Corresponding purified proteins with different doses were used to immunize the New Zealand rabbits of different ages. RESULTS When using 0. 3mg·L- 1purified proteins to immunize six-month-old New Zealand rabbit,the polyclonal antibodies possessed higher titer and specificity. CONCLUSION Our results establish a dependable basis for the large-scale production of the related polyclonal antibodies.
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Chitinase
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Purpose: In addition to conventional antibody Ig G1 in serum of camel,there are two subtypes of heavy chain antibody Ig G2 and Ig G3,they have the advantages of small molecular weight and strong stability,but the research and application of camel heavy chain antibody was certainly restricted since lacking of efficient detection techniques. It has some application prospect to prepare the polyclonal antibody that specific for camel heavy chain antibody. Method: In this experiment,camel plasma was obtained by Mararating lymphocytes from the camel whole blood with lymphocytes isolation kit,and then the three subtype antibody of camel was Mararated and purified through Protein G / A Resin column. After three times immunization of rabbit with camel Ig G2,the rabbit polyclonal antibodies were prepared in allusion to heavy chain antibody of camels,and its activity was identified. Result: The result of SDS- PAGE gel electrophoresis showed that camel subtype antibodies were Mararated and purified successfully. Indirect ELISA results showed the titer of polyclonal antibody was 1: 4 096. By combining Indirect ELISA and Western- blot determine the specificity of the antibody,we got that the polyclonal antibody has no any cross reaction with cattle Ig G and camel Ig G1,but has reaction with Ig G3. Conclusion: The experimental results show that we prepared camel heavy chain antibody specific polyclonal antibody successfully,and it will provide strong support for the development and utilization of camel antibody resources,as well as camel disease detection and research.
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Antibody titer
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Objective To express the human recombinant NSPc1 protein and prepare specific polyclonal antibody against it.Methods The recombinant expression plasmid pET43.1a-HIS-NSPc1 was constructed and then transformed into E.coli.(BL21),the recombinant fusion protein HIS-NSPc1 was expressed and purified.Four New Zealand rabbits were immunized with purified recombinant HIS-NSPc1 protein and polyclonal antibody against NSPc1 was prepared.The specificity of the anti-sera was analyzed by Western Blot.Results The purity of the recombinant NSPc1 protein is up to about 95%.Rabbit against NSPc1 antibody was obtained.Western blot result showed that the antibody was able to detect both endogenous and/or exogenous NSPc1.Conclusion The NSPc1 polyclonal antibodies prepared by using recombinant NSPc1 protein as antigen has high specificity to NSPc1.It can be used for functional analysis of NSPc1 in vivo and in vitro.
Polyclonal antibodies
Myc-tag
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Yolks from fresh eggs produced by Broad Breasted White turkeys were blended and analyzed. Proximate composition was determined and components were separated in 7% polyacrylamide gel. Turkey egg yolk was compared with chicken egg yolk. Turkey egg yolk contained a higher percentage of solids, fat, and protein than chicken egg yolk. When separated electrophoretically, components of chicken egg yolk migrated faster than those in turkey egg yolk. Turkey yolks could be resolved into 10 or 11 distinct components; chicken yolk, into 11 or 12 components, depending on electrophoretic technique.
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Previously we quantitated plasma Abeta40 and Abeta42 levels using a combination of mouse monoclonal antibody 6E10 as a capture antibody and rabbit polyclonal antibodies specific to Abeta as detection antibodies. Recently, we have produced rabbit monoclonal antibodies (Rabmab) to Abeta40 and Abeta42. Currently there is a great interest in the measurement of plasma Abeta levels at intervals in AD and elderly non-demented controls. Reports showed conflicting data and the reason is not known. We hypothesize that differences in antibody epitopes and affinity may have contributed to the variable findings. We examined 40 AD or control plasma samples using 1) a combination of Rabmab to Abeta as capture antibodies and 4G8 mouse monoclonal antibody as detecting antibody; and 2) mouse monoclonal antibody 6E10 as a capture antibody and Rabmab to Abeta as detecting antibodies in sandwich ELISA. There was a significant relation between Abeta40 levels of Rabmab to Abeta40 and rabbit polyclonal antibody to Abeta40 using 4G8 as detecting antibody (r = .67; p < 0.001), and Rabmab to Abeta42 and rabbit polyclonal antibody to Abeta42 using 4G8 as detecting antibody (r = .97; p < 0.001). Similarly there was a relation between Abeta levels using mouse monoclonal antibody 6E10 as capture antibody and Rabmab or polyclonal antibody as detecting antibodies. When the relationship between the levels using 4G8 and 6E10 were compared, there was a significant relation in Abeta40 levels (r = .63; p < .001) but not in Abeta42 levels (r = .19; p < .24). The data show that differences in capture or detecting antibodies may contribute to different findings among published studies.
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Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.
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Myc-tag
Immunofluorescence
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In order to get insights into the structural and molecular interaction mechanisms between PPIase and PP1, the recombinant plasmid pGEX-5X-1-Fpr3 was constructed and overexpressed in E. coli as a soluble protein. The fusion protein was purified by GSTrap affinity chromatography and the GST tag was removed by the method of on-column cleavage with Factor Xa. The recombinant GST-Fpr3 and Fpr3 was assayed and identified by SDS-PAGE and Western blot, respectively. The recombinant GST-Fpr3 was used to immunize rabbits for preparing polyclonal antibody. The polyclonal antibody with high titer and high specificity against the GST-Fpr3 has been successfully prepared.
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Myc-tag
Protein purification
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