Purification of duck immunoglobulins: an evaluation of protein A and protein G affinity chromatography (original) (raw)
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Protein A Reactivity of Various Mammalian Immunoglobulins
Scandinavian Journal of Immunology, 1978
Serum samples and immunoglobulin fraciions of eight mammalian species were applied to a Sepharose-protein A column. As with the human immunoglobulin subclasses IgGl, lgG2 and IgG4, all examined animal IgG classes and subclasses were bound to a greater or lesser extent lo protein A. However, the binding of IgGl of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reaciive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.
Differential binding properties of protein A and protein G for dog immunoglobulins
Journal of Immunological Methods, 1991
We studicd the binding of dog immunoglobulins G, A, M and E to protein A and protein G. Passivc cutaneous anaphylaxis (PCA) testing was used for the measurement of dog IgE and enzyme-linked immunosorbent assays (ELISA) were used for the measurements of dog IgG, IgA and IgM. Protein A from lyophilized cells of Staphylococcus aureus bound 97% of lgE,987o of IgG, SIVo of IgA, and 9Jo/o of IgM. Protein A-Sepharose CL-48 bound SlVo of lgF,, l00Vo of IgG and IgA, and 98Vo of IgM. In a stepwise elution with varying pH, a small amount of IgE was eluted at pH 5 and pH 6 and all the rcmaining lgs were eluted at pH 3 from the protein A column. In contrast to protein A, dog IgE was not bound to Protein G-Sepharose, while r007o of lgG,95o/o of IgA, and 447o of IgM were bound to Protein G-Sepharose.
The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera). These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.
Toxicon, 2012
We used high sensitivity and resolution fluorescence microscopy to study the interaction of ostrich IgY, horse F(ab') 2 and horse IgG with mice lymphocyte and erythrocyte plasma membrane. The immunoglobulins were labeled with fluorescin isotiocyanate (FITC). Our results show an interaction of IgY with lymphocyte plasma membrane which does not result in endocytosis of the labeled protein. Less IgG and its F(ab') 2 fraction bind to lymphocytes, and this binding seems to be followed by endocytosis producing a diffuse cytoplasmic fluorescence in most lymphocytes exposed to FITC-IgG or FITC-F(ab') 2 . Cytoplasmic fluorescence resembling FITC was not observed in lymphocytes exposed to FITC-IgY. Receptors in the erythrocyte membrane also differentiate between avian and horse Ig; while erythrocytes exposed to horse Igs became intensely fluorescent for at least 5 h, no erythrocyte labeling occurred when FITC-IgY was used. Our results suggest that IgY may be a stronger activator of adaptive immunity than horse IgG in mammals. Adaptive immunity against IgY is detrimental to its IV therapeutic use in humans and other mammals.
Binding of immunoglobulin G to peripheral blood lymphocytes
Immunology Letters, 1988
The specific and saturable binding of FITC conjugate of aggregated goat IgG to goat peripheral blood lymphocytes was studied in PBS containing 1% BSA. The polar nature of the specific interaction of heterologous aggregated IgG, IgG monomer and its fragment F(ab2') with the cells was studied by ELISA using the peroxidase conjugated ¥{3b£t of anti-human IgG under different conditions of pH and ionic strength.
Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera
Journal of Immunological Methods, 1983
The use of protein A from S. aureus (SPA) as an anti-IgG reagent in immunological techniques has extended in recent years, together with knowledge about its interaction with immunoglobulins of different species. Current data with respect to the binding of protein A to immunoglobulins and to the levels of immunoglobulins in the sera of some mammalian species are reviewed.
APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY
Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are nowadays used in biotechnology for affinity sorbents production. In the work comparative characteristics of proteins A, G, L, affinity sorbents on the basis of them, advantages and disadvantages of these proteins and their application as ligands in the affinity chromatography were conducted.
Studies on the Interaction between Protein A and Immunoglobulin G
The Journal of Immunology
Staphylococcal protein A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1̄. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but w...
Binding of immunoglobulins and immune complexes to erythrocytes of vertebrates
Immunochemistry, 1978
Previous mvestigations which have referred to red cell Fc receptor m rabbit. guinea pig. sheep and man. have been extended to other vertebrates. such as reptiles. amphihia. birds and mammalians. In these investigations. red cells from different animal spectes have been analyzed. As ligand DNP-BSA anti-DNP non-precipitating antibody, aggregated IgG by the bis-diazotized henzidine method. IgM 7s and non-modified IgG. IgA and IgM were used. The ligand binding to red cells was detected by Coomhs test with specific anti-immunoglohulin serum. The analyzed red cells showed exposed Fc receptor. In the case of human red cells. to render such receptor evident it is necessary to submit the cells to trypsinization. All the analyzed erythrocytes hind IgM 7s. Ab-Ag complex and aggregated IgG. and some of these cells hind non-modified immunoglobulins. * Members of the Scientific Researcher Career. National Research Council of Argentina. MATERIALS AND METHODS t+~~rhroc~~~lc~.s o/' tliffiwnt nnimul ~spccics These were obtained from heparinized blood. The cells were washed 5 times with 0. I5 M NaCI. 0.01 M phosphate, pH 7.6. phosphate buffered saline (PBS) before using. These were obtained by the Morton and Pickles (1947) method. The trypsin treatment time was carried out for 10 min. Antigen-antihocij~ wntple.ws ! Ag-Ah J These were prepared by mixing 2 mg ml of antibody w'ith 250 ng,ml of antigen.
Separation and reactivity of avian immunoglobulin Y
Immunoglobulin Y (IgY) is the mayor protein present in the avian egg yolk. This antibody fulfils important functions in the protection of the embryo against several challenging stimuli. Separation of IgY from the egg yolk of several birds was carried out by the Polson method. Their capacity to react with immunoglobulin-binding bacterial protein: protein A, L or LA was investigated. The cross-reactivity of an anti-chicken-IgY-HRP conjugate with different avian IgY was tested by ELISA. The results showed that protein L reacts with bantam hem IgY; and proteins A and LA react with ostrich, bantam hen or duck IgYs. These findings are important for the development of methods of detection and purification of avian IgY proteins.