23. Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre

Harboe, Niels; Ingild, A.

doi:10.1111/j.1365-3083.1973.tb03798.x

Cited by 803 publications

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“…Further injections of antigen without adjuvant were given weekly by subcutaneous injection at multiple sites. Subsequently, sera from four consecutive bleedings were pooled, and immunoglobulins were partially purified by precipitation with ammonium sulfate and dialysis against acetate buffer, pH 5.0 (9), and then concentrated by ultrafiltration to one-tenth of the original serum volume.…”

Section: Materials and Methods Preparation Of Protoplasts And Membranmentioning

confidence: 99%

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Owen¹,

Saltón²

1975

Proc. Natl. Acad. Sci. U.S.A.

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on the surface. Of these antigens the major one has been identified as a succinylated mannan. Five of the principal immunoprecipitates unaffected by absorption with protoplasts were shown by zymograms to possess the following enzymic activities: succinate dehydrogenase (EC 1.3.99.1), ATPase (EC 3.6.1.3), NADH dehyrogenase (EC 1.6.99.3)(two separate components), and malate dehydrogenase (EC 1.1.1.37). These enzymes or enzyme-complexes are, therefore, not expressed on the outer surface of the protoplast membrane.Bacterial membranes perform many of the functions found in separate membranous organelles in the eukaryotic cell. Thus functions of electron transport, of active transport, and the biosynthesis of phospholipids and cell walls all occur in the bacterial membrane (1). In membranes of Gram-positive bacteria two principal regions, the plasma or cytoplasmic membrane and the mesosome, are recognizable (2) and these two entities have been isolated as homogeneous fractions from several species (3). Our investigations have been directed towards the elucidation of the structure-function relationships of the membranes of Micrococcus lysodeikticus by a multidisciplinary approach combining biochemical, immunochemical, and electron microscopic techniques. With conventional immunochemical methods, earlier studies in this laboratory showed the presence of three principal immunoprecipitates when membrane fractions of M. lysodeikticus reacted with membrane antiserum (4). These results did not reflect the anticipated complexity of the membrane, considering the multiplicity of the functions it performs and the number (about 30) of individual polypeptides found by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (5, 6). Methods for the preparation of "total" membranes (i.e., plasma and mesosomal membranes), plasma membranes, and purified mannan from M. lysodeikticus are detailed elsewhere (5,8). Triton X-100 extracts of plasma membranes were obtained by treatment with detergent (final concentrations of 4%, w/v) for 1 hr at 220. Membrane residues (about 20%) sedimentable at 17,500 X g for 45 min were removed and the supernatant fractions (19 mg of protein per ml) were retained for analysis.Preparation of Antisera. Rabbits were immunized with "total" membranes (2 mg) given initially into both footpads and intradermally with Freund's complete adjuvant. Further injections of antigen without adjuvant were given weekly by subcutaneous injection at multiple sites. Subsequently, sera from four consecutive bleedings were pooled, and immunoglobulins were partially purified by precipitation with ammonium sulfate and dialysis against acetate buffer, pH 5.0 (9), and then concentrated by ultrafiltration to one-tenth of the original serum volume.Absorption of Anti-Membrane Serum with Protoplasts. Concentrated immunoglobulins (2.0 ml) were dialyzed against Buffer II (0.8 M sucrose, 10 mM Mg2+, 70 mM NaCl, 50 mM Tris'HCl, pH 8.6) and the final volume was adjusted to 6.0 ml. Aliquots (1.0 ml) were incubated for 1 hr at 22°with 0-3....

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Section: Materials and Methods Preparation Of Protoplasts And Membranmentioning

confidence: 99%

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Owen¹,

Saltón²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…Immunoglobulin G (IgG) fractions were isolated from the pre-immune and immune sera (P2X 1±7 ) following the method of Harboe and Ingild [20]. The protein concentration was determined at 280 nm using an extinction factor of 1.43 for 1 mg/ml.…”

Section: P2x Antibodiesmentioning

confidence: 99%

Age‐related changes in the localization of P2X (nucleotide) receptors in the rat adrenal gland

Afework¹,

Burnstock

2000

Intl J of Devlp Neuroscience

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Observation of the changes in the occurrence and distribution of nucleotide (P2X) receptors in the adrenal gland during development and ageing, and correlation with the changes in adrenal status at similar stages may give morphological insights into the functions of purine nucleotides in the gland. Age-related changes in the localization of all seven subtypes of the P2X receptor in the adrenal gland of rat were therefore investigated immunohistochemically. In the adrenal glands of prenatal rats, immunoreactivity to P2X receptor subtypes was not observed. In glands of the postnatal rat at the developmental stages studied, only immunoreactivity for the P2X 5 receptor subtype was observed. A small number of faintly P2X 5 -immunoreactive chroma n cells were found in the adrenal glands of 1-day-old rats; the frequency of localization and intensity of staining of immunoreactive cells had increased by day 4 and was further increased at day 7. P2X 5 immunoreactivity was not observed in the adrenal glands from 14-and 21-day-old rats. At 8 weeks of age, immunoreactivity with a speci®c distribution for each of the seven receptor subtypes was observed. Except for the P2X 4 receptor, adrenal glands at 24 months showed a similar pattern of immunoreactivity for the receptor subtypes as that observed at 8 weeks. Immunoreactivity for P2X 4 was ®rst observed in the adrenal cortical cells of the zona reticularis at 8 weeks, but was absent in 24-month-old rats. However, several P2X 4 -immunoreactive chroma n cells appeared at 24 months. Such immunoreactive cells were not seen in rats of any of the other ages studied. It was concluded that the greater expression of P2X 5 receptor at an early developmental stage and of P2X 4 in ageing might re¯ect functional roles for purines in cellular proliferation and/or di erentiation, and in cellular degeneration, respectively, in adrenal glands of rat. 7

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“…Immunization with foetal brain was performed on 5 rabbits for more than 1 year, using extracts of 3 foetal brains sequentially. The immunization protocol and the immunoglobulin purification method were those of Harboe and Ingild (1973).…”

mentioning

confidence: 99%

Antigens in human glioblastomas and meningiomas: search for tumour and onco-foetal antigens. Estimation of S-100 and GFA protein

Dittmann¹,

Axelsen²,

Nørgaard‐Pedersen³

et al. 1977

Br J Cancer

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Summary.-Extracts of glioblastomas and meningiomas were analysed by quantitative immunoelectrophoresis for the presence of foetal brain antigens and tumourassociated antigens, and levels of 2 normal brain-specific proteins were also determined. The following antibodies were used: monospecific anti-S-100 (glia specific); monospecific anti-GFA (glial fibrillary acidic protein), (astroglia specific); polyspecific anti -foetal brain (12-16th week of gestation); a polyspecific anti-glioblastoma antiserum, absorbed with insolubilized serum, haemolysate and normal brain extract; polyspecific anti-meningioma antiserum, absorbed as for glioblastoma antiserum; monospecific anti-a-foetoprotein; and monospecific anti-ferritin. Using the antibodies raised against the tumours, several antigens not present in foetal or adult normal brain were found in the glioblastomas and the meningiomas. These antigens cross -reacted with antigens present in normal liver and were therefore not tumour-associated. S-100 was found in glioblastomas in approximately one tenth the amount in whole brain homogenate, whereas GFA was found 2-4 times enriched. The 2 proteins were absent in meningiomas. The possible use of the GFA protein as a marker for astroglial neoplasia is discussed.Five foetal antigens were found in foetal brain, but none in the tumours. a-Foetoprotein could only be demonstrated in foetal tissue extracts, including foetal brain, but not in tumours. Ferritin was detected in all tumour extracts, although the amounts determined were unrelated to histological tumour type.

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23. Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre

Cited by 803 publications

References 0 publications

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

Age‐related changes in the localization of P2X (nucleotide) receptors in the rat adrenal gland

Antigens in human glioblastomas and meningiomas: search for tumour and onco-foetal antigens. Estimation of S-100 and GFA protein

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