Biomedical subjects
Publications and source records attributed to R Lindmark.
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.
Staphylococcus aureus Cowan I containing protein A were rapidly fixed in hot 5% (w/v) trichloroacetic acid (TCA) for 6 min in order to remove the negatively charged cell wall polymer teichoic acid. This resulted in a stable IgG adsorbent able to bind 1.4 mg of human IgG/ml 10% (v/v) suspension for at least 5 months. This IgG binding ability is 25% less than for formalin-fixed bacteria, which were stable for at least 1 year. Preincubation of the bacterial adsorbents in 0.5% (v/v) Tween 20 after fixation prolonged stability and rendered prewashes prior to use unnecessary. IgG could be quantitatively eluted from TCA-fixed bacteria at pH 3.0, but not from formalin-fixed bacteria unless 80 mM MgCl2 was included in the acid buffer. This is explained by ionic interaction between IgG and teichoic acid at the bacterial surface at low pH. Non-specific binding of different proteins to the bacterial adsorbents was also studied and buffers for reducing this effect are suggested.
Explore the source record for details and available documents.
The secondary structure of protein A(SpA) was estimated to 31% alpha-helix, 13% beta-structure and 56% random coil by CD-spectroscopy, which is close to the sum of the secondary structures of the SpA fragments. This supports that the SpA molecule is composed N-terminally of four Fc-binding units each consisting of two antiparallel alpha-helices interconnected by random coil segments and C-terminally of an region mainly in random coil.
A technique was developed for the identification and separation of cells according to their surface antigenic markers, based on immobilization of protein A by entrapment in polyacrylamide microbeads. This method is further development of the previous techniques with SpA-containing S. aureus bacteria, but has the advantage of having less unspecific binding to the cells. We show that this technique might be a useful tool for the separation of cells, exemplified by separation of cells on Petri dishes coated with a monolayer of SpA-microbeads. The viability of separated cells was unaffected and adherent cells were easily removed by trypsin.
Radioiodinated protein A (SpA) from Staphylococcus aureus was used to quantitate specific antibodies of the IgG class at the nanogram level in rabbit serum by a standardized radioimmunoassay with the antigen covalently bound to paper discs. Quantitation of IgG in micrograms of IgG per millilitre of serum was done by using a standard curve with purified anti-bovine serum albumin (BSA) IgG or by means of the calculated molar SpA to rabbit IgG ratio of 1:1, thus converting counts per minute of 125I-SpA to nanograms of IgG. The molar ratio for SpA to human IgG was also 1:1. The unspecific binding of IgG to the paper discs was studied and could be depressed by hen serum or by adsorption of the serum sample to cellulose before the assay. The affinity constants of the extracellular SpA and human, rabbit and guinea-pig IgG, respectively, were determined and found to be close to the values previously calculated for cell-wall-bound SpA. The applicability of the assay was demonstrated by quantitation of the specific IgG response on different days after immunization of rabbits with BSA and 3,4-dinitrobenzene (DNP)-BSA.
Protein A has been isolated from a methicillin-resistant strain of Staphylococcus aureus, A676, which produces only extracellular protein A. The material is homogeneous after a one-step separation and has a molecular weight of 41000. Its physicochemical and immunochemical properties have been studied.