Biomedical subjects

A Frostell

Publications and source records attributed to A Frostell.

7 recordsLinked to original sources

PubMed1993

Introducing a biosensor based technology for real-time biospecific interaction analysis.

This report describes a system for real-time biospecific interaction analysis, using biosensor technology based on the optical phenomenon surface plasmon resonance. The biospecific interface is a sensor chip consisting of a thin gold film deposited on a glass support and covered with a hydrogel matrix. One component of the interaction being studied is attached covalently to the hydrogel, and other interactants are passed over the chip in solution. The interaction is followed in real time in terms of changes in the mass concentration of biomolecules at the sensor surface. Surface concentrations down to 10 pg/mm2 can be measured. The technique does not require molecular labels such as isotopes or spectroscopic markers, and purification of interacting components can often be avoided. Repeated analyses can be performed on the same sensor chip. With this system, the same general procedure can be used for a wide range of different applications, including concentration determination, kinetic measurements and multi-site binding studies. The sensitivity of the technique can be adjusted by choice of reagents and experimental procedure: determination of specific proteins in serum down to 20 ng/ml and macromolecular association constants from 10(7) M-1 up to 4 x 10(11) M-1 are documentated examples. No other single analytical system has the same versatility and general applicability to biospecific interaction analysis. The system is developed and marketed by Pharmacia Biosensor AB, Sweden.

Biosensing Techniques↗

PubMed1991

Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay.

The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Mitochondrial autoantigens in primary biliary cirrhosis. Association of disease-specific determinants with a subunit of complex I (NADH-ubiquinone reductase) of the inner mitochondrial membrane.

Anti-mitochondrial autoantibodies (AMA) from patients with primary biliary cirrhosis (PBC) were analysed for fine specificity by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA showed that complex I (NADH-ubiquinone reductase) from beef heart mitochondria completely inhibited the binding of AMA to mitochondrial inner membranes (SMP), indicating that the major mitochondrial antigens are located in complex I. Immunoblot analysis of beef heart SMP, complex I and the iron sulphur (IP) subfraction of complex I revealed several antigens, one of which (75 kDa) reacted with all PBC sera but not with the additional autoimmune sera tested. Resolution of SMP or complex I by two-dimensional electrophoresis yielded in both preparations a polypeptide of 75 kDa with an isoelectric point of 6.4, which reacted with PBC serum and with rabbit antisera raised against the 75,000 subunit of complex I. In immunoblot experiments, the antigenicity of the 75,000 polypeptide in SMP, complex I, and the IP subfraction is increased by prior reduction of the sample with mercaptoethanol. This suggests a similarity to the PBC-specific 'M-2' antigen, which is also sensitive to sulphur reagents. The data indicate that the 75 kDa polypeptide of complex I is a major mitochondrial antigen binding AMA in PBC sera, and allows us to identify the location and probable function of the PBC antigen.

Animals↗

PubMed1988

Evidence that the major primary biliary cirrhosis-specific mitochondrial autoantigen is a subunit of complex I of the respiratory chain.

Primary biliary cirrhosis (PBC)-specific antigens were purified from beef heart mitochondria by immunoaffinity chromatography. Three major polypeptides (75, 60, and 40 kDa) were detected in the purified antigen fraction both by Coomassie blue staining and by western blot analysis. The 75 kDa antigen was identified as a subunit of Complex I (NADH-ubiquinone reductase) by the following criteria: (1) antibodies against the purified 75 kDa subunit of beef heart Complex I react with the immunoaffinity-purified 75 kDa antigen. (2) the 75 kDa subunit present in isolated Complex I, like that in the immunoaffinity-purified antigen, reacts with PBC sera only after reduction with mercaptoethanol, and (3) the 75 kDa antigen is enriched in isolated Complex I. A relationship between the 75 kDa and the 60 and 40 kDa antigens is suggested, since optimal binding of anti-mitochondrial autoantibodies (AMA) to the latter antigens also requires prior reduction with mercaptoethanol. A fourth major antigen (70 kDa) was also detected by western blot analysis, but only in samples that had not been boiled prior to electrophoresis. This antigen, which is also present in isolated Complex I, resembles the 75, 60, and 40 kDa antigens in its response to mercaptoethanol and its reaction with antibodies against the 75 kDa subunit of Complex I. A scheme is presented which relates all of the PBC antigens to the parent 75 kDa subunit of Complex I, probably as proteolytic products of the latter.

Adipose Tissue, Brown↗

PubMed1987

Primary biliary cirrhosis: antigenic specificity of IgM-type mitochondrial antibodies analyzed by immunoblotting and ELISA.

The antigenic reactivities of circulating IgM- and IgG-type antimitochondrial antibodies (AMA) from 18 patients with primary biliary cirrhosis (PBC) were compared by the use of immunoblotting and enzyme-linked immunosorbent assay (ELISA). In immunoblotting, the binding patterns of IgM and IgG were very similar when F1-ATPase and mitochondria were used as antigens. The major PBC-specific IgM-reactive antigen was identical with the dominating IgG-reactive antigen, sharing the same molecular weight of 70 kD and the same requirement for reduced thiol groups for expression of antigenicity. Other PBC-related mitochondrial proteins with variable antigenicity had the molecular weights of 60 and 43 kD. Depending on the IgM and IgG reactions in F1-ELISA, PBC patients can be grouped into three categories: patients with IgG and IgM (12/18), IgG alone (5/18) and IgM alone (1/18). By serum fractionation, the IgM reactivity was shown to be a true PBC-related antibody antigen reaction, and not due to interference of rheumatoid factors.

Adult↗

PubMed1986

Primary biliary cirrhosis: assessment of the quantitative importance of the adenine nucleotide translocator protein as a major mitochondrial antigen.

The adenine nucleotide translocator protein (ANT) is the first well-characterized mitochondrial polypeptide to be identified as an antigen for antimitochondrial autoantibodies (AMA) in PBC sera. Because of the potential use for a highly purified antigen as a tool in studying the aetiology of PBC, we have undertaken an assessment of the quantitative importance of ANT as a PBC-specific mitochondrial antigen. Immunoblotting and ELISA techniques were used. Both methods reveal PBC antibodies against isolated rat liver ANT. However, competitive ELISA experiments using purified rat liver ANT as the competing antigen show that anti-ANT antibodies in PBC serum comprise only a fraction of the total AMA. Furthermore, both ELISA and immunoblotting experiments show that rat liver ANT is not a specific antigen for PBC autoantibodies. Sera from patients with SLE, chronic active hepatitis, and sera from normal, control patients, have nearly the same, or higher, ANT antibody titres. Thus, ANT is not a good candidate as an antigen for the diagnosis of PBC.

Animals↗

PubMedDate not supplied

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.

Amino Acid Sequence↗