Development in electrophoretic and immunoelectrophoretic methodology. Applications to serum proteins analysis.
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Biomedical subjects
Publications and source records attributed to J M Fine.
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There have been several reports of human myeloma proteins exhibiting autoantibody activity. Such immunoglobulins represent therefore an appropriate system for investigating idiotypic diversity of human autoantibodies. Using this approach, we confirmed the marked idiotypic restriction of polyclonal anti-DNA autoantibodies and we showed that immunoglobulins without known binding affinity may express autoantibody-related idiotopes. These results are discussed in lights of recent concepts upon autoimmune processes and idiotypic interactions.
Sixty-one human myeloma proteins (HMP) from patients with multiple myeloma and Waldenström macroglobulinemia were tested for anti-idiotypic (Id) activity against autoantibodies to double-stranded DNA, small nuclear ribonucleoproteins, and human thyroglobulin (HTg), by competitive radioimmunoassays and enzyme immunoassays. An IgG1, lambda HMP from patient BEN with anti-Id activity against antibodies to HTg is reported. IgG1 BEN was not directed toward human Fc fragments and its activity was not related to allotypic determinants. IgG1 BEN molecules recognized Id determinants (idiotopes) on F(ab')2 anti-HTg fragments, but not idiotopes of F(ab')2 fragments of antibodies of other specificities. This observation supports the general significance of Id network interactions in regulation and diversification of immune responses in man.
The influence of a primary amine, hydroxylamine, on the interaction between alpha 2 macroglobulin (alpha 2M) and thrombin was analyzed by electrophoretic and enzymatic methods. Hydroxylamine (final concentrations 0.01 M and 0.1 M) was added to the alpha 2M solution 3 to 5 min before thrombin. In these conditions hydroxylamine had no direct influence on alpha 2M itself. The inhibition of thrombin activity by alpha 2M was still possible and alpha 2M/thrombin complexes were observed. However the rate of inhibition of the clotting activity of thrombin was diminished in function of the hydroxylamine concentration. The complexes obtained in the absence of the nucleophylic agent were resistant to SDS dissociation, whereas those obtained in the presence of hydroxylamine were dissociated by SDS. In both cases, the amount of alpha 2M polypeptide chains cleaved by thrombin was the same (50%). In conclusion, hydroxylamine does not prevent the formation of alpha 2M/thrombin complexes, but it reduces the covalent binding of the enzyme to the inhibitor in a concentration dependent fashion, leading to the formation of "abnormal" complexes.
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1. Isolated carbonic anhydrases CA I and CA II from different species (man, ox, sheep, pig, dog and rat) have been analyzed by immunodiffusion techniques using rabbit specific CA I or CA II antisera. 2. Immunodiffusion shows the absence of cross-reactivity between CA I and CA II isoenzymes. 3. Common antigenic determinants (cross-reactivities) were evidenced between the CA I of the different species under study. 4. To the contrary, cross-reactivity between CA II of the species mentioned above, can clearly be demonstrated only between bovine and sheep CA II.
In order to clarify the actual nomenclature of the albumin allotypes in French and Italian populations we compared the samples collected in our laboratory to those kindly supplied by Dr. Porta (Italy) with reference to albumin variants classified in starch gel electrophoresis using three buffer systems by Weitkamp. The inherited human albumin variants can be classified on the basis of their relative mobilities on cellulose acetate electrophoresis compatively to human transferrin mobility. The relative mobility of each variant can be expressed by the following ratio: migration distance of the variant versus migration distance of the normal albumin where zero represents the transferrin mobility. Using three buffers system at pH 8.6, 5.0 and 6.9, it is possible to distinguish some albumin variants having a same mobility at alkaline pH and different mobilities at acidic pH. In the european area, eleven albumin variants are distinguishable on the basis of their relative mobilities at different pH: four Fast moving variants: Gent, Vanves (a new variant described here), Reading and CN/BL, and seven Slow moving variants: MI/MI Slow, GE/CT, SO/BS (or D type), Pollibauer, Gainesville, Roma and B type. Thirty-six sera from unrelated subjects with genetic bisalbuminemia were analyzed in our laboratory. Their distribution was as follows: B type (22), Pollibauer (9), SO/BS (2), Gainesville (1), Gent (1) and Vanves type (1). The frequency of bisalbuminemia was 0.35 per 1,000 in a population of 19,949 blood donors.
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Low density lipoprotein (LDL + VLDL) concentrations, were measured in 48 patients with multiple myelomatosis, or Waldenström's macroglobulinaemia (malignant monoclonal gammopathies) and in 42 patients with "asymptomatic" benign monoclonal gammopathies (M.G.). In patients with malignant M.G., the level of LDL plus VLDL was significantly lower than in patients with "asymptomatic" M.G. who exhibit a normal level.
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Isolated albumin almost always contains polymerized forms which appear during preparation and storage of the protein. The proportion of polymerized forms reflects the degree of stability of the solution. The quantitative estimation of the polymers is usually performed by gel chromatography. In this work, the high resolution power of polyacrylamide gradient gel electrophoresis (Gradient PAGE) was used to separate the polymers present in the preparations of human serum albumin. The analysis of the different peaks obtained by gel chromatography allows to conclude that peak 1 contains aggregates and high polymers, peak 2 trimer and dimer and peak 3 the monomer of albumin. The aggregates of the peak 1 can be dissociated by SDS and correspond, in gradient PAGE, to the high polymers. By using gradient PAGE in the presence of SDS under the conditions described in this paper, it is possible to estimate the proportion of high polymers and aggregates present in albumin preparations. These results are similar to those obtained by chromatography followed by a protein assay but noticeably inferior to those resulting from measurements performed by absorbance at 280 nm.
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In the course of a study on the albumin variants in 36 unrelated subjects with genetic bisalbuminemia, we have observed, in the serum of a French blood donor, a new fast-moving variant. Cellulose acetate electrophoresis at pH 8.6 showed that this variant, so-called albumin 'Vanves', moved slowlier than albumin Gent and faster than albumin Reading.
An 85-year-old woman without personal history of haemorrhages was found to have qualitative and quantitative deficiency of Factor VIII persisting at least 6 months. Asymptomatic monoclonal IgG kappa gammopathy was also discovered in the same patient, together with a circulating inhibitor of ristocetin co-factor. The fact that the inhibitory effect was reduced after the patient's serum IgG's were bound to staphylococcal protein A suggests that the inhibitor belonged to that category of immunoglobulins, although the authors were unable to detect it after elution.
The atherogenic low density lipoproteins were evaluated using two turbidimetric methods: one based on the precipitation of LDL and VLDL by heparin-CaCl2 and the other on the precipitation of the VLDL by sodium dodecyl sulfate. In search of hyperlipoproteinemia, both tests were applied to a population of blood donors aged from 18 to 60 years. when both tests were negative, hyperlipoproteinemia can be excluded without quantitative evaluation of cholesterol and triglycerides levels. If the heparin-CaCl2 test is high and the SDS normal it can be concluded that it is a case of type IIa hyperlipidemia. When both test are high, further investigations are required. In the present study, corresponding to the screening of the sera from 2656 blood donors (including 1280 males and 1376 females), the high level of atherogenic lipoproteins is most frequently observed in the males (10 per cent for men and 2,5 per cent for women). This frequency increases with age.
Simultaneous occurrence of "myasthenia gravis" and monoclonal "asymptomatic" gammapathy has been observed in a 83 years old patient. Antistriated muscle antibodies could be detected at a level of 1/1,000 but no antiacetylcholine receptors antibodies. The antibody activity is located both in the monoclonal IgG kappa and in the polyclonal IgG. This case illustrates the frequent occurrence of several auto-immune disorders in a same patient.