Isoelectric focusing in agarose.

We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 X 10(6) mol.wt. were easily and rapidly focused.     

Isoelectric focusing studies of transferrin and haptoglobin subtypes in an Australian white population

This investigation presents the results of Hp and Tf subtyping of sera from 307 Australian whites by means of isoelectric focusing. Five Hp alleles were detected, these being Hp1F = 0.168, Hp1S = 0.235, Hp2FF = 0.003, Hp2FS = 0.561 and Hp2SS = 0.033. In the Tf system six alleles were found, TfC1 = 0.761, TfC2 = 0.176, TfC3 = 0.054, TfC6 = 0.002, TfB = 0.006 and TfD = 0.002. The usefulness of IEF together with modifications was highlighted for differentiating Tf and Hp subtypes; in particular, the absence of Hp2FF and Hp2SS in some earlier studies could be related to the technique used. The implication of the simultaneous presence of B and D transferrin variants in Caucasian populations is discussed.     

Isoelectric focusing of Chlamydia trachomatis.

HeLa-229 cells and the elementary bodies of Chlamydia trachomatis had a net negative electrical surface charge at neutral pH when measured by isoelectric focusing. Inclusion-forming and non-inclusion-forming elementary bodies focused in one band at pI 4.64.     

Isoelectric focusing of herpes simplex virus

Herpes simplex virus was inserted into a preformed stable pH gradient and electrofocused for about one hour. Dextran was used as the density gradient forming agent. Virions banded at pH 4.9+/-0.1 and nucleocapsids at 4.1+/-0.05. About 10-20 per cent of infective virus was recovered.     

Isoelectric focusing and 2D-analysis of poliovirus proteins.

The polypeptides of poliovirus were isoelectrically focused in urea containing polyacrylamide gels after dissociation of virus particles in urea. The bands obtained were correlated to the known four structural polypeptides of poliovirus by the two-dimensional technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The measured isoelectric points are: VP1, 8.1; VP2, 6.4; VP3, 6.0; VP4, 7.3. Additional bands caused by charge modification of the polypeptides during storage of the virus can be found.     

Isoelectric focusing spectra of antibodies which activate mutant -galactosidases

Antibodies raised against wild type β-galactosidase of Escherichiu coli activate certain inactive mutant β-galactosidases to enzymic activity. We have developed a technique to detect 7 S activating antibody after isoelectric focusing in thin layers of polyacrylamide gels. Enzymic activity bound to antibodies is detected using the histochemical substrate 5-bromo-4-chloro-3-indoxyl-β-D-galacto-pyranoside. When split, this substrate produces insoluble, blue-green indigo stain at the sites of enzymic reaction. With this technique antisera raised in five rabbits against the wild type enzyme have been analyzed for their capacity to activate inactive mutant enzyme. Each rabbit produces a characteristic spectrum of activating antibodies when tested with a given mutant enzyme, which is different from that produced by another rabbit. A restricted population of antibodies, e.g. the product of one to five clones of antibody-forming cells activate a mutant enzyme. The spectrum of activating antibodies obtained with a mutant enzyme can be correlated to the position of the mutation in the gene for β-galactosidase which gives rise to this inactive enzyme. Three genetically different groups of mutant enzymes are activated by three distinct populations of antibodies within the serum of one rabbit. Mutation at a fourth site within the gene yields a mutant enzyme which is activated by one of the three populations of specific antibodies. This indicates that one antibody, in binding to one antigenic site, can restore the activity of two mutant enzymes altered at two different sites within the polypeptide chain of the enzyme.     

Isoelectric focusing of ureases from Campylobacter pylori and related organisms.

Agarose gel isoelectric focusing was used to determine the isoelectric points of ureases from strains of gastric campylobacterlike organisms isolated from ferrets (pI 5.4), baboons (pI 5.7), and pigs (pI 5.9) and from isolates of Campylobacter pylori in humans (pI 5.9). This technique may help differentiate these closely related bacteria.     

Isoelectric focusing of bovine major histocompatibility complex class I molecules

The products of the major histocompatibility complex (MHC) loci regulate an individual's immune response to pathogens. Cattle provide an important model to study the relationship between disease susceptibility and MHC haplotype since large half-sibling families are common. The definitive demonstration, however, of a firm relationship between MHC phenotype and disease susceptibility in cattle will require a precise definition of the bovine MHC allelic products. Available reagents for serological characterization of the bovine MHC gene products have not been adequate for these purposes. We have shown that existing mouse monoclonal antibodies and rabbit anti-human antisera precipitate bovine class I molecules, that these structures separate well by one-dimensional isoelectric focusing (1-D IEF), and that immunoprecipitation followed by 1-D IEF allows the detection of bovine class I MHC allelic products. Through this technique, we have identified previously undetected class I products. This approach will facilitate a detailed characterization of the bovine MHC class I gene products.     

Isoelectric focusing of bovine major histocompatibility complex class II molecules

Serological approaches have been relatively unsuccessful in defining the allelic products of the bovine major histocompatibility (MHC) class II loci. We demonstrate that bovine class II allelic products can be characterized by precipitation with a polyclonal antiserum and separation using one-dimensional isoelectric focusing. Polymorphic beta chains were present in immunoprecipitates from both biosynthetically and surface-labeled lectin-stimulated bovine T cells. Precipitates from biosynthetically labeled but not surface-labeled T cells contained a basic invariant chain and a non-polymorphic structure. The non-polymorphic structure appears to be a beta chain. The polymorphic class II beta chain co-segregated with bovine MHC class I allelic products in a half-sibling family, providing evidence for linkage between bovine class I and class II loci. This approach to the biochemical analysis of the bovine class II structures should facilitate the investigation of the association between the bovine products and disease susceptibility.     

Isoelectric focusing of serum in genetic counselling of families with cystic fibrosis

The high incidence of carriers of cystic fibrosis in the general population allows application of a less than perfect test to genetic counselling of relatives of children with the disorder and their spouses. In the absence of a definitive carrier detection test, we employ isoelectric focusing of serum in this way and include the a priori chance of carrier status in calculating the risk. The test will eventually be replaced but for the present is preferable in our hands to counselling based simply on the known gene frequency.     

Isoelectric focusing spectra of anti-bacterial alpha-amylase antibody unique for antigen-induced suppression.

The effect of intravenous (i.v.) administration of bacterial alpha-amylase (B alpha A) on the IgG antibody response to a subsequent challenge with B alpha A in incomplete Freund's adjuvant (IFA) varied with the difference in responsiveness of the parental strains. High-responder C3H/He (C3) mice given injections of either 200 or 4 micrograms of B alpha A, which alone were unable to trigger a detectable IgG antibody response, generated an enhanced response to an immunogenic challenge given 25 days after the last i.v. injection. The response of low-responder C57BL/6 (B6) mice previously exposed to B alpha A, following a different kinetic course depending on the exposing dose, reached a plateau lower than the levels of control responses (tentatively designated as high- and low-zone suppression). Prior exposure of (B6 X C3)F1 hybrids to 200 micrograms led to the enhanced response, whereas pretreatment with 4 micrograms rendered them partially tolerant to a subsequent challenge. These results suggest that the capacity to achieve low-zone suppression is inherited as a dominant trait. Isoelectric focusing (IEF) analysis revealed that these enhanced responses expanded antibody heterogeneity in a strictly restricted, strain-specific manner as observed during the normal antibody response, although the rate of expansion was accelerated. The specific antibodies produced by individual high-zone suppressed B6 mice were focused as a limited set of bands in a narrow pH range where the specific antibodies produced early in the normal response were focused. In contrast, the response of low-zone suppressed B6 and F1 hybrid mice was characterized by a unique process of heterogeneity expansion.     

Isoelectric focusing of human and guinea-pig C2: polymorphism of guinea-pig C2

The isoelectric point (pI) of human and guinea-pig C2 was determined by electrofocusing. The results showed that human C2 and C2 of some guinea-pigs had a pI of approximately pH 5.5–5.6; in some guinea-pig sera C2 activity was resolved into two fractions, one with a pI of about 5.2 and the other of about 5.5. The data suggest the possibility that guinea-pig C2 may exist in at least two allotypic forms.     

Isoelectric focusing and immunochemical analysis of proteins of the “Diaferm-3” antitetanus serum

The isoelectric points (PIs) of proteins present in the Diaferm-3 antitetanus serum, a mixture of partially purified [F'ab]₂-fragments of native molecules of antitetanus immunoglobulins, their antitoxic activity, and their immunochemical properties were investigated by isoelectric focusing in a pH gradient of ampholines. The PI values and electrophoretic mobility of the preparation were compared. Proteins with antitoxic activity were shown to have PI values within the range 9.1–5.15, and from their electrophoretic mobility they can be classed as G- and -globulins. No antitoxic activity was found in macroglobulins with the mobility of -globulins.Laboratory of Pathophysiology of Toxicoinfections, Institute of Normal and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 10, pp. 41–45, October, 1974.     

Isoelectric focusing spectra of rabbit antibodies to Salmonella abortus-equi detected by anti-idiotypic sera

Serum antibodies from rabbits hyperimmunized with Salmonella abortus-equi (S.a-e) were examined by isoelectric focusing in polyacrylamide gel, followed by the coating of the gel with iodinated polysaccharide extracted from bacterial cell walls. The antibody populations in these sera were highly heterogeneous. Anti-idiotypic sera, prepared in allotypically matched rabbits, recognized only a fraction of these antibody populations (generally from one to three clonal antibodies per serum). Different rabbits subjected to the same anti-idiotypic immunization can recognize different antibodies in the same serum to S.a-e.

Homologous and heterologous idiotypes recognized by the same anti-idiotypic serum have shown very similar but never identical pI spectra. It appears that the same group of idiotypic determinants can be markers for several clonal antibodies and are not unique to a single clone.

Two rabbits were studied throughout an immunization course lasting 2 years; we did not observe any change in the clonal product of antibody-producing cells carrying the idiotypic determinants.