Liquid Isoelectric Focusing Research Articles

Heterogeneity of factor VIII antibodies: further immunochemical and biologic studies

Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

Studies on allergens of Dermatophagoides pteronyssinus by direct and indirect RAST.

Dermatophagoides pteronyssinus (DP) extract was fractionated by Sephadex G-75 and liquid isoelectric focusing (IEF) and the allergenic activity of different fractions was monitored by direct and indirect RAST. The fractionation on Sephadex G-75 showed that the allergenic activity of DP extract was related to wide molecular weight spectrum components, even though the maximum amount was recovered in effluent that contained protein with a molecular weight ranging between 25,000 and 12,500 daltons. By fractionation of the mite extract on IEF, three main peaks of allergenic activity (pI less than 3.0; pI = 4.3 +/- 0.25; pI = 6.4 +/- 0.25) were found. Cross-inhibition experiments showed a high degree of cross-reactivity between allergenic material eluted in very distant regions of molecular weight or isoelectric point. The allergenic activity of unfractionated mite extract and of its IEF fractions was destroyed by pronase - but not by neuraminidase - treatment. These results suggest that DP extract probably contains one main allergen existing in multiple molecular forms rather than several distinct allergens and that a protein moiety of the allergen is necessary for the combination with IgE.

Accelerated liquid isolectric focusing using a direct current source that maintains constant power

A power supply has been designed to improve liquid isoelectric focusing by automatically maintaining constant power applied to the column. The main advantages are (a) a reduced exposure time which is particularly important when sensitive proteins are being resolved; (b) a reduction in the loss of resolution due to diffusion or denaturation of the proteins; and (c) a considerable increase in efficiency of the use of the equipment for preparative purposes.

Studies on hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits—III: Fractionation of restricted rabbit anti-SIII and -SVIII antibodies from partially inbred rabbits by affinity chromatography and electrophoretic properties of these antibodies

Sixty-three homozygous rabbits from five different partially inbred strains were hyperimmunized with either type III or VIII pneumococcal vaccine for 1–3 courses of immunization, Fifty-six percent of the ACEP strain rabbits and 46 per cent of the New Zealand White type III strain demonstrated the presence of either electrophoretically restricted or partially restricted antibody molecules during the primary or secondary courses of immunization. None of the type III c, AX or ACCR(B) strains of rabbits gave either restricted responses when immunized with the pneumococcal vaccines. Three of the ACEP strain rabbits and two of the type III strain produced large quantities of highly restricted antibodies and were used to initiate structural studies on the antigen-combining site of antibody molecules. The rabbit antibodies were purified initially by affinity chromatography. The anti-SVIII antibodies were all eluted in a single fraction from an SVIII immunoadsorbent. The anti-SIII antibodies were all eluted in two fractions (one in various aqueous buffers and the other in guanidine-HCl) from an SIII immunoadsorbent. The specificially purified antibodies were examined analytically by isoelectric focusing in polyacrylamide gels and then were further fractionated by preparative liquid isoelectric focusing. Each purified antibody preparation from a restricted responder yielded 2–4 major electrofocused antibody fractions. The isoelectric point difference between the first and last eluted focused antibody fractions varied between 0·2–0·8 pH unit. The affinity chromatography and the isoelectric focusing procedures provide a successful means of preparatively obtaining different antibody fractions even from a single highly restricted antiserum.

The Characterization of Human Anti-IgG Autoantibodies by Liquid Isoelectric Focussing

A series of human anti-IgG autoantibodies (AGA) have been analyzed by liquid isoelectric focusing in order to a) determine their isoelectric points (pI) and b) assess their heterogeneity. The 13 AGA examined include five IgM, five IgA, and three three IgG AGA. All demonstrate a single major peak indicative of restricted heterogeneity and each is characterized by an acidic pI. This anodal pI is distinct from other immunoglobulins isolated from the same serum that do not exhibit anti-IgG activity. It is speculated that the acidic pI is the result of acidic amino acid substitutions in the variable regions of immunoglobulin chains. The restricted heterogeneity and distinctive pI of the AGA suggest the selection of structurally similar antibody molecules from the large repertoire available in the human genome.

The use of liquid and gel isoelectric focusing as a means of further evaluating the degree of restriction of rabbit antibodies

Antibodies from a homozygous rabbit hyperimmunized with type VIII pneumococcal vaccine was examined by isoelectric focusing in polyacrylamide gels. Significant quantities of electrophoretically restricted pneumococcal polysaccharide antibodies were obtained from single rabbit bleedings by preparative liquid isoelectric focusing of total immunoglobulins, obtained by salt precipitation, in a sucrose gradient using 1% w/v ampholytes covering 3 pH units. The method was modified to obtain the best resolution possible. The electrophoretic mobility of the electrofocused antibodies was compared by gel isoelectric focusing. The degree of restriction of the heavy and light polypeptide chains of the electrofocused antibody fractions was examined both by alkaline disc gel electrophoresis in urea and by isoelectric focusing in polyacrylamide gels in urea following complete reduction and alkylation of the focused antibody fraction. Isoelectric focusing in polyacrylamide gels in urea was found to be the best and easiest method to study the number and electrophoretic mobility of the fully reduced and alkylated light and heavy chains. Attempts were made to show that no molecular sieving occurred with the antibody heavy chains in 6.7% polyacrylamide gels. A number of electrofocused anti-pneumococcal polysaccharide antibody fractions yielded single light chain zones with different electrophoretic mobilities from a single antiserum suitable for comparative primary sequence analyses.

Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques.

AbstractFour heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.

Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits. II. Allotypes and idiotypes in sera and electrofocused fractions.

AbstractThe expression of allelic forms of immunoglobulin during immunization of heterozygotes with pneumococcal vaccines was monitored using anti‐allotype antisera specific for the genetically different forms of rabbit kappa chains (b‐locus allotypes). We also used the a‐locus (VH) allotypic markers of rabbit heavy chains and idiotypic determinants associated with a restricted subpopulation of anti‐polysaccharide antibodies of an individual rabbit. Analysis of fractions obtained by preparative liquid isoelectric focusing (IEF) of antisera from heterozygous rabbits exhibiting restricted antibody responses to type 3 or type 8 pneumococcal polysaccharides, indicated an enrichment for one a or b locus allotype. Enrichment of a specific idiotype from ∼ 11 % in the starting serum up to as high as 65 % in a focused fraction was also obtained. The method of preparative liquid IEF can thus be used to isolate antibodies restricted in allotype and idiotype from antisera. The quantities obtained are sufficient for further chemical and immunological study.

Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations: III. NH2-TERMINAL LIGHT CHAIN SEQUENCE ANALYSES OF FOCUSED ANTI-p-AZOPHENYLTRIMETHYLAMMONIUM AND ANTI-p-AZOBENZENEARSONATE ANTIBODY FRACTIONS

Abstract The structural heterogeneity of light polypeptide chains derived from rabbit antibodies directed against p-azophenyltrimethylammonium (Ap) and p-azobenzenearsonate (Rp) haptens from individual heterozygous rabbits was investigated. Antibodies were purified by adsorption on specific solid immunoadsorbents, followed by preparative liquid isoelectric focusing at either pH 5 to 8 or pH 7 to 10. The focused anti-Ap antibody fractions were refocused in pH gradients extending only 0.5 pH unit on either side of their respective isoelectric points. A number of focused antibody fractions demonstrated electrophoretic, structural, and functional features characteristic of homogeneous antibody populations. Amino-terminal sequences of the kappa light chains, obtained by mild reduction and alkylation followed by gel filtration, were determined for nine focused fractions of anti-Ap antibody from one rabbit, seven focused fractions of anti-Rp antibody from a second rabbit, and two focused fractions of anti-Rp antibody from a third rabbit. Amino acid sequence analyses extended for 6 to 39 positions (a few included the first hypervariable region) depending upon the quantity of light chain obtained from the focused fractions. Sequences obtained from light chains isolated from serum prior to immunization immunoglobulins or from specifically purified unfractionated antibodies were heterogeneous (2 to 4 residues at most positions), while those from the major focused antibody fractions were as homogeneous, for most of the first 25 to 30 positions, as human kappa Bence-Jones proteins sequenced by the same methods. The light chains from the focused antibodies demonstrated significant microheterogeneity and nonidentity within limited areas (positions 31 to 34). Focused antibody light chains had sequences representative of either the Vkii or Vkiii subgroups. This approach, therefore, permits structural studies to be performed on the homogeneous antibody constitutents of heterogeneous antibody preparations and demonstrates that the heterogeneity is relatively restricted to limited areas.

Substrate-derived reversible and irreversible inhibitors of the multi-enzyme I of gramicidin S biosynthesis

1. 1. The heavy fraction (Fraction I enzyme) which activates proline, valine, ornithine and leucine in gramicidin S biosynthesis has been purified by preparative liquid isoelectric focusing. 2. 2. l-Isoleucine, an amino acid not found in gramicidin S, supported PP i-ATP exchange to 43% that of l-leucine. 3. 3. Of the ring analogues of proline tested, l-azatidine-2-carboxylic acid and l-pipecolic acid supported PP i-ATP exchange to the extent of 64 and 13% that of proline, respectively. 4. 4. In the ring substituted analogues of proline, trans-3-methylproline and trans-4-fluoroproline inhibited exchange in the presence of l-proline by 29 and 93%, respectively. 5. 5. The d-isomers of the constituent amino acids of gramicidin S did not support PP i-ATP exchange and at equimolar concentrations inhibited the exchange supported by the l-isomers by 80%. 6. 6. An analysis of the slope-intercept effects on double reciprocal plots obtained when ATP is the variable substrate and l-proline is the changing fixed substrate gives results which are in accord with a random order of addition for ATP and l-proline to the heavy fraction. 7. 7. The chloromethyl ketone of l-proline (Pro-Cmk) inhibited the PP i-ATP exchange occuring in the presence of l-proline of Fraction I enzyme 95% in 60 min. Preincubation for 60 min with Pro-Cmk resulted in a residual activity of 9.7% whereas the native enzyme alone and in the presence of the reversible inhibitor l-2-hydroxymethyl pyrrolidine gave a residual activity of 73 and 79%, respectively. The exchange activity supported by l-valine, l-ornithine and l-leucine remained essetially unchanged.

Structural and immunological studies of two IgGl-λ myeloma proteins from a single patient

Serum from a patient with multiple myeloma contained two monoclonal IgGl-λ M-components of different electrophoretic mobility. Melphalan therapy reduced the serum concentration of the slow M-component (average pI 7·1) but had no effect on the fast M-component (average pI 6·5). Conversion of the slow M-component into the fast M-component could not be accomplished by in vitro incubation of the patient's serum. Both components had identical sedimentation coefficients of 6·5 S ruling out the possibility of aggregation of one component to another serum protein. Neither M-component showed antigen-combining specifity. Both isolated chains belonged to the λl subgroup with Gm a −b −f + allotypes and were indistinguishable in their acidic and alkaline urea starch gel electrophoretic patterns. The isolated light chains of both M-components were V λIII[St(−) II, Oz(−)] and were indistinguishable in the first five NH 2-terminal residues ([]-Tyr-Val-Leu-Thr), circular dichroism spectra (240–310 nm), amino acid composition and mobility in acidic urea starch gel[ electrophoresis. However, the light chains had different moobility in alkaline urea starch gel electrophoresis. Since both light chains lacked sialic acid, the differences in electrophoretic mobility can be primarily attricuted to the fact the light chain of the fast M-component had 5–7 more dicarboxylic acid residues than the light chain of the slow M-component, and this difference in primary sequence was also reflected un the peptide maps. The liquid isoelectric focusing elution profile of both M-components indicated that each component resolved into four subfractions in decreasing concentration from cathode to anode and any two successive subfractions differed by 0·1 pH unit. This microheterogeneity may also be due to a variable dicarboxylic acid content and could have arisen by post-synthetic deadmidation. The two M-components differed in their average pI values by 0·6 pH units suggesting at least six charge differences between the two M-components. The presence of the two M-components differing in primary sequence of the light chains could have resulted from either a divergent clone or two clones.

The use of preparative liquid isoelectric focusing for the further purification of rabbit antihapten antibodies

Preparative liquid isoelectric focusing has been used to separate specifically purified rabbit antihapten IgG antibodies into a number of fractions. Antibody samples were fractionated in a sucrose gradient using 1% w/v carrier ampholytes covering 7, 3, and 1 pH units. A number of the antibody fractions have been shown to be both structurally and functionally homogeneous. These antibodies are sufficiently homogeneous for sequence studies to be performed on the variable regions of the light and heavy polypeptide chains. This method is independent of the type of immunogen used and the kind of animal immunized. The fractionation is much more efficient if specifically purified antibodies (purified on a solid immunoadsorbent) are used rather than whole immunoglobulin samples. The amount of antibody recovered in the major antibody fractions was 65 to 90% of the specifically purified antibodies applied to the column. The eluted antibody fractions full biological activity. A number of factors have been discussed which often improved the resolution of the antibody fractions.

Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations: II. A SURVEY OF THE ISOELECTRIC PROPERTIES OF ANTIHAPTEN ANTIBODIES DIRECTED AGAINST CHARGED AND UNCHARGED HAPTENS

Abstract Liquid isoelectric focusing profiles of a number of conventionally raised rabbit antibodies directed against acidic, neutral, and basic haptens have been determined. All antibody preparations showed a degree of heterogeneity similar to that already reported for antibodies to the basic hapten p-azophenyltrimethylammonium. There was little difference in the degree of heterogeneity found in the focusing profiles of antibody from different rabbits directed against the same hapten. There appear to be between 10 and 30 individual antibody species in each case, but some of this complexity may be a result of variable, postsynthetic deamidation. Isoelectric focusing profiles of the antigens also showed considerable heterogeneity. The mean isoelectric point of both rabbit and bovine γ-globulins was approximately pH 6.0, and the distribution of the isoelectric point, pI, values of the focused specifically purified rabbit antibodies about this point was reciprocally related to the pI of the focused antigens. It appears that a single charge unit causes a displacement of the mean pI value by approximately 0.1 pH unit. The displacement found for various antibody preparations indicates antibodies to acidic and basic antigens may differ by as many as 19 charged residues.

Studies on Human Myeloma Proteins with Antibody-Like Activity

Abstract More than 500 human sera containing one or more M-components were screened for antibody-like activity, using both double diffusion in agarose and modified Farr binding studies. Antibody activity was detected in two IgG myeloma proteins and two IgM macroglobulins using a number of negatively charged, positively charged, and neutral haptens. We have at least one of these M-components growing in cell culture producing two different cell lines, and we have obtained small crystals with two of the M-components. Equilibrium dialysis of the purified M-components yielded K0 values with low affinity. We have isolated, using liquid isoelectric focusing, a homogeneous IgG(γG1, K) protein from a leukemic patient 2 months after a bone marrow transplant operation. The IgG was not directed against the histocompatibility antigens. We detected significant binding with this protein and 3H-hydrocortisone. (Supported by MRC grant MA-3001 and the Ontario Treatment and Research Foundation).

A Method for Further Separation of Specifically purified Antibodies by Liquid Isoelectric Focusing

Abstract Specifically purified anti-p-azophenyltrimethylammonium antibodies from individual rabbits have been separated by isoelectric focusing on a preparative scale over a pH range of 5 to 8 into as many as 30 components differing in isoelectric properties and demonstrating reduced heterogeneity. A number of these components have many of the properties characteristic of homogeneous antibodies. The isolated antibodies are electrophoretically discrete and show homogeneous binding properties towards the p125I-iodophenyltrimethylammonium hapten, giving two combining sites/mol protein with a heterogeneity index of 1.00 ± 0.05. On alkaline urea disc electrophoresis, the fully reduced and alkylated antibody light chain patterns were monodisperse. Several specifically purified rabbit and goat antibodies directed against neutral, positively charged and negatively charged haptens have been purified. This procedure appears to afford a general method for the further purification of antibodies to a state suitable for sequence studies and detailed structural studies on the nature of the combining sites.

Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations: I. SEPARATION AND PROPERTIES OF HOMOGENEOUS ANTI-p-AZOPHENYLTRIMETHYLAMMONIUM ANTIBODIES

Abstract A method is described by which liquid isoelectric focusing over a pH range 5 to 8 has been used on a preparative scale for the further purification of specifically purified antibodies directed against the positively charged p-azophenyltrimethylammonium hapten (anti-Ap antibodies). The anti-Ap antibodies separated into 4 to 8 major fractions and 10 to 16 minor fractions. More than 95% of the antibodies directed against the positively charged hapten had isoelectric points between pH 5 and 8. The charge, structural, and functional homogeneity of several of the major fractions was assessed by a number of independent criteria. The charge homogeneity of three of the electrofocused antibody fractions was demonstrated by the presence of monodisperse zones in electrophoresis, by focusing the major fractions over a narrow pH range, and also by acrylamide disc electrophoresis of fully reduced and alkylated light chains in alkaline urea gels. The structural homogeneity of two of the electrofocused antibody fractions was demonstrated by the finding of single NH2-terminal residues on the isolated, fully reduced and alkylated light chains. Sequence studies on the isolated light chains of these fractions showed each to have a single coherent sequence compared with the parent antibody which was typically heterogeneous. These antibody fractions showed only the b4 allotypic marker on their light chains and 95 to 96% of the heavy chains contained the a1 allotypic marker, although the unfractionated antibodies were heterozygous (a1, a3) with respect to the heavy chain allotypic markers. The functional homogeneity of these electrofocused antibody fractions was determined by equilibrium dialysis using the 125I-labelled p-iodophenyltrimethylammonium hapten. The results of the binding plots, using both the Scatchard and Sips analyses, demonstrated that the specifically purified anti-Ap antibodies had a heterogeneity index of 0.6 and contained 2 moles of combining sites per mole protein, while each of the electrofocused anti-Ap antibody fractions had a heterogeneity index of 1.0 and contained 2 moles of combining sites per mole protein.