Region-selective labeling of antibodies as determined by electrospray ionization-mass spectrometry (ESI-MS)

The electrospray ionization-mass spectrometry (ESI-MS) analysis of three sets of monoclonal antibody-acridinium-9-carboxamide conjugates is described. The conjugates (nine total) were enzymatically digested using papain and the resulting fragments [Fc heavy chain, Fab, or F(ab')(2)] were analyzed using liquid chromatography/ESI-MS. The average number of labels per fragment were calculated using Sigma nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative area of the corresponding peaks in the mass spectrum. When these values were normalized against the molecular weight of their respective region, antibody-dependent labeling patterns were observed. For antibodies T (anti-L-T(4)) and F (anti-FITC), there was a preference for conjugation of the Fab region over the Fc region. For antibody B (anti-biotin), the trend was reversed.     

Simultaneous production of immunoaffinity membranes

We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3, haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids.     

Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states.

Commercial 14C-labeled KB cell DNA, widely used to assay sera for anti-DNA antibodies, was chromatographed on benzoylated-naphthoylated-DEAE-cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaC1 (KB fraction I) characteristic of ds-DNA. Fifty-five percent of the label eluted with 50% formamide-1 M NaC1 (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss-DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds-DNA. after pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C-KB-cell DNA preparation was ds-DNA with ss regions which was undetectable by HAP chromatography. 3H-ds-DNA and circular 3H-ss-DNA prepared from T7 and phiX174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non-SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti-ds-DNA antibodies on the basis of KB cell DNA testing and detectable antibodies against KB fraction 1 or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non-SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phiX174 DNA, KB fraction II DNA and alkali-denatured T7 DNA. The data support the conclusions that 1) false positive tests for anti-ds-DNA antibodies can result from contamination of ds-DNA with ds-DNA having ss regions, and 2) non-SLE sera do not contain antibodies specific for ds-DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.     

Analysis of antibody cross-reactivity in experimental American trypanosomiasis.

Susceptible C3H/He mice were immunized with the avirulent Corpus Christi strain of Trypanosoma cruzi and subsequently infected with virulent Brazil stain organisms. Seventy days after infection sera were isolated and analyzed on western blots of electrophoretically separated T. cruzi antigens prepared from culture-form parasites (primarily epimastigotes). More than 25 bands were identified. The antibodies were fractionated by elution from various regions of western blots corresponding to average molecular weights of approximately greater than 130, 77, 70, 60, 48, or 38 kDa. Each of these antibody preparations was then incubated with strips of nitrocellulose containing all of the electrophoretically separated T. cruzi, and cross-reactivity was determined. Antibodies isolated from the 130-, 77-, and 70-kDa regions all cross-reacted with each other. Antibodies eluted from the 60-kDa region bound antigens in the 60-, 70-, and the 77-kDa regions. More importantly, antibodies eluted from every region bound antigens in the 70-kDa region. Conversely, antibodies eluted from this region bound to antigens in all of the other regions. These data indicate the presence of (a) common antigenic epitope(s) in T. cruzi infections in these mice that is predominantly found in the 70-kDa antigen-antibody complex on western blots.     

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies

Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.     

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.     

Immunochemical Studies of Bovine and Human Choline-O-Acetyltransferase Using Monoclonal Antibodie

Abstract: Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline- O -acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations, although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.     

Novel separation method for serum immunoglobulins. Application to thyroid related antibodies

A dye-based affinity chromatographic system using Remazol yellow GGL-Sepharose is described for the fractionation of serum immunoglobulins. Immunoglobulins are sequentially eluted from the gel columns using gradients of pH and salt with greater than 88% recovery. Specific immunoglobulin activities were identified as discrete peaks and antibodies raised against the same antigen were separated. Biological properties of antibodies were retained following chromatography. The method is applicable to both human and animal immunoglobulins.     

Attempt to isolate from Ascaris suum an antigenic fraction for use in the immunologic diagnosis of onchocerciasis

For the purpose of immunological diagnosis of onchocerciasis, the authors attempted to isolate an antigenic fraction from Ascaris suum which would only detect anti-Onchocerca volvulus antibodies. Chromatofocusing was done on a crude antigenic preparation from the ascaris. Immunoelectrophoresis showed that the fraction eluted between pH 5.6 and pH 4.9 contained antigens which reveal anti-Onchocerca antibodies; anti-Loa loa antibodies are revealed by the fraction eluted at pH less than 4. From the fraction which reveal anti-Onchocerca antibodies, affinity chromatography isolated a system in which three components were detected by electrofocusing at a Pi of about pH 5.5. The specificity and sensitivity of this system are being studied.     

Targeted protein pullout from human tissue samples using competitive elution

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.     

Elution of antibodies from a Protein-A column by aqueous arginine solutions

Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.     

Utilization of antipeptide antibodies as affinity ligands in immunoaffinity purification

Anti-peptide antibodies against the C-terminal regions of chimeric α-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The proteins were purified to high purity using the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides.     

Preparative isoelectric focusing of immunologically reactive components of Aspergillus fumigatus mycelium

A water-soluble mycelial extract of Aspergillus fumigatus has been fractionated by preparative isoelectric focusing using carrier ampholytes in a layer of granulated gel. The separated components were located by staining paper prints from the gel. Within a narrow pH range of 2.5 units, multiple protein bands were visualized with Coomassie Brilliant Blue G. Periodate-Schiff-positive material was generally associated with the major protein zones. When these fractions were eluted the total recovery, calculated on the basis of protein and carbohydrate analyses of the isolated fractions, varied between 20 and 60% of the applied material. Low recoveries were associated with low recoveries of protein; recoveries of carbohydrate were higher and less variable. The immunological activity and specificity of the eluted fractions were assessed in an enzyme-linked immunosorbent assay for the detection of IgG antibodies to A. fumigatus.     

Anti-idiotypic T lymphocyte responsiveness in murine Schistosomiasis mansoni

Pooled sera from CBA/J mice infected for greater than or equal to 16 weeks with the blood fluke Schistosoma mansoni were immunoaffinity purified using soluble schistosome egg antigens (SEA) coupled to Sepharose 4B. The bound and then eluted fraction was shown to contain only immunoglobulins and to have anti-SEA activity. These anti-SEA antibodies stimulated proliferation of lymph node cells from mice infected with S. mansoni for 8, 12, or greater than or equal to 16 weeks but not from uninfected mice. The cells stimulated by anti-SEA antibodies were nylon wool adherent, Thy-1.2+, L3T4+, Lyt-2-lymphocytes. Immunoglobulins without anti-SEA activity isolated from the sera of syngeneic uninfected mice were not stimulatory for cells from normal or infected animals. Thus the responding T cells appear to be stimulated by the idiotypes expressed on the syngenic anti-SEA antibodies. These data present evidence for anti-idiotypic cellular reactions in murine schistosomiasis that could play important immunoregulatory roles in this disease.     

Isolation of antiserum against rat transcortin and characteristics of specific antibodies

Repeated chromatography of rat plasma protein on DEAE-cellulose, hydroxylapatite and subsequent gel-filtration through Sephadex G-200 were used to obtain a pure rat transcortin homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of transcortin was about 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Immunization of a rabbit with the homogeneous preparation of rat plasma transcortin caused development of antibodies to transcortin. It was shown that the antibodies of rabbit antisera in the experiments made in vitro and in vivo neutralized 60 and 65% of 3H-corticosterone-transcortin complexes, respectively. Specific antibodies to the transcortin were isolated from the homogeneous fraction of IgG by affinity chromatography on transcortin-sepharose 4B. 125J-labelled antibodies were adsorbed by protein A-sepharose; IgG can be eluted by IM acetic acid as a sharp peak. The SDS-polyacrylamide gel electrophoresis demonstrated that affinity-eluted material contains 25 and 50 kDa polypeptides.     

Solid phase immunoadsorption for therapeutic and analytical studies on neuropathy-associated anti-GM1 antibodies

Autoimmune neuropathies including Guillain-Barré syndrome are frequently associated with anti-GM1 ganglioside antibodies. These are believed to play a pathogenic role and their clearance from the circulation would be predicted to produce therapeutic benefit. This study examines the conditions required for effective immunoadsorption of anti-GM1 antibodies using glycan-conjugated Sepharose as a matrix. In solution inhibition studies using a range of GM1-like saccharides in conjunction with mouse and human anti-GM1 antibodies, the whole GM1 pentasaccharide beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1-4)-beta-Glc was the favored ligand for maximal inhibiton of antibody-GM1 interactions in comparison with monosaccharides, Gal-(1-3)-beta-GalNAc-betaOMe, and synthetic GM1 mimetics. Immunoadsorption studies comparing binding of mouse monoclonal anti-GM1 antibodies to GM1-Sepharose and beta-Gal-(1-3)-beta-GalNAc-Sepharose confirmed the preference seen in solution inhibition studies. GM1-Sepharose columns were then used to adsorb anti-GM1 immunoglobulin G and immunoglobulin M antibodies from human neuropathy sera. Anti-GM1 antibodies subsequently eluted from the columns often showed a striking monoclonal or oligoclonal pattern, indicating that the immune response to GM1 is restricted to a limited number of B-cell clones, even in the absence of a detectable serum paraprotein. These data support the view that immunoadsorption plasmapheresis could potentially be developed for the acute depletion of serum anti-GM1 antibodies in patients with neuropathic disease, and also provide purified human anti-GM1 antibodies for analytical studies.