Multiple Sclerosis: Enzymatic Cross Site-Specific Hydrolysis of H1 Histone by IgGs against H1, H2A, H2B, H3, H4 Histones, and Myelin Basic Protein.

Histones play a key role in chromatin remodeling and gene transcription. Further, free histones in the blood act as damage-associated molecules. Administration of histones to animals results in systemic inflammatory and toxic effects. Myelin basic protein is the principal constituent element of the myelin-proteolipid sheath of axons. Abzymes (antibodies with catalytic activities) are the original features of some autoimmune diseases. In this study, electrophoretically homogeneous IgGs against H1, H2A, H2B, H3, and H4 histones and myelin basic protein (MBP) were isolated from the blood sera of multiple sclerosis (MS) patients by several affinity chromatographies. Using MALDI mass spectrometry, the sites of H1 histone cleavage by IgGs against H1, H2A, H2B, H3, H4, and MBP were determined. It was shown that IgGs against H1 split H1 at 12 sites, while the number of cleavage sites by abzymes against other histones was lower: H2A (9), H2B (7), H3 (3), and H4 (3). The minimum rate of H1 hydrolysis was observed for antibodies against H3 and H4. A high rate of hydrolysis and the maximum number of H1 hydrolysis sites (17) were found for antibodies against MBP. Only a few sites of H1 hydrolysis by anti-H1 antibodies coincided with those for IgGs against H2A, H2B, H3, H4, and MBP. Thus, the polyreactivity of complexation and the enzymatic cross-activity of antibodies against H1, four other histones, and MBP have first been shown. Since histones act as damage molecules, abzymes against histones and MBP can play a negative role in the pathogenesis of MS and probably other different diseases as well.

Antibodies against H3 and H4 histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

Histones and their posttranslational modified forms play pivotal roles in chromatin functioning and gene transcription. Also, histones are harmful when they enter the intercellular space; their administration to animals results in systemic inflammatory and toxic responses. Autoantibodies having enzymatic activities (abzymes) are the specific feature of several autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical proteases were purified from sera of HIV-infected patients by using several affinity chromatographies. In contrast to known canonical proteases, Abs from HIV-infected patients hydrolyzed exclusively only histones but no other control globular proteins. The H3 and H4 histone cleavage sites by antihistone IgGs were determined by matrix-assisted laser desorption/ionization mass spectrometry for the first time. Two clusters of H3 hydrolysis contain major (↕) and minor (*) cleavage sites: 18-K*Q*LA↕TK*A↕AR*KS↕A*P-30 and 34-G*VK*KPHR*YRPGTVA*L*R-50. H4 histone has only 1 cluster of cleavage sites containing additionally moderate (↓) cleavage sites: 15-A↕KR↕HR↕KVLR↓D*NIQ↓GIT*K-31. Sites of these histones cleavage correspond mainly to their known epitopes. It was surprising that most of the cleavage sites of histones are involved in the interaction with DNA of nucleosome core. Because histones act as damage-associated molecules, abzymes against H3 and H4 can play important role in pathogenesis of AIDs and probably other viral and immune diseases.

Hydrolysis and Dissolution of Amyloids by Catabodies.

Catalytic antibodies (catabodies) hold potential for superior immunotherapy because of their turnover capability and no or minimal induction of inflammatory responses. Catabodies neutralize and remove target antigens more potently than conventional antibodies. Depending on the catalytic rate constant, a single catabody molecule degrades thousands to millions of target molecules over its useful lifespan, whereas conventional antibodies only form reversibly associated, stoichiometric complexes with the target. Thus, removal of the antibody-bound target requires accessory phagocytic cells that ingest the immune complexes, which is usually accompanied by release of inflammatory mediators. In comparison, catabodies bind the target only transiently, and the rapid and direct target destruction reduces the concentration of immune complexes that can activate inflammatory processes. These features are especially pertinent when large target amounts at anatomically vulnerable sites must be removed, e.g., amyloids. We reported specific catabodies to misfolded transthyretin (misTTR) amyloid and amyloid ß peptide (Aß). Accumulation of the oligomeric and fibrillized amyloid TTR forms causes diverse systemic pathologies, including cardiomyopathy, polyneuropathy, and skeletal diseases. Brain Aß aggregates are thought to cause central nervous system degenerative disease, chiefly Alzheimer's disease. We describe methods for testing catabody-mediated degradation and dissolution of Aß and TTR.

New Genetic Constructs for Generation of Stable Therapeutic Antibodies to Organophosphorus Toxins in Methylotrophic Yeasts Pichia Pastoris.

We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product.

A novel method of preparing the monoform structure of catalytic antibody light chain.

Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.

Desialylation of dying cells with catalytically active antibodies possessing sialidase activity facilitate their clearance by human macrophages.

Recently we reported the first known incidence of antibodies possessing catalytic sialidase activity (sialidase abzymes) in the serum of patients with multiple myeloma and systemic lupus erythematosus (SLE). These antibodies desialylate biomolecules, such as glycoproteins, gangliosides and red blood cells. Desialylation of dying cells was demonstrated to facilitate apoptotic cell clearance. In this study we assessed the possibility to facilitate dying cell clearance with the use of F(ab)2 fragments of sialidase abzymes. Two sources of sialidase abzymes were used: (i) those isolated from sera of patients with SLE after preliminary screening of a cohort of patients for sialidase activity; and (ii) by creating an induced sialidase abzyme through immunization of a rabbit with synthetic hapten consisting of a non-hydrolysable analogue of sialidase reaction conjugated with bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Antibodies were purified by ammonium sulphate precipitation, protein-G affinity chromatography and size exclusion-high performance liquid chromatography (HPLC-SEC). Effect of desialylation on efferocytosis was studied using human polymorphonuclear leucocytes (PMN), both viable and aged, as prey, and human monocyte-derived macrophages (MoMa). Treatment of apoptotic and viable prey with both disease-associated (purified from blood serum of SLE patients) and immunization-induced (obtained by immunization of rabbits) sialidase abzymes, its F(ab)2 fragment and bacterial neuraminidase (as positive control) have significantly enhanced the clearance of prey by macrophages. We conclude that sialidase abzyme can serve as a protective agent in autoimmune patients and that artificial abzymes may be of potential therapeutic value.

Catalytic antibodies and their applications in biotechnology: state of the art.

Catalytic antibodies are immunoglobulins endowed with enzymatic properties. Discovered in the second part of the 1980s, the enthusiasm they initially aroused was counterbalanced by the difficulty of their production and their low catalytic rates. Nevertheless, improvements in expression systems and engineering technologies, combined with various studies suggesting that catalytic antibodies play a role in the immune system, have opened the way to new applications for these proteins. Herein we review catalytic antibodies from a biotechnological point of view, focusing our study on the different production methods, expression systems and their potential clinical applications dedicated to these proteins.

Proteolytic activity of IgGs from blood serum of Wistar rats at experimental rheumatoid arthritis.

The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

Strategies for the selection of catalytic antibodies against organophosphorus nerve agents.

Among the strategies aimed at biocompatible means for organophosphorus nerve agents neutralization, immunoglobulins have attracted attention in the 1990's and 2000's both for their ability to immobilize the toxicants, but also for their ability to be turned into enzymatically active antibodies known as catalytic antibodies or abzymes (antibodies--enzymes). We will present here a critical review of the successive strategies used for the selection of these nerve agent-hydrolyzing abzymes, based on hapten design, namely antibodies raised against a wide variety of transition state analogs, and eventually the strategies based on anti-idiotypic antibodies and reactibodies.

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus.

In contrast to canonical proteases, myelin basic protein (MBP)-Sepharose-purified IgG from multiple sclerosis (MS) and systemic lupus erythematosus (SLE) patients efficiently hydrolyze only MBP, but not many other tested proteins. It was shown that anti-MBP SLE IgGs cleave nonspecific tri- and tetrapeptides with an extremely low efficiency and cannot efficiently hydrolyse longer oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. To identify all sites of IgG-mediated proteolysis corresponding to two AGDs of MBP, we have used a combination of reverse-phase chromatography (RPhC), MALDI spectrometry, and TLC to analyze the cleavage products of two (17- and 19-mer) encephalytogenic oligopeptides corresponding to these AGDs. Both oligopeptides contained several clustered major and minor sites of cleavage. The active sites of anti-MBP abzymes are localized on their light chains, while the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high specificity of MBP hydrolysis. The affinity of anti-MBP abzymes for intact MBP was ∼10(3)-fold higher than for the oligopeptides. The data suggest that both oligopeptides interact mainly with the light chain of different monoclonal abzymes of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific.

Anti-integrase abzymes from the sera of HIV-infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides.

In contrast to canonical proteases, total immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies (Abs) from HIV-infected patients hydrolyze effectively only HIV integrase (IN), reverse transcriptase (RT), human casein, and serum albumin. Anti-IN IgG and IgM isolated by chromatography on IN-Sepharose hydrolyze specifically only IN but not many other tested proteins. Total Abs from HIV-infected patients hydrolyze not only globular proteins but also different specific and nonspecific tri-, tetra-, and 20- to 25-mer oligopeptides (OPs) with a higher rate than anti-IN Abs isolated using IN-Sepharose. A similar situation was observed for IgG from patients with multiple sclerosis and HIV-infected patients, which after purification on myelin basic protein (MBP)-Sepharose and RT-Sepharose specifically hydrolyze only MBP and RT, respectively. The active sites of all anti-protein abzymes are localized on their light chains, whereas the heavy chain is responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide the specificity of protein hydrolysis. The affinity of anti-IN and anti-MBP abzymes for intact IN and MBP is approximately 10(2)- to 10(5)-fold higher than for short and long specific and nonspecific OPs. The data suggest that all OPs interact mainly with the light chain of different Abs, which possesses a lower affinity for substrates, and therefore, depending on the OP sequences, their hydrolysis may be less specific or completely nonspecific. The data indicate that the relative activity of Abs not fractionated on specific protein sorbents in the hydrolysis of specific and nonspecific OPs can correspond to an average proteolytic activity of light chains of polyclonal Abs directed against many different proteins.

Affinity and catalytic heterogeneity and metal-dependence of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with systemic lupus erythematosus.

It was shown using enzyme-linked immunosorbent assay (ELISA) that titers of antibodies against human myelin basic protein (hMBP) in systemic lupus erythematosus (SLE) patients 4.2-fold higher than in healthy individuals, but 2.1-fold lower than in patients with multiple sclerosis (MS). Approximately 86% electrophoretically and immunologically homogeneous SLE immunoglobulin Gs (IgGs) purified using several affinity resins including Sepharose with immobilized hMBP specifically hydrolyze only hMBP but not many other tested proteins. Several rigid criteria were applied to show that the hMBP-hydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors. In contrast to MS IgGs, abzymes from SLE patients are more sensitive to ethylenediaminetetraacetic acid and less sensitive to specific inhibitors of serine-like proteases. We present the first evidence demonstrating a significant diversity of different fractions of SLE IgGs in their affinity for hMBP-Sepharose, the ability of IgGs to hydrolyze hMBP at different optimal pHs (5-10) and be activated by different metal ions: Ca(2+) > Mg(2+) ≥ Co(2+) ≥ Fe(2+) ≥ Ni(2+) ≥ Zn(2+) ≥ Cu(2+) ≥ Mn(2+) . Combinations of Ca(2+) + Mg(2+) and Ca(2+) + Co(2) lead to a significant increase in the antibody proteolytic activity as compared with Ca(2+) , Co(2+) , or Mg(2+) ions taken separately. Our findings suggest that the immune systems of individual SLE similar to MS patients can generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons and play an important role in pathogenesis not only MS but also SLE patients.

Metal dependent hydrolysis of ß-casein by sIgA antibodies from human milk.

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A-Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe approximately Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the ß-casein-hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine-Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease-like ß-casein-hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ∼1.2-1.9-fold after addition of external metal ions (Mg(2+) > Fe(2+) > Cu(2+) ≥ Ca(2+) ≥ Mn(2+)) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high ß-casein-hydrolyzing activity in the presence of individual external metal ions (Fe(2+) > Ca(2+) > Co(2+) ≥ Ni(2+)) and especially several combinations of metals: Co(2+) + Ca(2+) < Mg(2+) + Ca(2+) < Ca(2+) + Zn(2+) < Fe(2+) + Zn(2+) < Fe(2+) + Co(2+) < Fe(2+) + Ca(2+). The patterns of hydrolysis of a 22-mer oligopeptide corresponding to one of sIgA-dependent specific cleavage sites in ß-casein depend significantly on the metal used. Metal-dependent sIgAs demonstrate an extreme diversity in their affinity for casein-Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ- and κ-type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed.

Catalytic digestion of human tumor necrosis factor-α by antibody heavy chain.

It has long been an important task to prepare a catalytic antibody capable of digesting a targeting crucial protein that controls specific life functions. Tumor necrosis factor-α (TNF-α) is a cytokine and an important molecule concerned with autoimmune diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn's disease. A mAb (ETNF-6 mAb) raised against human TNF-α was prepared, and the steric conformation was created by using molecular modeling after the cDNA was sequenced. The heavy chain (ETNF-6-H) of the mAb was considered to possess a catalytic triad-like structure in the complementarity determining regions (CDRs). As a result, ETNF-6-H exhibited a peptidase and a protease activity. In fact, ETNF-6-H predominantly cleaved the Ser5-Arg6 bond of TNF-α at the first step, resulting in the generation of a fragment of ∼ 17 kDa. This fragment was digested to a smaller molecule of 15 kDa by scission of the Gln21-Ala22 bond. The intermediate product was further converted into a fragment of 13.3 kDa by successive cleavage of the Leu36-Leu37 and Asn39-Gly40 bonds. The heavy chain possessed a protease activity against TNF-α with a multicleavage site.

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients.

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.

Immunoglobulin A with protease activity secreted in human milk activates PAR-2 receptors, of intestinal epithelial cells HT-29, and promotes beta-defensin-2 expression.

Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.

Improving GPX activity of selenium-containing human single-chain Fv antibody by site-directed mutation based on the structural analysis.

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.

Detection and characterization of IgG- and sIgA-Abzymes capable of hydrolyzing histone H1.

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity.

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol.