The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.     

Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,)

The neonatal Fc receptor (FcRn) performs two distinct but related functions: transport of maternal immunoglobulin G (IgG) to pre- or neonatal mammals, thus providing passive immunity, and protection of IgG from normal serum protein catabolism. FcRn is related to class I MHC proteins but lacks a functional peptide binding groove. The crystal structure of human FcRn has been determined at 2.7 A resolution and compared to the previously described structure of rat FcRn [Burmeister et al. (1994) Nature 372, 336-343] and to the structures of MHC and MHC-related proteins. Human FcRn is structurally similar to the rat receptor but does not form receptor dimers in the crystals as observed in crystals of rat FcRn. The interaction between human FcRn and IgG was characterized by determining the binding stoichiometry using equilibrium gel filtration and by deriving binding affinities for the different human IgG subclasses using a surface plasmon resonance assay. Like rat and mouse FcRn, human FcRn interacts with IgG with a 2:1 receptor:ligand stoichiometry. The binding of human FcRn to the four human IgG subclasses shows subclass and allotype variations but no clear subclass affinity differences that correlate with serum half-lives. The structure of human FcRn and studies of its ligand binding are relevant to current efforts to use FcRn-mediated regulation of IgG half-life in serum to increase the lifetimes of antibody-based therapeutics.     

Quantitative measurement of mouse IgG subclasses with the use of heteroantisera: the importance of allotype considerations.

Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.     

Plasma proteins and immunoglobulins in penguins (Pygoscelis adeliae).

Total and fractionated proteins were evaluated in the serum of normal penguins. Four proteins with antibody activity, determined by passive haemagglutination, were isolated by Sephadex G-200 filtration and DEAE-cellulose chromatography, from the serum of penguins (Pygoscelis adeliae) inoculated with dinitrophenylated human gamma-globulin. Immunoelectrophoresis and immunodiffusion showed that all these proteins exhibited both their own and shared antigenic determinants. On account of their electrophoretic mobility, elution conditions and characteristics of the precipitation arcs, they have been tentatively denominated IgG2 (gamma2), IgG1 (gamma1), IgM (gammam) and IgX (probably IgA).     

Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.     

Characterization of tyrosyl kinase activity in human serum

Tyrosyl kinase activity was detected in 1.0 microliter or less of human serum with the substrates angiotensin II, polyamino acid polymer Glu-Tyr (4:1), anti-pp60src IgG, and endogenous serum proteins. Most (about 84%) of the tyrosyl kinase activity was in the 100,000 X g soluble fraction from serum and a similar level of activity was found in the soluble fraction from plasma. Expression of tyrosyl kinase activity in individual serum samples differed more than 15-fold. The different levels of tyrosyl kinase activity were not likely due to phosphatases, proteases, ATPases, or kinase inhibitors and activators in serum. The normal serum and plasma tyrosyl kinase activities were not stimulated by epidermal growth factor, platelet-derived growth factor, insulin, or growth factors from fetal calf serum. Investigations of samples from patients with malignant disorders indicated that those with malignant melanoma contained the highest levels of serum tyrosyl kinase activity.     

Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo

Schistosomula of Schistosoma mansoni are known to be killed in vitro by complement and IgG (lethal antibody). To investigate whether this mechanism reflects the in vivo situation, we isolated IgG subclasses from sera of infected rats and assayed their ability to promote the complement-mediated killing of schistosomula in vitro as well as to protect normal recipients from a challenge infection. We found that a serum fraction containing only IgG2a + IgG2b has lethal activity to schistosomula in vitro, whereas a fraction containing only IgG1 + IgG2c fails to kill schistosomula in the presence of complement. The assay of protective activity has shown that the same fraction containing the lethal activity (IgG2a + IgG2b) was able to reduce the number of schistosomula recovered from lungs. These results provide evidence of the participation of IgG2a and/or IgG2b, but not IgG1 or IgG2c, in protective immunity to S. mansoni in rats, possibly through a complement-mediated mechanism.     

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants

Summary Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.     

Anomalous Raji cell activity related to DNA binding in some patients with autoimmune disease

We examined sera from patients with autoimmune disease that showed a discrepancy between the Raji cell assay and other tests for circulating immune complexes where the Raji cell activity was highly elevated. Using gel filtration chromatography, we found that the Raji cell activity was associated with monomeric IgG and little evidence of aggregates in the samples. Samples elevated for circulating immune complexes by all tests showed aggregates with associated Raji cell activity. The activity in discrepant samples was decreased by up to 40% by absorption of the IgG fraction with DNA-cellulose prior to the Raji cell assay. It is suggested that binding by autoantibodies to the Raji cell membrane is due to a variety of mechanisms.     

Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea.

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.     

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti‐streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti‐streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV‐AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8‐Anilino‐1‐naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near‐UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β‐sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ～2.4‐fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4‐fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

FCgammaRII (CD32)-dependent induction of interferon-alpha by serum from patients with lupus erythematosus

Interferon-alpha (IFN-alpha) is detected in the serum of 70-80% of patients with systemic lupus erythematosus (SLE). Furthermore, soluble factors in SLE serum can induce peripheral blood mononuclear cells (PBMC) to produce IFN-alpha. The purpose of this work was to investigate the mechanism of this IFN-alpha induction. In eleven of fifteen SLE serum samples, an IFN-alpha inducing activity was detected, whereas serum from healthy controls, patients with other autoimmune disease and patients with viral infections were ineffective under the same conditions. After gel filtration of the serum, the inducing activity was found in the same fraction as IgG. The IFN-alpha inducing activity was inhibited by native monoclonal antibodies to the receptors for the Fc portion of IgG: FcgammaRIIA/C and FcgammaRIIB subclasses (CD32) and by their F(ab)'2 fragments. Purified Fc fragments of human IgG were also effective in abolishing the IFN-alpha-inducing activity. Since no anti-CD32 autoantibodies were found in SLE serum, this IFN-alpha-inducing activity may be due to immune complex antibodies. Such results may allow better understand the origin of endogenous IFN-alpha, which has a deleterious effect on the course of this autoimmune disease. The inhibition of this function by the CD32 antibody could lead to new therapeutic approach in SLE.     

Characterization and Partial Purification of AIM: A Plasma Protein That Induces Rat Cerebral Type 2 Astroglia from Bipotential Glial Progenitors

Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte-type 2 astrocyte (O-2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia-inducing molecule (AIM). Using cultures enriched in O-2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240-fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin-agarose, followed by gel filtration chromatography. In crude preparations, AIM activity migrated at 50 kDa by gel filtration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of greater than 9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O-2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects. AIM may be a novel differentiation factor.     

Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides.

Inhibitor antibodies of blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic patients. anti-FVIII antibodies may also develop in a variety of disease-associated autoimmunity. Mapping of human FVIII inhibitors in haemophilia A or autoantibody origin have delineated three major clusters of B-cell inhibitory epitopes (domain A2, A3 and C2). Inhibitory and non-inhibitory FVIII antibodies have also been described in plasma of healthy donors and pools of immunoglobulins. The purpose of this study was to use synthetic FVIII-peptides to more closely define regions of the molecule targeted by natural anti-FVIII antibodies. Predictive algorithms were used for defining the positions of potential continuous epitopes. To investigate the presence of peptide-reactive antibodies in normal plasma pools of healthy donors, a plasma fraction (Cohn fraction II+III) containing all IgG subclasses was purified by affinity chromatography on peptide-Sepharose columns. The results of ELISAs and Western blotting experiments (with the selected peptides and well-defined recombinant FVIII thrombin fragments) confirmed the reaction specificities of the affinity-purified human antibodies. For each IgG preparation, the isotopic subclass was also determined. In the clotting assay, several IgG preparations showed neutralising activity in a dose-dependent manner. Our observations support the recent hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.     

IgG subclass distribution among antibodies to varicella-zoster virus in human varicella/zoster immunoglobulin preparations and the corresponding donor plasma

IgG subclasses to varicella-zoster virus (VZV) were detected in plasma from different sources used for the production of varicella/zoster immunoglobulin (VZIG). IgG1 and IgG3 were the principal virus antibodies in plasma from healthy donors as well as from convalescents after primary and reactivated disease. Anti-VZV IgG3 antibodies were predominant among varicella convalescents while IgG1 antibodies dominated among zoster convalescents. IgG4 antibodies were present in zoster convalescents and healthy donors but were rarely detected in varicella convalescents. Antiviral IgG2 antibodies were found only in a few cases. Studies of plasma samples collected from one varicella convalescent during a period of seven months following an outbreak of disease, demonstrated a rapid fall in antiviral IgG1 and IgG3, while IgG4 increased to reach a maximum six months after the onset of symptoms. The relative distribution of VZV-specific subclasses in a plasma pool was conserved during a fractionation procedure combining polyethyleneglycol 6000 precipitation with ion exchange chromatography, thus suggesting that the protective efficacy is maintained in the resulting immunoglobin preparations.     

Trypanosoma brucei: some properties of the cytotoxic reaction induced by normal human serum.

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α₂-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.