Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Tritiation of protein antigens of Plasmodium knowlesi schizont-infected erythrocytes using pyridoxal phosphate-sodium boro[3H]hydride

The protein antigens of Plasmodium knowlesi schizont-infected red blood cells (SI-RBCs) and normal RBCs were compared using the pyridoxal phosphate/NaB³H₄ method. Permeation of the outer SI-RBC membrane by the pyridoxal phosphate anion was enhanced since unlike normal RBCs it was not possible to exclusively label surface membrane proteins without concurrent haemoglobin labelling. Under conditions of minimal haemoglobin labelling a subset of total susceptible SI-RBC proteins (M_r 125 000, 50 000, 45 000 and 30 000) were labelled that were absent from normal RBCs and which may be surface proteins. The M_r 125 000 band labels much more readily than Band 3, the normal anion transporter, suggesting that it may be a new anion transporter in the SI-RBC membrane. At higher pyridoxal phosphate concentrations additional bands (M_r 230 000, 180 000, 165 000, 145 000, 107 000 and 72 000) were labelled exclusively with SI-RBCs. The new pyridoxal phosphate-labelled proteins had altered electrophoretic mobility and reduced Coomassie Blue staining, both properties assisting in their identification. Antigen analysis using Protein A-Sepharose and sera from infected monkeys demonstrated that all new ³H-labelled proteins were SI-RBC-specific antigens. One very high M_r antigen (> 250 000) recognized only by homologous strain antisera may represent a strain-specific antigen.     

Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates

Promastigotes of Leishmania adleri were submitted to an extraction procedure providing different carbohydrate-containing extracts. The purified aqueous extract showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a complex peptide pattern but carbohydrate was present only in bands of M_r ≈ 45 000–50 000 and 13 500. Methylated derivatives of the hexose components in this extract, analysed by mass spectrometry, suggest the presence of short sugar chains of α-d-mannopyranose and a branched α-d-mannan. The phenol extract, released in the aqueous layer a chloroform/methanol/water soluble complex contained 25% protein, 17% phosphate, 11% glucosamine, uronic acid and 61% neutral carbohydrate, and a chloroform/methanol/water insoluble fraction consisting of a glycoprotein M_r ≈ 22 000 and a proteic doublet M_r ≈ 58 000–66 000. A polysaccharide, showing galactose as predominant sugar, was released through alkaline extraction corresponding to a branched, mainly 1→3 linked galactan associated with α-d-mannopyranosyl units.     

The dependence on external cations of the oxygen consumption of mammalian non-myelinated fibres at rest and during activity

1. A study has been made of the effect of potassium and other cations on the oxygen consumption of rabbit desheathed vagus nerves at rest and during activity.2. In normal Locke solution (containing 5·6 mM-K) the resting oxygen consumption, Q_r, was 0·0903 μmole/g wet wt. min. The extra oxygen consumed as a result of stimulation, Q_s, at 3 stimuli/sec was about 600 p-mole/g.impulse; at 30 stimuli/sec it was less, being 240 p-mole/g.impulse.3. Only a fraction of Q_r (18% at 5·6 mM-K) was sensitive to ouabain (1 mM). The ouabain-sensitive component, however, increased as the external potassium concentration was increased, in the range 0-100 mM. Q_s was virtually abolished by ouabain.4. Reduction of the external potassium concentration from 5·6 mM to zero reduced Q_r (by 10%) and increased Q_s, but the changes were scarcely significant statistically.5. The conclusions were drawn: that Q_s reflected the pumping of cations to restore the ionic imbalance following activity, particularly reflecting the extrusion of sodium ions from the fibre; that this pumping was normally absolutely dependent on the presence of potassium externally and that no pumping could occur in its absence; and that Q_s was not reduced to zero in ostensibly potassium-free solutions because enough potassium was released into the periaxonal space during activity to maintain pumping.6. Thallium, rubidium, caesium and lithium could replace potassium and allowed pumping to occur.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

Large-scale isolation of the two major polypeptides of the hepatitis B surface antigen (HBsAg) by gel filtration in 6 M guanidinium chloride

Purified hepatitis B surface antigen (HBsAg) of subtype ay was solubilized in guanidinium chloride and submitted to chormatography on Sepharose 4B in the presence of guanidinium chloride. The polypeptides P1 (M_r = 24,000) and P2 (M_r = 29,000) were eluted in the same fraction with a minor contaminant (M_r = 40,000). Large amounts of these two polypeptides were obtained in a single step. This technique which constitutes a method for large-scale purification of the P1 and P2 polypeptides should permit more complete characterization of the P1 and P2 polypeptides and of their antigenic determinants.     

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. III. Changes in zygote surface proteins during transformation to mature ookinete

Proteins expressed on the surface of zygotes of Plasmodium gallinaceum during their development from fertilization to mature ookinetes have been examined by lactoperoxidase catalysed surface radioiodination and immunoprecipitation with stage-specific immune rabbit sera and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Surface-labelled proteins of apparent Mr equal to or greater than 55 000 on the female gametes and newly fertilized zygotes were shed during transformation and were recovered quantitatively and apparently intact from the culture supernatant. Zygote surface proteins of Mr 50 000, 19 000 and 17 000 remained bound to the surface throughout the transformation. Three proteins were expressed de novo on the surface of the mature ookinete of which Mr 26 000 and 28 000 represented major surface components and Mr 52 000 a relatively minor component.     

Plasminogen activator inhibitor induced by lipopolysaccharide injection in the rat Zymographic analysis

It has been shown that endotoxin injection induces formation of Plasminogen Activator Inhibitor (PAI). The present study shows that a decrease in euglobulin fibrinolytic activity 4 h after lipopolysaccharide (LPS) injection is associated with an increase in urokinase neutralisation, either by euglobulin or plasma. After 30 μg/kg LPS this inhibitor reaches 6 U/ml in euglobulin and 41 U/ml in plasma. Zymographic analysis of post LPS euglobulin shows evidence of an increased PAI resulting in a reduction of tissue plasminogen activator (tPA) (M_r 68 000) and formation of higher complexes (M_r, 105 000), but development time is critical for detecting these modifications simultaneously. Increase in a urokinase binding factor in post-LPS plasma or euglobulin is clearly demonstrated from reduction in urokinase lytic bands (M_r, 34 000 and 54 000) with a parallel generation of a large complex (M_r 80 000 to 105 000). From this shift, the mol. weight of PAI may be estimated to be 46 000–51000. Combination of Polymyxin B or Colimycin with LPS could prevent the decrease in euglobulin fibrinolytic activity in low endotoxemia.     

Inhibitory effects of rat alpha2 macroferoprotein (alphaMFP), an acute phase globulin, on galactosamine hepatitis.

Refractoriness to Gal N toxicity occurs especially in fetal rats, newborn rats, and in rats after partial hepatectomy. An injury however (laparotomy, incision on the back or ip BaSO₄ suspension), prior to Gal N administration, also inhibits Gal N toxicity. In all these circumstances high levels of rat α₂-macrofetoprotein (α_MFP) occur. This protein is an acute phase reactant and is identical to rat α₂-macroglobulin. α_MFP isolated from the serum of injured rats and then administered to normal rats strongly inhibits Gal N toxicity. When time interval between the preceding injury, provoking α_MFP production and Gal N administration shortens, the inhibiting effects are less and α_MFP production remains low.During resistance to Gal N, the primary and secondary biochemical lesions of Gal N persist and the protecting effect of α_MFP must be due to another mechanism, operating in later phases of cell injury. Very probably this is attributable to the stabilizing effect on membranes of hepatocytic organelles and the plasma membranes. As α_MFP is an acute phase reactant the importance of these proteins to the course of hepatitis must be considered.     

Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.     

Altered protein patterns in brains of children with neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative diseases characterized by massive intralysosomal accumulation of storage materials. We have studied the protein patterns in 5 NCL, 5 control, and one Alzheimer disease brains. When protein patterns in NCL and control brain gray matter homogenates were examined by SDS-PAGE, NCL brains showed an absence or greatly reduced amounts of the M_r 160–180 kDa component and reduced amounts of the M_r 29–36 kDa component. Concomitantly, an increase in several components with M_rs of 45–50 kDa was noted. The 180 kDa polypeptide appears to be a glycoprotein because it was bound to the lectins concanavalin A and Ulex europaeus. Recently, the abnormal processing of amyloid protein precursor (APP) and its potential role in NCL have been suggested. Possible defects in tissue proteases and protease inhibitors may be considered responsible for the presence of these amyloid beta protein precursor fragments. To examine this possibility we are using polyclonal antibodies to the C terminal 672–695 (APP) and monoclonal antibodies to inter-α-trypsin inhibitor. Polypeptides with molecular weights of approximately 35–38 kDa were detected in the NCL brain, but not in controls in both cases. These findings suggest abnormal protein processing in NCL brain tissue, disturbances in protein and glycocon-jugate metabolism, impaired lysosomal function (i.e., metabolic enzyme and/or proteases/ proteinase inhibitor abnormalities), and the involvement of improperly processed APP.     

The oxygen consumption of mammalian non-myelinated nerve fibres at rest and during activity

1. A study has been made of the oxygen consumption of non-myelinated nerve fibres of rabbit desheathed cervical vagus nerves at rest and during activity.2. The average resting oxygen consumption (Q_r) was 0·0924 μmole/g. min at 21° C. Stimulation for 1-3 min at 3/sec caused an extra oxygen consumption (Q_s) of 816 p-mole/g.shock.3. When the frequency of stimulation was increased, to 10/sec and 30/sec, Q_s fell. When the frequency was decreased, to 1/sec and 0·3/sec, Q_s increased slightly.4. When the temperature was decreased, Q_r fell; when the temperature was increased, Q_s also increased. Temperature similarly affected Q_s with high frequencies of stimulation, but had relatively little effect on Q_s at low frequencies of stimulation.5. An isolated single shock seemed to produce an increase in oxygen consumption of about 1200 p-mole/g, and this value was largely independent of temperature.6. When part of the sodium in the Locke solution was replaced by barium, Q_r decreased (by 12%) whereas Q_s increased (by 87%).7. Veratrine (1 μg/ml.) increased both Q_r (by 142%) and Q_s (by 361%).8. Acetylcholine (1·7 mM) increased Q_r (by 32%).9. When nerves were transferred to potassium-free solutions there was little change in Q_r, and Q_s fell slightly (by 8%).10. When the potassium concentration in the Locke solution was increased 4-fold, Q_r increased (by 27%).11. Salicylate (1-10 mM) increased Q_r (by 24%) and abolished Q_s.12. When the sodium of Locke solution was replaced by lithium, Q_r decreased (by 19%) and Q_s was abolished.13. In sodium-Locke solution ouabain (100 μM) decreased Q_r (by 26%) and abolished Q_s. In lithium-Locke solution ouabain also decreased Q_r (by 28%).14. All or nearly all of the oxygen consumed at rest or during activity seemed to be used to pump potassium ions into, and sodium ions out of, the axoplasm.15. The K/O₂ ratio during pumping was about 5·0.     

Deletion of the Central Proline-Rich Repeat Domain Results in Altered Antigenicity and Lack of Surface Expression of the Streptococcus mutans P1 Adhesin Molecule

Members of the family of surface adhesins of oral streptococci, including P1 of Streptococcus mutans, contain two highly conserved repeat domains, one rich in alanine (A region) and the other rich in proline (P region). To assess the contribution of the P region to the biological properties of P1, an internal deletion in spaP was engineered. In addition, the P region was subcloned and expressed as a fusion partner with the maltose binding protein of Escherichia coli and liberated by digestion with factor Xa. Results of Western blot experiments in which recombinant polypeptides were probed with a panel of 11 monoclonal antibodies indicated that the P region is a necessary component of conformational epitopes within the central portion of P1. Antibodies reactive with the P region were detected in a polyclonal rabbit antiserum generated against whole S. mutans cells but not in two rabbit antisera generated against purified P1 (M_r ~ 185,000), suggesting that this domain is immunogenic on the surface of intact bacteria but not as part of a soluble full-length molecule. Finally, transformation of a spaP-negative mutant with a shuttle vector containing an internally deleted spaP lacking P-region DNA resulted in a complete absence of surface-localized P1 and substantially less P1 in sonicated cells compared to the case for the mutant complemented with the full-length gene. These results suggest that the P region is an integral component contributing to the conformation of the central region of P1 and indicate that its presence is necessary for surface expression of the molecule on S. mutans.     

Intracellular protein degradation in Leishmania tropica promastigotes

A labile class of proteins in the range of M_r = 30 000–60 000 which turn over rapidly have been demonstrated in Leishmania tropica promastigotes. The rate of protein degradation is increased by exhaustion of nutrients or inhibition of energy metabolism. Proteolysis is reduced when the parasites are provided with a readily utilizable carbon and energy source such as glucose, glutamate or proline. The breakdown of proteins in L. tropica is dependent on continuous protein synthesis probably for protease synthesis. It is suggested that the relatively high rate of intracellular protein degradation is an auxiliary means for generating carbon and energy sources during nutritional stress.     

N-terminal amino acid sequence of the histidine-rich protein from Plasmodium lophurae

We have determined the N-terminal amino acid sequence of the first 25 amino acids of the histidine-rich protein (HisRP) isolated from granules of the avian malaria parasite Plasmodium lophurae. The protein was purified from cytoplasmic granules and shown to be 65.2 mol % histidine, close to the previously described value of 73 mol % histidine (Kilejian (1974) J. Biol. Chem. 249, 4650–4655). Ten of the first 25 residues were histidine, five of which formed the sequence His-His-His-His-His (positions 14–18). Also notable was the presence of eight acidic residues within the N-terminal 25 residues. HisRP contained no detectable carbohydrate. When the HisRP was biosynthetically labeled in cultured infected erythrocytes, incorporation of [³H]His greatly exceeded [³H]Ile. Labeled HisRP was not solubilized with 1% w/v Triton X-100 but could be solubilized with ? 1% w/v sodium dodecyl sulfate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the [³H]His labeled protein migrated as a doublet (M_r 53 000 and 50 000). Only one of these bands (M_r 53 000) comigrated with the Coomassie Blue stained protein isolated by the acid-extraction procedure from purified granules. The amino acid composition of HisRP and presence of five contiguous histidine residues in the sequence studied here suggests that other sequences of several contiguous histidine residues must exist in this molecule.     

Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

ABSTRACT: In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium-3,5-diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona-IgG was raised by immunization of female rabbits with 500 heat-solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (M_r = 42,000), ZP II/1 (M_r = 67,000), ZP II/2 (M_r = 32,000), ZP III/1 (M_r = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high-pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O-glycosidic-glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.     

TechLab and Alexon Giardia Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1

A Giardia lamblia antigen detected by the TechLab Giardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (M_r 22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (M_r 26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases and N- and O-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins with M_rs of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.     

The use of protein A and concanavalin A to examine the possible role of the carbohydrate of IgG in the binding of Clq

The suggestion that Fc-carbohydrate of rabbit IgG becomes accessible to conA on cross-linking by Staphylococcal protein A and can play a role in fixing complement (Langone et al., 1978a) was examined specifically for its relevance to Clq binding.On chromatography of pooled rabbit IgG on conA-Sepharose, 80 ± 5% of IgG shows no interaction with conA—Sepharose. Five per cent of IgG is bound and eluted with 0.1 M 1-O-methyl-α-d-glucopyranoside.The apparent K_d of the unretarded fraction for conA was less than 140 μM, whereas that of pooled IgG was 14 μM, as measured by competition for ¹²⁵I-labelled conA with guinea pig erythrocytes.No change in the K_d of the unretarded fraction of IgG for conA could be detected in the presence of protein A. A change of up to two-fold was observed in pooled IgG, and must be a result of heterogeneity of the carbohydrate in either the Fab or the Fc region of IgG.The fraction of IgG which did not bind conA showed the same Clq-binding activity as pooled IgG in ¹²⁵I-labelled Clq binding assays, so that there can be no direct relation between conA-binding activity and Clq binding.The binding of Clq to IgG was not affected by concentrations of conA (1mg/ml) and human transferrin glycopeptide (2 mM) under conditions where inhibition would be expected if complete exposure of the Fc-carbohydrate to solution was important for binding Clq.     

The interaction of immune serum globulin and immune globulin intravenous with complement

The in vitro anticomplementary activity of untreated and heat-aggregated (63°C, 10 min) immune serum globulin (ISG) and immune globulin intravenous (IGIV) prepared by partial reduction and alkylation have been evaluated by three assays, C3 activation, binding to Clq and enhancement of alternative pathway lysis of rabbit erythrocytes. Crossed immunoelectrophoresis was used to quantitatively measure the ability of ISG and IGIV to activate endogenous C3 in normal serum. Binding to Clq was determined according to the ability to inhibit binding of ¹²⁵I-Clq to solid phase IgG. ISG and IGIV enhancement of lysis of rabbit erythrocytes by normal human serum adsorbed with rabbit erythrocytes in the presence of MgEGTA was used to determine activity in the alternative complement pathway. Unheated IGIV at 10mg/ml only marginally activated endogenous C3 in normal serum, had about a 5-fold lower affinity for ¹²⁵I-Clq (K_i = 138 to 356 μM vs K_i = 62.5 μM for ISG), but was very similar in ability to ISG on a weight basis in enhancing complement alternative pathway activity (RCH50 = 0.23 to 0.40 mg for IGIV vs 0.17mg for ISG). Heat-aggregated IGIV at 5 mg/ml in normal human serum was about 2-fold less effective than heat-aggregated ISG in the activation of C3 in normal serum and had approximately 2- to 3-fold lower affinity in the Clq binding assay (K_i = 45 to 83 nM for heat-aggregated IGIV vs K_i = 14.6 nM for heat-aggregated ISG). These data suggest that IGIV prepared by chemical modification retains sufficient specific receptor activity to allow in vivo efficacy in complement-mediated amplification of host defense reactions, but is safe for intravenous use due to a lower capacity to initiate nonspecific complement activation.