Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.     

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports

Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modern proteomics, the detection of very weak protein-protein interactions.     

The immunogenicity of soluble haptenated polymers is determined by molecular mass and hapten valence

T cell-independent Ag are believed to stimulate antibody formation in the relative absence of Ag processing and T cell help. Previous studies on the type 2 T cell independent (TI-2) Ag DNP-polyacrylamide, have shown that when one systematically varies the molecular mass and hapten valence, the immunogenic potential of this type of molecule depends on definable molecular characteristics. It was found that to be immunogenic, these molecules had to exceed a threshold molecular mass of 100,000 Da and a threshold hapten valence of 20. The present study was undertaken to determine whether such findings could be generalized to other molecules belonging to the TI-2 class of Ag. The molecular characteristics of five chemically different fluoresceinated (FL)-polymers were systematically varied, and their ability to stimulate an IgM antihapten immune response was measured. The polymers used as carriers were carefully size-fractionated and consisted of one natural polymer (dextran), one modified natural polymer (carboxymethyl cellulose), and three synthetic polymers (Ficoll, polyvinyl alcohol, and polyacrylamide). The carriers varied in physical structure from the highly cross-linked Ficoll, to the somewhat branched dextran, to the linear polyacrylamide, carboxymethyl cellulose, and polyvinyl alcohol. Polymers were haptenated with FL and size-fractionated so as to yield a panel of molecules with varying molecular mass, hapten valence, and hapten density. Anti-FL IgM response to these haptenated polymers was measured in vivo after i.p. injection of the FL-polymer in saline, and measured in vitro after culture with unfractionated spleen cells from naive mice. In agreement with the previous studies on DNP-polyacrylamide, it was found that to be immunogenic, each of the FL-polymers had to exceed a comparable threshold value of molecular mass and of hapten valence. Optimal immunogenicity occurred when the FL-polymers had values of mass and hapten density lying within a predictable range. Immunogenicity decreased when these optimal parameters were substantially increased or decreased. We conclude that the immunogenicity of soluble haptenated polymers depends on predictable physical molecular characteristics, and is relatively independent of the chemical composition and conformation of the carrier polymer.     

Photo-cross-linking between polymers derivatized with photoreactive ruthenium-1,4,5,8-tetraazaphenanthrene complexes and guanine-containing oligonucleotides

We have shown previously that complexes containing 1,4,5,8-tetraazaphenanthrene (TAP) ligands are able to form photoadducts with the guanine bases of DNA and oligonucleotides. In this work, we have exploited this specific photoreaction for carrying out photo-cross-linkings between guanine-containing oligonucleotides (G-ODNs) and biodegradable polymers derivatized with the photoreactive Ru(II) compounds. The aim in the future is to use these polymer conjugates as vectorizing agents of the metallic compounds inside the cells. Thus, photooxidizing Ru(II) complexes such as [Ru(TAP)3]2+ and [Ru(TAP)2phen]2+ (phen = 1,10-phenanthroline) have been derivatized by an oxyamine function to attach them, via an oxime ether linkage, to a soluble 6 or 80 kDa poly-[N-(2-hydroxyethyl)-l-glutamine] polymer that contains pendent aldehyde groups. It is demonstrated that the resulting Ru-labeled polymers exhibit photophysical properties and a photochemistry that are comparable with those of the free, nonattached complexes. The photo-cross-linkings with the G-ODNs are clearly detected by gel electrophoresis with the 6 kDa Ru conjugates upon illumination.     

Studies of reticuloendothelial function in the mouse with model immune complexes. II. Serum clearance, tissue uptake, and reticuloendothelial saturation in NZB/W mice.

We have extended the findings in the accompanying paper by characterizing the serum clearance and tissue uptake of model soluble immune complexes and the saturation of the reticuloendothelial system (RES) by these complexes in normal mice and in mice with murine lupus (NZB/W F1 females). Adult NZB/W or young C3H mice were injected with radiolabeled stable site-specifically cross-linked mouse anti-DNP oligomers as model immune complexes to probe RES function. Blood clearance and uptake by liver, spleen, and kidney were unimpaired in NZB/W mice. To determine if the RES exhibits partial saturation in the NZB/W mice, we deliberately induced a state of RES blockade with heat-aggregated human gamma-glubulin (HAG). With increasing doses of HAG (1 to 4 mg/20 g body weight) both strains similarly showed a progressive increase in RES saturation as measured by reduced liver uptake of model oligomers. Recovery from saturation was complete by 120 min in both strains. At maximal liver saturation there was only a small increase in oligomer uptake by kidney or spleen, the majority of complexes remaining within the circulation. Thus, RES capacity for handling soluble model immune complexes appears unimpaired in NZB/W mice.     

An efficient binding chemistry for glass polynucleotide microarrays.

A variety of methods have been described for making synthetic polynucleotide microarrays. These include in situ synthesis directly on the array surface, for example, by photolithography or ink-jet printing technologies, and the application of presynthesized polynucleotides that are derivatized with various nucleophiles or electrophiles. In the latter case, a variety of surface chemistries have been developed, and several are available commercially. These chemistries must be compatible with nanoliter-scale volumes of polynucleotide reagents, which contact the array over a small portion of their surface. We reasoned that a three-dimensional polymer coating could potentially offer greater surface contact and higher binding efficiency. Here we describe a polyethylenimine-based coating chemistry that provides exceptional binding and hybridization characteristics. In our preferred process, size-fractionated polyethylenimine polymers are cross-linked onto an aminopropylsilanated glass surface in the presence of cyanuric chloride. The resulting three-dimensional coating binds polynucleotides through a mixture of covalent and noncovalent interactions as evidenced by comparisons between 5'-aminoalkyl modified and unmodified polynucleotides. Binding and hybridization comparisons are presented including analogous two-dimensional electrophilic and electrostatic chemistries.     

Neurofilaments from ox spinal nerves. Isolation, disassembly, reassembly and cross-linking properties.

An isolation procedure for neurofilaments from ox spinal nerves is described where the triplet polypeptides (which have molecular weights of 205 000, 158 000 and 72 000) constitute more than 80% of the preparation. Soon after purification, the neurofilaments form a gel that is stable for many weeks. The purified neurofilaments disassemble in low-salt buffers at pH greater than 7.0 into soluble particles that contain all of the triplet polypeptides. Greater than 90% of the protein can reassemble to form filaments. The thiol-containing residues in the filaments can be cross-linked. Analyses of the complexes formed show that in the filament the 205 000-mol.wt. components are arranged to that they can be cross-linked to themselves and to the 158 000-mol.wt. polypeptides, and that the 72 000-mol.wt. components are arranged so that their thiol groups can be cross-linked together.     

Only the initial binding of cationic immune complexes to glomerular anionic sites is mediated by charge-charge interactions

The role of charge-charge interactions between cationic immune complexes and the anionic sites on the glomerular basement membrane was examined. For this purpose, soluble immune complexes at fivefold antigen excess were prepared with human serum albumin and cationized rabbit antibodies to this protein. When unrelated cationic proteins, protamine sulfate or cationized rabbit serum albumin, were given 1 min before the cationized immune complexes, glomerular immune deposits did not form. Cationic immune complexes allowed to deposit in glomeruli could readily be displaced by protamine sulfate or cationized rabbit serum albumin injected 1 min after the immune complexes. If the same cationic molecules were injected 1 hr after the immune complexes, the complexes could not be displaced from glomeruli. In contrast, cationic complexes that were deposited in glomeruli in the presence of a very high degree of antigen excess in circulation to prevent their condensation into larger complexes in glomeruli were readily displaced at 1 min and 1 hr with protamine sulfate or with cationized rabbit serum albumin. On the basis of these results, we concluded that the initial binding of cationic immune complexes to glomeruli occurs by charge-charge interactions. Once the immune complexes in glomeruli condense to larger deposits, forces other than charge-charge interactions are responsible for their retention in glomeruli.     

Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate.

A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

Stimulation of reactive oxidant production in neutrophils by soluble and insoluble immune complexes occurs via different receptors/signal transduction systems

Abstract Cell-free synovial fluid from patients with rheumatoid arthritis contains soluble and insoluble IgG-containing immune complexes which activate reactive oxidant production in human neutrophils. In this report we have measured the effects of inhibitors of signal transduction pathways on neutrophil activation by these complexes and also following activation by synthetic soluble and insoluble immune complexes made from human serum albumin (HSA) and anti-(HSA) antibodies. In all aspects studied, the soluble rheumatoid complexes and the soluble synthetic complexes were indistinguishable in the ways in which they activated neutrophils. Activation of reactive oxidant production in response to these soluble complexes was completely inhibited by pertussis toxin (indicating G-protein coupling of receptor occupancy), completely insensitive to staurosporine (indicating that oxidant production did not require protein kinase C activity), only marginally (<30%) inhibited by butanol (indicating that dependence upon activity of phospholipase D was minimal), and completely inhibited by chloracysine, an inhibitor of phospholipase A₂. In contrast, activation of reactive oxidant production in response to the insoluble rheumatoid or insoluble synthetic immune complexes was largely pertussis toxin insensitive, inhibited by >50% by staurosporine, inhibited by >50% by butanol, and completely inhibited by chloracysine. These results show that the receptor-mediated signal transduction systems activated by the soluble and insoluble immune complexes are different. Because the soluble complexes activate a transient burst of reactive oxidant secretion from primed neutrophils, the mechanisms regulating either the release or the intracellular production of oxidants within rheumatoid joints are distinct and hence may be pharmacologically modified independently of each other.     

Affinity separation of proteins in aqueous three-phase systems

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.     

Interleukin-1 receptor-like proteins synthesized by translation, in vitro, of immunopurified polysomal messenger RNA

Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons.     

Ability of modified forms of phenylalanine tRNA to stimulate guanosine pentaphosphate synthesis by the stringent factor-ribosome complex of E. coli

tRNA(Phe) of E. coli, modified at its 4-thiouridine ((4)Srd) and 3-(3-amino-3-carboxypropyl)uridine (nbt(3)Urd) residues, was tested for its ability to induce (p)ppGpp synthesis. The (4)Srd residue was derivatized with the p-azido-phenacyl group, cross-linked to Cyd(13), and the borohydride reduction product of the cross-link was prepared. The nbt(3)Urd residue was derivatized with the N-(4-azido-2-nitrophenyl)glycyl group. None of these derivatives had more than a minor effect on the affinity of the tRNA for the stringent factor-ribosome complex, and no effect at all on the maximum velocity of (p)ppGpp synthesis, either at 2 or 82 mM NH(4)Cl. These two regions of the tRNA which are on opposite faces of the tRNA molecule do not appear to be structurally important for recognition by the stringent factor-ribosome complex. They may provide useful sites, therefore, for the introduction of photoaffinity or fluorescent probes with which to study tRNA-stringent factor recognition.     

High affinity binding proteins for pancreatic polypeptide on rat liver membranes.

We report here the identification on rat liver plasma membranes and microsomes of proteins that bind pancreatic polypeptide (PP) with high affinity and specificity (plasma membranes: KD = 4.6 nM, Bmax = 3.28 pmol/mg protein; microsomes: KD = 3.45 nM, Bmax = 18.7 pmol/mg protein). These binding proteins appeared coupled to a G-protein, since 0.1 mM guanosine 5'-O-(3-thiotriphosphate) decreased the affinity by half. When 125I-labeled PP-binding protein complexes covalently cross-linked with disuccinimido suberate were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two radioactive bands with M(r) values of 52,000 and 38,000 were demonstrated. Both bands were inhibited by unlabeled PP with an IC50 of approximately 5 nM (but not by neuropeptide Y or peptide YY). After the cross-linked complexes were solubilized from liver microsomes with 0.2% Triton X-100 and gel-filtered, they did not interact with the lectins wheat germ agglutinin, Ulex europaeus agglutinin, Ricinus communis agglutinin, and soy bean agglutinin. That these binding proteins may not be glycosylated was further supported by the failure of either peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F to alter the size of the PP-binding protein complexes on gel electrophoresis. These PP-binding proteins may serve as receptors and mediate a hepatic effect of PP.     

In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA-binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal-mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78-kDa protein could be cross-linked to poly(A).     

Identification of five human lymphocyte subpopulations by their differential binding of various strains of bacteria.

The ability of human peripheral blood lymphocytes to kill antibody-coated Chang liver cells in antibody-dependent cell-mediated cytotoxicity (ADCC) can be blocked with aggregated IgG (agg-IgG) or by soluble immune complexes. Dissociation of aggregates of IgG or immune complexes from the cell surface, however, resulted in partial recovery of the ability both to bind agg-IgG and to kill in the ADCC assay. Our results indicate that “unblocking” of effector cells could occur in vivo when the concentration of circulating immune complexes is lowered.     

Two distinct mechanisms of cytotoxicity by porcine alveolar macrophages in antibody-dependent and immobilized immune complex-dependent cellular cytotoxicity

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.     

The complement-mediated binding of soluble antibody/dsDNA immune complexes to human neutrophils

The complement-mediated binding of soluble antibody/3H-dsDNA immune complexes (prepared in vitro) to human polymorphonuclear leukocytes (PMN) has been investigated quantitatively. Studies with isolated complement components in conjunction with experiments on the binding of these complexes to human red blood cells suggest that the binding to both cell types is mediated predominantly by CR1 (C4b-C3b) receptors but that CR3 (iC3b or C3d-g) receptors may play a role in binding to PMN but probably not to RBC. Our results also indicate that under the standard conditions of these assays (37 degrees C, 20 to 40 min incubations) there is no significant internalization of the soluble antibody/dsDNA immune complexes after they are bound by the PMN.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.