protein-G Affinity Chromatography Research Articles

Macroprolactin Molecular Heterogeneity and Variable Immunoassay Reactivity

Circulating prolactin (PRL) exhibits molecular heterogeneity the clinical significance of which is not known. Macroprolactin (mPRL) is widely reported, however, its heterogeneity is unknown. In addition to determining its molecular nature, the relative impact of the various forms on routinely available immunoassays needs to be examined. This study, applied various proteomics techniques to define the nature of mPRL and examined the effect of those forms on its measurement. Methods: Samples suspected of false hyperprolactinemia were subjected to; 1) precipitation by polyethylene glycol, 2) gel permeation chromatography, 3) protein-G affinity chromatography to identify presence of PRL-antibody complex and, 4) samples negative for antibody by step (3) were applied onto a lectin concanavalin A column to examine the presence of glycosylated PRL forms. Analysis was according to manufacturer’s instructions. All study samples and their chromatography fractions were measured using highly sensitive ELISA (DuoSet, R&D Systems, MN). Samples identified above as containing different mPRL forms were analyzed using four immunoassay analyzers in routine clinical use, namely; Advia Centaur® and Atellica® (Siemens, PA), Alinity-ci (Abbott Laboratories, IL), and Cobas 6000® (Roche Diagnostics, IN). Results: A total of 13 samples were entered into the study. PRL levels ranged from 21.4 to 1,469 ng/mL (median 48.8). Samples positive for mPRL (n=8) (with <40% recovery by PEG) exhibited predominant (52.3 to 95.0%) PRL activity in the (H) range (≥150kDa), with significant but relatively lower amount (3.6 to 34.1%) in the (M) range (≥30<150kDa) and (1.4 to 34.5%) at the (L) range (<30kDa). Samples negative for mPRL exhibited little PRL activity (1.2 to 5.1%) in (H) range, predominantly (60.0 to 79.4%) in the (M) range and moderate presence (15.4 to 38.9 %) in the (L) range. Two samples indeterminate for mPRL contained prolactin forms at all molecular weight levels, (H) (7.9, 27.1%), (M) (67.0, 40.7%), and (L) (5.9, 51.4%). Samples with mPRL exhibited significant binding to protein G affinity column indicating presence of PRL-antibody complex. Samples with (M) range mPRL forms exhibited significant lectin affinity binding. Samples with (H) mPRL showed markedly high PRL by two of the analyzers (Atellica®, Abbott Laboratories) and Cobas 6000®, (Roche Diagnostics). The deviation was more marked when using the Atellica® compared to Cobas 6000®. Samples with (M) mPRL showed marked deviation using the Roche Cobas 6000® compared to the others. In conclusion: Macroprolactin is heterogenous with antibody-PRL complex and aggregated glycosylated forms. Those forms exhibit variable immunoassay reactivity in different routinely used assays. Knowledge of circulating form as well as of their immunoreactivity is important when suspecting false hyperprolactinemia due to macroprolactin.

Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. Recombinant polymeric anti-G IgG1 (IgG1μtp) and IgG3 (IgG3μtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. The recombinant IgG1μtp and IgG3μtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. The enhanced opsonic properties of the polymeric anti-G IgG1μtp and IgG3μtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

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.

Production of monoclonal antibody against FimA protein from Porphyromonas gingivalis in rice cell suspension culture

Major fimbriae (FimA) of Porphyromonas gingivalis play an important role in the adherence of the bacterium to oral surfaces, making them a target for the development of a vaccine against periodontal disease. In our previous study, monoclonal antibodies to FimA were expressed in tobacco plants using the agroinfiltration method. In this report, for easy purification, monoclonal antibodies to FimA were produced and accumulated in rice callus suspension culture. The accumulated antibodies were purified by protein G-affinity chromatography. The plant-produced monoclonal antibodies inhibited the binding of P. gingivalis to saliva-coated hydroxyapatite beads, as well as the invasion of oral epithelial KB cells due to the bacterium. The antibodies enhanced the intracellular killing of P. gingivalis by polymorphonuclear neutrophils. These results suggest that FimA-specific monoclonal antibodies produced in a rice suspension culture were easily purified and biologically active against P. gingivalis, and thus may be used for passive immunization to prevent P. gingivalis-induced periodontal disease.

Proteomic Selection of Immunodiagnostic Antigens for Human African Trypanosomiasis and Generation of a Prototype Lateral Flow Immunodiagnostic Device

BackgroundThe diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.Methodology/Principal FindingsWe took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.Conclusions/SignificanceUsing a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

Allergen-Specific IgG Antibodies Purified from Mite-Allergic Patients Sera Block the IgE Recognition ofDermatophagoides pteronyssinusAntigens: AnIn VitroStudy

One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%–51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.

Comparative studies on thermoresistance of protein G-binding region and antigen determinant region of immunoglobulin G in acidic colostral whey

The kinetics of the denaturation of antigen determinant (AGD) region and protein G-binding (PGB) region of bovine immunoglobulin G (IgG) in acidic colostral whey were studied by single radial Immunodiffusion (SRID) and protein G affinity chromatography (PrGAC) during the same courses of heat treatment. The kinetic and thermodynamic parameters for heat-induced denaturation of AGD and PGB regions of IgG were determined over a temperature range of 69–81°C. The denaturation reactions of both AGD and PGB regions of IgG were best described with apparent reaction order of 1.2, the activation energy values of the denaturation reactions were 159.42 kJ/mol and 235.37 kJ/mol respectively. This work suggested that the AGD region was more heat-labile than the PGB region of IgG in acidic colostral whey, Moreover, the higher values of the constant for the AGD region meant that, once the unfolding of the region started, denaturation would occur more quickly than the PGB region on IgG molecule. All these factors contributed to the fact that generally the IgG contents in the products of bovine colostrum determined by PrGAC would be higher than those by SRID.

The pathogenetic role of immunoglobulin G from patients with systemic lupus erythematosus in the development of lupus pleuritis.

To investigate the pathophysiological effect of immunoglobulin G (IgG) from systemic lupus erythematosus (SLE) patients on pleural mesothelial cells and related mechanisms. Serum IgG from 28 lupus patients and 13 healthy controls was purified by protein-G affinity chromatography. The concentrations of anti-dsDNA-, anti-histone- and/or anti-nucleohistone-containing IgGs were determined by enzyme-linked immunosorbent assay (ELISA). Lupus patients were divided into an active (n = 12) and an inactive group (n = 16) on the basis of the SLE Disease Activity Index (SLEDAI). The binding of IgG to a human pleural mesothelial cell line (MeT-5A) under different conditions, including pretreatment with DNase and preincubation with exogenous histone, DNA or nucleohistone, was examined using flow cytometry and cellular ELISA. The effect of IgG on MeT-5A cell proliferation was studied using an MTT assay. Gene expression and protein synthesis for interleukin 1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor beta1 (TGF-beta1) in MeT-5A cells were determined using reverse transcription-polymerase chain reaction and ELISA. The binding of IgG to MeT-5A cells was higher in the active lupus group than the inactive lupus group (P = 0.047) and controls (P = 0.003). The binding decreased in both lupus groups following pretreatment of MeT-5A cells with DNase. The binding of IgG to MeT-5A cells was greater by 112% in the active lupus group after preincubation with histone (P < 0.001), but not with DNA or nucleohistone. Exposure of MeT-5A cells to IgG from either lupus group induced cell proliferation when compared with IgG from healthy controls (P = 0.04). Gene expression and protein synthesis of MCP-1, TGF-beta1 and IL-1beta in MeT-5A cells were significantly increased after incubation with IgG from patients with active lupus when compared with IgG from the inactive lupus and control groups (P < 0.01). The concentration of anti-dsDNA antibodies correlated with the binding of IgG to MeT-5A cells and the synthesis of cytokines by MeT-5A cells. The serum level of anti-histone antibodies in the active lupus group was higher than that in the inactive group (P = 0.015) and the serum concentration correlated with cell binding and MCP-1 production. IgG from lupus patients can bind to MeT-5A cells and the binding is modulated by DNA or histone. Binding of anti-dsDNA-containing IgG to MeT-5A cells induces the synthesis of proinflammatory cytokines. Our findings suggest that the binding of anti-dsDNA antibodies, particularly the IgG isotype, to pleural mesothelium plays a direct pathogenetic role in inducing inflammatory injury in the serositis of SLE.

Immunogenicity of interferon-? in multiple sclerosis patients: Influence of preparation, dosage, dose frequency, and route of administration

A total of 754 consecutive patients with relapsing-remitting multiple sclerosis were investigated for interferon-beta (IFNbeta) antibodies by protein-G affinity chromatography and antiviral neutralization bioassay during 24 months on 6 MIU (22 microg) of subcutaneous IFNbeta-1a once weekly (n = 143) or three times weekly (n = 160), 6 MIU (30 microg) of intramuscular IFNbeta-1a once weekly (n = 140), or 8 MIU every other day of IFNbeta-1b (n = 311). The proportion of binding antibodies was higher in those receiving IFNbeta-1b compared with 6 MIU of IFNbeta-1a three times weekly (97 vs 89% at 12 months), and fewer became positive if 6 MIU of IFNbeta-1a was administered once weekly (58 vs 89%). Fewer patients on intramuscular than subcutaneous IFNbeta-1a became positive (33 vs 58%). The binding and neutralizing capacities were higher in the IFNbeta-1b group than in the IFNbeta-1a groups; these differences, however, were not significant after 12 months. The number of positive patients varied considerably and depended on the amount of IFN added to the bioassay; adding 10 LU/ml or more masked antibody detection. Antibodies induced by either preparation neutralized both IFNbeta species but not IFNalpha. In conclusion, IFNbeta-induced antibodies are frequently found in multiple sclerosis patients, and IFNbeta-1b is more immunogenic than IFNbeta-1a. The immunogenicity of IFNbeta-1a increases with the frequency of administration and if it is given subcutaneously.

Antiphospholipid antibodies permeabilize and depolarize brain synaptoneurosomes.

Antiphospholipid antibodies (aPL) are associated with neurological diseases such as stroke, migraine, epilepsy and dementia and are thus associated with both vascular and non-vascular neurological disease. We have therefore examined the possibility that these antibodies interact directly with neuronal tissue by studying the electrophysiological effects of aPL on a brain synaptosoneurosome preparation. IgG from patients with high levels of aPL and neurological involvement was purified by protein-G affinity chromatography as was control IgG pooled from ten sera with low levels of aPL. Synaptoneurosomes were purified from perfused rat brain stem. IgG from the patient with the highest level of aPL at a concentration equivalent to 1:5 serum dilution caused significant depolarization of the synaptoneurosomes as determined by accumulation of the lipophylic cation [3H]-tetraphenylphosphonium. IgG from this patient as well as IgG from two elderly patients with high levels of aPL were subsequently shown to permeabilize the synaptosomes to labeled nicotinamide adenine dinucleotide (NAD) and pertussis toxin-ADP-ribose transferase (PTX-A protein) as assayed by labeled ADP-ribosylation of G-proteins in the membranes. No such effects were seen with the control IgG. aPL may thus have the potential to disrupt neuronal function by direct action on nerve terminals. These results may explain some of the non-thromboembolic CNS manifestations of the antiphospholipid syndrome.

Novel Tumor-Associated Surface Antigen: Broad Distribution among Neuroectodermal, Mesenchymal and Epithelial Tumors, with Restricted Expression among Normal Tissues † 785

Tumor-selective surface antigens may be targets for diagnosis and immune-based therapies. Monoclonal antibody 8H9 is a murine IgG1 hybridoma derived from the fusion of SP2/0 mouse myeloma cells and lymphocytes from BALB/c mice immunized with human neuroblastoma (NB). 8H9 reacts with a surface antigen on NB cell lines NMB7 and LAN1 by immunofluorescence. By immunohistochemistry, it is nonreactive with normal human tissues, including bone marrow, colon, stomach, heart, lung, muscle, thyroid, testes, pancreas, kidney and brain (frontal lobe, cerebellum, pons and spinal cord), but moderately reactive with human hepatocytes. Reactivity with normal tissues from cynomolgus monkey was similarly restricted. 8H9 is reactive with human brain tumors, childhood sarcomas, NB and less so with adenocarcinomas. Among brain tumors, 14/18 glioblastomas, 3/6 high-grade gliomas, 3/3 astrocytomas, 3/6 oligodendrogliomas, 2/3 meningiomas, 1/1 of medulloblastoma and 1/1 schwannoma. Among sarcomas, 12/12 Ewing's/PNET, 8/8 rhabdomyosarcomas, and 6/7 osteosarcomas also tested positive with 8H9. 20/26 NB and 8/16 adenocarcinomas(2 metastatic to the brain) were positive. 8H9 could be purified by protein-G affinity chromatography and radiolabeled with 99mTc via hydrazino nicotinamide derivative; human NB xenografts in nude mice showed selective tumor uptake. When injected intravenously with 99mTc-8H9, normal cynomolgus monkey demonstrated uptake in the liver as well as ends of long bones. This tumor-selective antigen expressed on a broad range of pediatric and adult cancers may have clinical utility in diagnosis and therapy. Further biochemical characterization is warranted.

Purification of Immunoglobulin G

IgG can be purified, as described here, by ammonium sulfate precipitation followed by size-exclusion (SE) chromatography. This is the least expensive option available for purification of antibodies. Protein A- and protein G-affinity chromatography are the fastest methods for purifying antibodies, but they are not effective for all subclasses of rat antibody. A protocol for affinity chromatography using anti-rat antibody is provided for purification of rat antibodies. Ion-exchange (IEX) chromatography is described for purifying intact monoclonal and polyclonal antibodies and antibody fragments.

Dermal Mast Cell Activation by Autoantibodies Against the High Affinity IgE Receptor in Chronic Urticaria

Previous studies identified autoantibodies against the IgE high affinity receptor alpha-chain, Fc epsilon RI alpha, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-Fc epsilon RI alpha autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti-Fc epsilon RI alpha autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4-34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG-mediated. Preincubation of sera or the IgG fraction with a recombinant alpha-chain of Fc epsilon RI inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human Fc epsilon RI alpha antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of Fc epsilon RI, with 0.01-1.0 micrograms/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-Fc epsilon RI alpha autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-Fc epsilon RI alpha autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients.

Chromatographic and electrophoretic studies of circulating immune complexes in plasma

The protein nature of soluble immune complexes from fresh plasma was studied by combining several analytical biochemical techniques. Free immunoglobulins (Ig) G were separated from larger immune complexes by gel permeation chromatography. In a second step, immune complexes, free IgA and IgM were isolated by protein-A and protein-G affinity chromatography and analysed by two-dimensional gel electrophoresis. Sixteen plasma samples from healthy donors were analysed and evaluated visually. Their protein profiles on the gels turned out to be similar, showing only slight quantitative differences. In one case, additional proteins were detected. To prove the ability of the method, immune complexes were analysed from four plasma samples that showed macro creatine kinase type 1, a complex formation between creatine kinase BB and IgG. This methodology can be used for the examination of immune complexes of unknown protein composition in serum or plasma.

Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.

Humoral modulation of lymphokine‐activated killer (LAK)‐cell induction in humans: IgG‐related and non‐IgG inhibitors in sera from cancer patients

Soluble inhibitors of lymphokine-activated killer (LAK) cell induction were characterized and purified from serous fluids from healthy donors or from patients with advanced cancer. Inhibitory activity in sera from cancer patients was partially absorbed with protein A agarose or anti-human IgG agarose. Following absorption, residual inhibition varied with individual sera, suggesting the presence of IgG-related and non-IgG inhibitory factors, and the proportions varied in the patient population. IgG, purified by affinity chromatography from ascitic fluids, from plasma of cancer patients, and from plasma of healthy donors, significantly inhibited IL-2 induction of LAK cells in a dose-dependent manner. Irrespective of the sources, inhibitory activity resided in the high-molecular-weight IgG fraction composed of IgG aggregates and immune complexes, but not monomeric IgG. Commercially prepared human IgG in aggregated form, but not in monomeric form, inhibited LAK cell induction in a dose-dependent manner. In contrast, neither bovine IgG nor human albumin affected LAK cell induction, even at higher concentrations. Aggregated human IgG inhibited LAK cell induction in unfractionated peripheral blood mononuclear cells (PBMC) but not in monocyte-depleted peripheral blood lymphocytes (PBL). Despite extensive (greater than 99%) depletion of IgG by protein-G affinity chromatography, the serous fluid from cancer patients displayed significant inhibitory activity. Fractionation of the IgG-depleted inhibitory materials by Sephacryl S-300, high-pressure ion-exchange column (HPIEC) or gel-permeation chromatography (HPGPC) demonstrated a 65-kDa inhibitor, distinct from IgG. Affigel-blue affinity chromatography of the 65-kDa fraction depleted albumin but did not remove the inhibitory activity, suggesting that the 65-kDa inhibitor is not serum albumin nor an albumin-bound component. These results suggest that serous fluids from patients with advanced-stage cancer contain 2 distinct regulators for LAK cell induction: (I) aggregated IgG and (2) a 65-kDa inhibitor, distinct from albumin.