Immusorba TR and Immusorba PH: Basics of Design and Features of Functions

Abstract: Guest Editor's Introduction: Selective adsorption of target substances can be achieved based upon the application of physical and chemical interactions as well. Asahi Medical Co. developed 2 types of selective adsorbent columns, Immusorba PH‐350, and Immusorba TR‐350. These adsorbents consist of crosslinked polyvinyl alcohol gel beds with microporous structures for the base material, which immobilized a specific amino acid with hydrophobic property. Immusorba PH‐350 has phenylalanines as the ligand, which adsorbs the pathogenic substances such as immune complexes and anti‐DNA antibody. Tryptophan is immobilized in the Immusorba TR‐350, which adsorbs antiacetylcholine receptor antibodies. This paper describes the basic designs, adsorption characteristics, and summary of clinical applications of these adsorbents. This paper was printed in Therapeutic Apheresis, vol 2, page 185–192 (1998), and reprinted here with permission. Immusorba was reported by Yamazaki et al. to be the world's first practical immunoadsorbent in 1982. Since then, this immunoadsorbent has accumulated an abundance of clinical achievements. Immusorba has such unique functions that it is used in treating various diseases and holds possibilities for application to more diseases. Immusorba was designed as an artificial receptor for rheumatoid factor (RF) based on structural analysis of heat‐denaturated globulin. Subsequently, new substances that it can adsorb have been found as seen in reports on the adsorption performance of Immusorba to anti‐acetylcholine receptor antibodies (anti‐AChR Abs) and antiganglioside antibodies. Along with this, Immusorba has been used in treating a wide range of diseases. The greatest characteristic of Immusorba is that its adsorption capability is selective rather than specific, making it effective against a great number of diseases.     

Immusorba TR and PH

Immusorba TR (IM-TR) and PH (IM-PH) were developed as adsorbents with non-biological materials as affinity ligands to remove pathogenic autoantibodies. The adsorbents of IM-TR and IM-PH are polyvinyl alcohol gel immobilized with tryptophan and phenylalanine as ligand, respectively. IM-TR is clinically applied for treatment of autoimmune neurological diseases such as myasthenia gravis and Guillain-Barre syndrome. IM-PH is used for not only neurological diseases such as GBS and multiple sclerosis but also collagen diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). As many autoantibodies with different specificities have been found to have similar affinities to the ligand of Immusorba, it is expected that Immusorba will be applied to more diseases and contribute to the clarification of the mechanisms of the development of diseases by the identification of adsorbed unknown pathogenic substances with Immusorba.     

Development of a technique of immunoadsorption of LDL-cholesterol

An immunoadsorption system for lowering plasma cholesterol was optimized. Several polyclonal and monoclonal antibodies were compared and the best results were obtained with goat polyclonal antibodies. The optimum quantity of antibodies to be immobilized on the gel was 5 mg/ml. Taking into account two variables, i.e., 1) that the regeneration must be as complete as possible and, 2) that immunoadsorbents must be used several times without a loss of adsorption capacity, desorption was achieved with 0.3 M glycine adjusted to pH 2.8. Antibody release from the immunoadsorbent was determined and can be minimized by glutaraldehyde treatment of the immunoadsorbent. Each phase, adsorption and desorption, respectively, was well-defined and synchronized, so that two columns could be used in parallel in an automated procedure. The kinetics of plasma protein removal demonstrated the efficiency and the specificity of the procedure.     

The multichain interleukin-2 receptor: a target for immunotherapy.

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.     

Cytokine adsorptive property of various adsorbents in immunoadsorption columns and a newly developed adsorbent: an in vitro study

BACKGROUND/AIMS: Cytokines play important roles in the pathophysiology of systemic inflammatory response syndrome (SIRS) and sepsis. Therefore, some effective measures to remove cytokines from the bloodstream could be effective in the treatment of SIRS and sepsis. The aim of this study was to evaluate the cytokine adsorptive property of various adsorbents for the purpose of the development of new selective cytokine adsorption columns. METHODS: The cytokine adsorptive property of adsorbent in a CF-X column, which consists of cellulose beads cross-linked with hexamethylene-di-isocyanate, was compared with those of various adsorbents in currently available immunoadsorption columns, such as Immusorba TR, Immusorba PH, Selesorb, and Lixelle, in vitro batchwise test using patients' plasma. A newly developed adsorbent, MPCF-X, which was modified by coating the surface of the adsorbent in CF-X with 2-methacryloyloxyethyl phosphorylcholine (MPC), was also tested for its cytokine adsorptive property. RESULTS: The adsorbent in CF-X showed a significantly higher adsorption rate for TNF-alpha, interleukin (IL)-6 and IL-10 compared with other adsorbents (p < 0.05). Adsorbent in Lixelle showed good affinity to TNF-alpha and IL-8. Especially, the adsorbent in CF-X almost completely removed TNF-alpha, whereas it also had considerable affinity to normal IgG. MPCF-X showed decreased affinity to IgG with considerable adsorptive properties to cytokines. CONCLUSION: Selective cytokine adsorption columns could be developed with improvement of currently available adsorbents. Such a new selective cytokine adsorption column could be clinically applied for the treatment of SIRS/sepsis.     

Specific Immunoadsorbent for Myasthenia Gravis Treatment: Development of Synthetic Peptide Designed to Remove Antiacetylcholine Receptor Antibody

Abstract: We have developed Medisorba MG, a new immunoadsorbent column for myasthenia gravis (MG). The a 183–200 segment of the Torpedo Californica acetylcholine receptor (AChR) is recognized as the acetylcholine binding site by the blocking antibody, which is one of the anti-AChR antibodies involved in the pathogenesis of MG. As a specific affinity ligand to remove the blocking antibody, Torpedoα 183–200 was synthesized and immobilized covalently to porous cellulose beads. This immunoadsorbent showed specific removal of the blocking antibody without reducing IgG and albumin levels significantly in clinical evaluation and in vitro study. Clinical improvement was found in 78% of the cases, and no adverse effects were observed in any case. The Medisorba MG column has been confirmed as a useful device for the treatment of MG.     

Neutrophil Activation in Immunoadsorption

Abstract: Immunoadsorption therapy (IAT) is used in the treatment of autoimmune diseases. Although IAT has been reported to modify humoral immunity by inducing chemokines and activating complements, much remains unknown about the biological effects of IAT on cellular components in peripheral blood. To define the influence of IAT on leukocytes, we determined leukocyte l ‐selectin (CD62L) and Mac‐1 (CD11b) as parameters for activation of leukocytes in peripheral blood during IAT. Peripheral leukocyte l ‐selectin and Mac‐1 were determined continuously by flow cytometry in 6 patients with neuroimmunological disorders in whom IAT was conducted using a Plasma Flow OP‐05 (Asahi Medical Corp., Tokyo, Japan) as a plasma separator and Immusorba TR‐350 (Asahi Medical Corp., Tokyo, Japan) as an adsorption column. Expression of neutrophils (PMN) l ‐selectin was decreased 30 min after starting IAT, with the decreases particularly marked at the end of IAT, while expression of mononuclear cells (MNC) l ‐selectin slightly increased during IAT. Expression of PMN Mac‐1 was markedly increased at the end of IAT, whereas expression of MNC Mac‐1 did not change during IAT. Leukocyte counts decreased 30 min after starting IAT, and then increased to the initial level or higher in parallel with l ‐selectin downregulation and Mac‐1 upregulation on PMN. l ‐selectin downregulation and Mac‐1 upregulation on PMN suggested that activation of PMN associated with changes in peripheral leukocyte counts occurred during IAT and might play some role in modulating the human circulating blood and immune systems.     

One-step purification of chicken growth hormone from a crude pituitary extract by use of a monoclonal immunoadsorbent

The immunization of mice with an affinity-purified glycoprotein preparation from chicken pituitary tissue yielded several monoclonal antibodies towards the recently described glycosylated variant of chicken GH. As all these antibodies recognize the classical (non-glycosylated) GH molecule equally well, they provide a suitable tool for the development of both a specific immunoadsorbent and an assay method. This paper deals with the surprising purification power of the immunoadsorbent that was produced with one of the monoclonal antibodies. The resulting preparation was more than 99% pure as assessed by reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, so that no further purification steps were needed before the determination of the amino acid sequence of the material. The efficiency of the purification protocol as determined by a homologous, monoclonal antibody-based radioimmunoassay was virtually absolute. Moreover, the affinity-purified GH preparation was a mixture representing the multiple molecular forms of pituitary chicken GH, including both oligomeres and glycosylated GH. The purified preparations were finally used to demonstrate the hepatic 5'-monodeiodinase-stimulating activity of GH in the chicken embryo (results not shown), in order to prove that the biological activity of the molecule had not been damaged by elution from the immunoadsorbent.     

Immunoadsorption in Systemic Connective Tissue Diseases and Primary Vasculitis

Abstract: Immunoadsorption offers some advantages over plasmapheresis; until recently the primary advantage has been avoidance of substitution fluids. In collagen vascular disorders, immunoadsorption is performed for the same indications as plasma exchange; most often adsorbers with binding capacities for IgG and circulating immune complexes are used. Tested ligands are protein A, anti-IgG antibodies, Clq, phenylalanine, and tryptophan. Human IgG was utilized to adsorb rheumatoid factor and dextran sulfate, DNA, or specific anti-idiotypes for anti-DNA antibodies in systemic lupus erythematous (SLE). Most applications have used immunoadsorbent columns in pre-transplantation treatment of patients with high panel reactivity and in patients with idiopathic thrombocytopenic purpura (ITP). For these indications, as for systemic connective tissue diseases, randomized trials have yet to be conducted. SLE controlled trials have been completed for IMPH-350 and Ig-Therasorb. Results indicated excellent biocompatibility and good clinical responses. Using protein A in primary systemic vasculitis, histologically proven inactivation of renal involvement was demonstrated, but the patients were also treated with immunosuppressive drugs. Randomized controlled trials are mandatory to provide continued support to the therapeutical opportunities offered only by immunoadsorption.—     

Development of Selective Low‐Density Lipoprotein (LDL) Apheresis System: Immobilized Polyanion as LDL‐Specific Adsorption for LDL Apheresis System

Abstract: Guest Editor's Introduction: The binding site of the human LDL receptor is rich in anionic amino acids, and interacts with apolipoprotein‐B by the electrostatic force. Therefore, anionic ligands could be used for the selective adsorption of LDL without HDL adsorption. Based upon this concept, Kaneka Co. developed LDL adsorbent. The adsorbent named Loposorber consists of microporous cellulose beads which immobilized dextrane sulfate with a molecular weight of several thousands. This paper describes the basic study for the selection of polyanions, in vitro adsorption characteristics, and the references of initial clinical applications. This paper was printed in Artificial Organs, vol. 20, page 922–929 (1996), and reprinted here with permission. Low‐density lipoprotein (LDL) is widely recognized as one of the major risk factors for developing coronary heart diseases. Despite intensive development of LDL‐lowering drugs, there still exist those patients with refractory hyperlipidemia whose plasma LDL levels are not sufficiently lowered by drugs. LDL apheresis, direct removal of plasma LDL from circulating blood, is thought to be the most promising treatment for such refractory patients. Various techniques, such as the use of an immunoadsorbent utilizing an anti‐LDL antibody, have been used in an attempt to achieve the selective removal of LDL. However, none were widely used because of complications, poor selectivity, and so forth. To establish a safe and effective LDL apheresis system, we chose a synthetic affinity adsorbent as the LDL‐removing device. Synthetic polyanion compounds were used as the affinity ligands for LDL adsorbent to simulate the anion‐rich sequence of LDL binding sites in the human LDL receptor. Among various polyanion compounds, those polyanions with sulfate or sulfonate groups and hydrophilic backbone were found to have a strong affinity for LDL. In contrast, polyanions with carboxyl groups showed poor affinity. Dextran sulfate (DS) was selected as the affinity ligand of LDL adsorbent for its high affinity and low toxicity. The influence of its charge density and molecular weight on its affinity for LDL was suitable. The affinity rapidly increased as the charge density increased, then, reached a constant value. Little affinity was found for either the DS monomer (glucose sulfate) or DS with a molecular weight higher than 10⁴ daltons whereas DS with molecular weights in the midrange showed strong affinity. DS with a midrange molecular weight was immobilized on cellulose hard gel to give LDL adsorbent clinical application. The adsorbent demonstrated an excellent selectivity for LDL and very low density lipoprotein (VLDL) in vitro. Adsorption of high‐density lipoprotein and major plasma proteins was almost negligible. Additional study of the LDL‐binding mechanism revealed that DS directly interacts with positively charged sites on LDL, which demonstrates that the nature of the interaction is the same as that of the LDL receptor. An LDL adsorption column (Liposorber) packed with an LDL adsorbent and polysulfone hollow‐fiber plasma separator (Sulflux) was developed as an efficient LDL apheresis system. Clinical investigation proved that this system is capable of intensively lowering the plasma LDL level without affecting major plasma components.     

The proinflammatory cytokines interleukin-1 and tumor necrosis factor and treatment of the septic shock syndrome

Treating the septic shock syndrome with antibodies that block only endotoxin has its limitations. Other targets for treating septic shock include neutralizing antibodies to the complement fragment C5a, platelet-activating factor antagonists, and blockade of endothelial cell leukocyte adhesion molecules. Specific blockade of the proinflammatory cytokines interleukin-1 (IL-1) or tumor necrosis factor (TNF) reduces the morbidity and mortality associated with septic shock. Moreover, blocking IL-1 and TNF likely has uses in treating diseases other than septic shock. Use of neutralizing antibodies to TNF or to IL-1 receptors have reduced the consequences of infection and inflammation, including lethal outcomes in animal models. The IL-1 receptor antagonist, a natural-occurring cytokine, blocks shock and death due to Escherichia coli and ameliorates a variety of inflammatory diseases. Soluble TNF and IL-1 surface receptors, which bind their respective cytokines, also ameliorate disease processes. Current clinical trials are evaluating the safety and efficacy of these anticytokine therapies either alone or together.     

Preparation of monoclonal antibodies to the avian progesterone receptor

In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated alpha PR 6, 11, 13, 16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradients, and in recognizing receptor proteins that were resolved by denaturing gel electrophoresis and transferred to nitrocellulose (Western blots). Immunoisolation of receptor was also demonstrated using receptor labeled covalently with the synthetic progestin, R5020. The antibodies were specific for progesterone receptor and did not cross-react with estrogen receptor from the oviduct or glucocorticoid receptor from chick liver. Two antibodies, alpha PR 6 and alpha PR 22, also recognized some mammalian forms of the progesterone receptor. Both antibodies reacted with progesterone receptor from the rabbit uterus and alpha PR 6 recognized human progesterone receptor. Four of the antibodies recognized both A and B forms of the avian receptor while alpha PR6 was specific for the B form.     

High prevalence of antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) in AIDS and related conditions but not in other disease states.

A rapid, sensitive indirect immunofluorescence assay has been developed for detection of antibodies to the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV). The human T-cell line HUT-78 was chronically infected with ARV-2 and used to detect antibodies to virus-specific cytoplasmic antigens. Because the helper T-cell marker Leu-3 is substantially reduced in this cell line after ARV infection, it appears to be an important receptor for virus infection. Nearly all patients with AIDS and most cases with related conditions showed antibodies against ARV. Some healthy individuals in risk groups for developing AIDS also had antibodies to the agent. In contrast, no antibodies to the virus were found in any individuals outside the risk groups for developing AIDS or with diseases other than those associated with AIDS. The titers of antibodies to ARV and to Epstein-Barr virus varied independently from each other. The level of anti-ARV antibodies in a patient's serum was found to reflect the severity of the disease; it was lower in individuals with more severe manifestations. Taken together, these data support the role of ARV in AIDS and its related disorders.     

Comparison of the Adsorption Ability Between Tryptophan and Modified Tryptophan Columns

Abstract: Immunoadsorption therapy (IAT) using the tryptophan column (TR:TR-350, Asahi Medical Co., Tokyo, Japan) is used for patients with Guillain-Barré syndrome or Fisher's syndrome. Recently, a modified tryptophan column (modified TR:TR-350S, Asahi) was developed to reduce adsorption of fibrinogens. However, it is not clear whether the new column can effectively adsorb the antiganglioside antibodies. We treated 2 patients with Guillain-Barré syndrome, 1 patient with Fisher's syndrome, and 1 patient with Bickerstaff s brainstem encephalitis by IAT using the TR and the modified TR. Samples were taken from the inlet and outlet of the affinity column. We compared the adsorption ability of antiganglioside antibodies between the TR and modified TR. The modified TR adsorbed antiganglioside antibodies less effectively than the TR. No significant bleeding was observed in the patients during each session. Furthermore, results of the clinical study were confirmed by batchwise adsorption test. We propose that the modified TR should not be used for treating patients with Guillain-Barré syndrome and related disorders.—     

心脏肾上腺素受体及其自身抗体研究进展

作为交感神经递质的去甲肾上腺素(norepinephrine,NE)及内分泌激素的肾上腺素(adrenaline,Ad)参与体内多数器官功能的调节,而这种调节都要通过靶器官上的肾上腺素受体(adrenergic receptor,AR)来实现。此外,在所有G蛋白耦联的膜表面受体中,AR是目前相对了解最清楚的一种,因而AR又可作为研究整个G蛋白耦联受体家族的一个理想模型。自Friou(1957年)等首先在系统性红斑狼疮(systemic lupus erythematosus,SLE)病人血清中发现抗核抗体(anti-nuclear antibody,ANA)以来,人们在自身免疫性疾病,心脏病病人甚至健康人血清中检测出越来越多的抗不同抗原的自身抗体,并对其功能进行了一些研究。现综述近年来AR及其自身抗体的研究进展。     

Anti-F(ab')2 antibody response to the injection of anticlass II HLA alloantibodies in patients with rheumatoid arthritis.

Placenta eluted gamma globulins (PEGG) contain antibodies against class II HLA antigens and have been used for treating patients with rheumatoid arthritis (RA). In view of the potential use of antibodies to class II HLA for treating autoimmune diseases we looked for the immunobiological effects of PEGG injections in patients. No modulation of class II HLA was seen at the surface of circulating mononuclear cells after one week of daily PEGG injections. In some patients, antibodies to F(ab')2 fragments of PEGG-IgG were produced. These antibodies reacted against F(ab')2 of any IgG as well and did not prevent anticlass II HLA antibodies from binding to class II HLA, thus showing no characteristics of classical antiidiotypic antibodies. The appearance of anti-F(ab')2 antibodies was not correlated with the clinical course of the disease. Their significance is discussed.     

Immunoadsorption in Myasthenia Gravis Based on Specific Ligands Mimicking the Immunogenic Sites of the Acetylcholine Receptor

Abstract: A specific system for antibody removal from blood circulation in myasthenia gravis (MG) patients was devised by use of the immunoadsorbent bound to an acetylcholine receptor (AChR) peptide that was synthesized corresponding to the sequence of residues 183‐200 of the AChR alpha‐subunit (alpha 183‐200), antibodies which prevent the binding of ACh to AChR. The alpha 183‐200 peptide was confirmed to be immunogenic for induction of an animal model of the disease and for reactivity with MG autoantibodies. We then made use of these results for immunoadsorption therapy through the antigen‐antibody reaction on the molecular level, having given patients relief from myasthenic weakness. The greatest care was taken for the selection of an antigenic region in the molecular structure among various myasthenogenic domains of AChR and for the antigenic conformation of synthetic peptide as the adsorbent to react with antibodies raised against the native protein.     

Leukotriene synthesis inhibitors versus antagonists: The pros and cons

It has been recognized for many years that leukotrienes play an important role in mediating various effects of the allergic reaction. Recent evidence has shown that they play a role in other diseases. Leukotrienes can be separated into the fairly well-characterized cysteinyl leukotrienes and the less well-characterized leukotriene B(4). Effects of the leukotrienes are mediated through receptors that are expressed on a variety of cell types and can be modulated based on the inflammatory environment present. The pharmaceutical industry has long been interested in blocking leukotriene action. As such, two approaches have been developed that led to drugs approved for treating allergic disease. The most widely used class is the cysteinyl type 1 receptor antagonists, which block binding of the cysteinyl leukotrienes to the cell. The second class is an inhibitor of the 5-lipoxygenase enzyme that prevents synthesis of both the cysteinyl leukotrienes and leukotriene B(4). This review focuses on the role that leukotrienes play in various diseases, with the emphasis on allergic diseases, and considers the rationale for choosing either a leukotriene antagonist or synthesis inhibitor as a treatment option.     

Improvement in cholestasis associated with total parenteral nutrition after treatment with an antibody against tumour necrosis factor alpha

BACKGROUND: Many patients receiving long-term total parenteral nutrition (TPN) develop liver disease; cholestasis is common and may be severe. Antitumour necrosis factor alpha (TNFalpha) antibodies have recently been used in order to treat Crohn's disease, but their effect on cholestasis in humans has not been previously described. CASE REPORT: A 45-year-old woman had complicated Crohn's disease with multiple fistulae and only 1 m of residual small bowel. She had been receiving TPN for 2.5 years when she developed cholestasis which worsened despite adjustments to her TPN regimen. Infliximab, an anti-TNFalpha antibody, was given with the aim of treating an enterocutaneous fistula, but it also produced a marked biochemical and histological improvement in the TPN-related cholestasis. CONCLUSIONS: Anti-TNFalpha antibodies appeared in this case to improve TPN-related cholestasis. This implies that TNFalpha may play an important role in the development of this condition.     

Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.