RESUMEN
Hirudin, a potent and specific thrombin inhibitor, is a protein of nonhuman origin and therefore potentially immunogenic. The primary objectives of this investigation were to determine the incidence of antihirudin antibodies (ahir-ab) in patients with heparin-induced thrombocytopenia (HIT) who received lepirudin as parenteral anticoagulation and to determine the incidence of death, limb amputation, new thromboembolic complications (TECs), and major hemorrhage in patients who had ahir-ab, compared with patients who were ahir-ab negative. The investigation used data from 2 prospective multicenter studies with the same study protocol, in which HIT patients received 1 of 4 intravenous lepirudin dosage regimens. The treatment duration was 2 to 10 days. Ahir-ab were determined by a newly developed enzyme-linked immunosorbent assay (ELISA). Eighty-seven of 196 evaluable patients (44.4%) had ahir-ab of the IgG class. Development of ahir-ab was dependent on the duration of treatment (ahir-ab-positive patients 18.6 days vs ahir-ab-negative patients 11.8 days; P =.0001). Fewer ahir-ab-positive than ahir-ab-negative patients died (P =.001). Ahir-ab did not cause an increase in limb amputation (P =.765), new TECs (P >.99), or major bleedings (P =.549). In 23 of 51 (45.1%) evaluable patients in whom ahir-ab developed during treatment with lepirudin ( = 12% of all lepirudin treated patients), the ahir-ab enhanced the anticoagulatory effect of lepirudin. Ahir-ab are frequent in patients treated with lepirudin for more than 5 days. Ahir-ab are the first example for a drug-induced immune response causing enhanced activity of a drug. Therefore, during prolonged treatment with lepirudin, anticoagulatory activity should be monitored daily to avoid bleeding complications.
Asunto(s)
Anticuerpos/sangre , Anticoagulantes/uso terapéutico , Heparina/efectos adversos , Hirudinas/análogos & derivados , Hirudinas/inmunología , Tiempo de Tromboplastina Parcial , Trombocitopenia/tratamiento farmacológico , Amputación Quirúrgica , Anticoagulantes/inmunología , Extremidades , Hemorragia/inmunología , Terapia con Hirudina , Humanos , Inmunoglobulina G/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Trombocitopenia/inducido químicamente , Trombocitopenia/mortalidad , Tromboembolia/inmunología , Factores de TiempoRESUMEN
Recombinant hirudin (rH) is a highly specific thrombin inhibitor which is under clinical investigation for various thrombotic disorders. However, one of its potential risks during clinical use might be hemorrhage, especially when combined with other agents interfering with the coagulation system like antiplatelet or fibrinolytic agents. In this experimental study we investigated whether Haemate, a von Willebrand Factor (vWF) and factor VIII containing product, could correct rH/aspirin induced bleeding in an experimental pig study. Skin bleeding time was evaluated in three open, placebo-controlled, randomized studies following comparable designs. A total of 62 animals were given a short-term infusion of aspirin (20 mg/kg) followed by a three-hour infusion of a high or low dose (0.3 or 0.5 mg/kg/h) of rH. At cessation of rH infusion, animals were allocated to treatment with either Haemate (30 FVIII U/kg) or the recombinant factor VIII, Helixate, which is devoid of vWF. The skin bleeding time (SBT, given as times of baseline) as measured four hours after the start of the rH infusion was defined as the prospective endpoint. In study 1 (low dose rH + Haemate) 4 h SBT was 2.18 (placebo) and 1.61 (Haemate, p = 0.0111). In study No. 2 (high dose rH + Haemate) SBT was 2.58 in placebo and 1.73 in Haemate (p = 0.0001). No significant difference between placebo and treatment were detected in study No. 3 (low dose rH + Helixate). Haemate but not Helixate significantly decreased bleeding time as compared to placebo at termination of the study (7 hours) which was defined as the secondary endpoint. No effect on either aPTT nor rH plasma levels were observed with any of the study drugs. It was concluded that Haemate decreases excess bleeding induced by rH/aspirin treatment without altering rH's anticoagulant effect.
Asunto(s)
Aspirina/farmacología , Hemorragia/tratamiento farmacológico , Hirudinas/efectos adversos , Inhibidores de Agregación Plaquetaria/farmacología , Piel/irrigación sanguínea , Animales , Tiempo de Sangría , Factor VIII/farmacología , Hemorragia/inducido químicamente , Infusiones Intravenosas , Masculino , Ingeniería de Proteínas , Proteínas Recombinantes/efectos adversos , Porcinos , Factor de von Willebrand/farmacologíaRESUMEN
Percutaneous transluminal coronary angioplasty is often complicated by thrombotic abrupt vessel closure in patients with unstable angina pectoris. The present multicentre trial was performed to determine the feasibility of two-dose regimens of recombinant hirudin (r-hirudin) compared to standard heparin in patients undergoing coronary angioplasty for unstable angina, and to investigate the effects of the different treatment regimen on markers of coagulation activation. At five participating centres, 61 patients were randomly enrolled in one of two sequential groups of r-hirudin (group 1: 0.3 mg.kg-1 i.v. bolus, 0.12 mg.kg-1.h-1 i.v. infusion; 21 patients; group 2: 0.5 mg.kg-1 i.v. bolus, 0.24 mg.kg-1.h-1 i.v. infusion; 19 patients) or in a heparin control group (150 IU.kg-1 i.v. bolus, 20 IU.kg-1.h-1 i.v. infusion; 21 patients). Antithrombotic therapy was started immediately before coronary angioplasty and continued for 24 h. This was followed by a low-dose anticoagulant infusion for another 24 h (r-hirudin: 0.04 mg . kg-1 . h-1; heparin: 7 IU . kg-1 . h-1). Activated partial thromboplastin time, r-hirudin plasma concentrations by both immunological and functional assay, thrombin-hirudin complex, thrombin-antithrombin III complex, soluble fibrin, and prothrombin fragment 1 + 2 were closely monitored. The median partial thromboplastin time prolongations at 24 h vs baseline were found to be 1.9-fold and 2.3-fold in r-hirudin group 1 and dose group 2, respectively, and 3.0-fold in the heparin group. There was a dose-dependent correlation between partial thromboplastin time and the r-hirudin plasma levels (r = 0.61). In five of 21 patients of dose group 1, three of 19 patients of dose group 2, and 10/21 patients of the heparin group, partial thromboplastin time values exceeding the predefined target range prompted an interruption of the infusion. One major bleeding complication occurred in dose group 2. The functional assay for the estimation of r-hirudin plasma concentrations showed excellent correlations to the immunological technique (r = 0.99). Differences between the thrombin-hirudin complex levels could not be observed. Increased concentrations of thrombin-antithrombin III complex, soluble fibrin, and prothrombin fragment 1 + 2 were seen 4-8 h after coronary angioplasty and after reduction of the high-dose therapy in dose group 1 when compared with dose group 2 and the heparin group, respectively. Based on coagulation tests the present study showed the feasibility of a periprocedural antithrombotic regimen with r-hirudin for patients undergoing coronary angioplasty for unstable angina. In addition to the partial thromboplastin time the determination of r-hirudin plasma levels by a chromogenic substrate assay considerably improves the monitoring of therapy. The lower dose r-hirudin regimen seems to be suboptimal as periprocedural anticoagulation in coronary angioplasty patients as indicated by markers of thrombin generation and thrombin activity.
Asunto(s)
Angina Inestable/terapia , Angioplastia Coronaria con Balón , Fibrinolíticos/administración & dosificación , Hirudinas/administración & dosificación , Adulto , Anciano , Angina Inestable/sangre , Anticoagulantes/administración & dosificación , Biomarcadores/sangre , Coagulación Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas , Ensayo de Inmunoadsorción Enzimática , Heparina/administración & dosificación , Humanos , Infusiones Intravenosas , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Proteínas Recombinantes/administración & dosificación , Trombina/metabolismoRESUMEN
To prevent r-hirudin induced excess bleeding an animal model was established in pigs for the investigation of an anti-bleeding strategy. We used the Simplate® device to monitor skin bleeding time (SBT) at the inner site of the ear. r-Hirudin infused in a dose of 0.3 mag per h induced a prominent increase of SBT. The aim of our studies was to reverse r-hirudin induced bleeding by enhancing platelet adhesion to the endothelium via the administration of von Willebrand Factor (vWF). Pigs were treated with vWF containing solutions (Haemate® and a vWF-concentrate) at 3h after the start of the r-hirudin infusion. Both compounds suppressed SBT 1h after administration and significantly prevented bleeding until the termination of the experiment. SBT values (given in times of baseline) in the placebo group were 3.32 ± 0.9, 1.51 ± 0.14 in the Haemate® and 1.85 ± 0.42 in the vWF concentrate group (P = 0.008 or 0.032, in a two-sided Kruskall-Wallis-test). Coagulation parameters (aPTT, PT) were unaltered by the treatment, as were the r-hirudin plasma levels suggesting that vWF is not an antidote in its strict sense. It is concluded that vWF reverses bleeding without altering the anticoagulant effect of r-hirudin. Addition of 20 mg/kg per h aspirin resulted in a further increase of SBT. Aspirin, moreover, suppressed platelet aggregation but did not alter platelet counts. In a further study, bleeding induced by r-hirudin and aspirin was antagonized by Haemate® (66 Ukg i.v. bolus + 187 Ukg per h for 2 h infusion) and a significant reduction of bleeding time occurred.
RESUMEN
A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 microliters sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2-4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63% and 2.78% on the Hitachi 911 and 1.51% and 7.84% on the Cobas Mira, respectively and interassay coefficients of variation of 3.57% to 9.15% (Hitachi 911) and 3.72% to 12.99% (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x-0.003, n = 323).
Asunto(s)
Compuestos Cromogénicos , Colorimetría , Hirudinas/sangre , Oligopéptidos , Animales , Automatización , Calibración , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Proteínas Recombinantes/sangre , Valores de Referencia , Trombina/farmacologíaRESUMEN
The pharmacokinetics of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), induction of anti-GM-CSF antibodies, and clinical effects related to the induction of the antibodies were analyzed in patients with metastatic colorectal carcinoma (CRC) who were not on chemotherapy (n = 20, nonimmunocompromised patients). rhGM-CSF (250 micrograms/m2/d; Escherichia coli-derived) was administered subcutaneously for 10 days every month for 4 months. Eight patients with multiple myeloma (MM) on intensive chemotherapy followed by rhGM-CSF treatment were also included (immunocompromised patients). After a single injection of GM-CSF at the first cycle in CRC patients, the maximum calculated concentration (Cmax) was 5.24 +/- 0.56 ng/mL; the half life (T1/2) was 2.91 +/- 0.8 hours; and the area under the concentration curve (AUC) was 30.86 +/- 6.03 hours x ng/mL (mean +/- SE). No anti-GM-CSF antibodies were detected. During the subsequent cycles, 95% of the CRC patients developed anti-GM-CSF IgG antibodies, which significantly altered the pharmacokinetics of rhGM-CSF at the third and fourth cycles with decreased Cmax (2.87 +/- 0.57 ng/mL; P < .05), T1/2 (1.57 +/- 0.2 hours; P < .05), and AUC (14.90 +/- 4.10 hours x ng/mL; P < .005). The presence of anti-GM-CSF antibodies significantly reduced the GM-CSF-induced enhancement of granulocytes, and there was a clear tendency for a decreased increment of monocytes. Antibodies diminished systemic side effects of rhGM-CSF. Only 1 of 8 MM patients showed a very low anti-GM-CSF antibody titer after GM-CSF therapy, as shown by enzyme-linked immunosorbent assay and Western blot. Therefore, in nonimmunocompromised patients, exogenous nonglycosylated GM-CSF induced an anti-GM-CSF IgG antibody response in practically all patients, which seemed to be of clinical significance. In immunocompromised patients, virtually no significant antibody response was shown.
Asunto(s)
Neoplasias Colorrectales/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factores Inmunológicos/inmunología , Isoanticuerpos/biosíntesis , Adolescente , Adulto , Anciano , Complejo Antígeno-Anticuerpo/sangre , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Terapia Combinada , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacocinética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Inmunocompetencia , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/uso terapéutico , Inyecciones Subcutáneas , Isoanticuerpos/inmunología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Metástasis de la Neoplasia , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico , Terapia RecuperativaRESUMEN
Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Monoclonales , Separación Celular/métodos , Compuestos Férricos/química , Magnetismo , Compuestos de Sulfhidrilo/química , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Neoplasias/inmunología , Antígenos CD8 , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Microesferas , NeprilisinaRESUMEN
Mouse x mouse hybridomas secreting monoclonal anti-idiotypic antibodies have been prepared from BALB/c mice immunized with syngeneic monoclonal antibodies specific for IgE, AFP, CEA and TSH respectively. The hybridomas were selected on the basis that the secreted antibodies competed with antigen for binding to the immunizing idiotope. Using IgE and AFP as a model it could be shown that syngeneic antigen inhibitable monoclonal anti-idiotypic antibodies can act well as antigen surrogate. Thus a complementary idiotype-anti-idiotype antibody pair could be used in a competitive assay wherein the extend of idiotype-anti-idiotype complex formation is inversely proportional to the amount of antigen in the sample. Apart from this a lot of human and animal studies have shown that this interaction between idiotype and anti-idiotype could be utilized to prevent certain infectious diseases and to treat some kinds of cancers.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Idiotipos de Inmunoglobulinas/inmunología , Neoplasias/terapia , Animales , Especificidad de Anticuerpos , Humanos , Técnicas para Inmunoenzimas , Ratones , Neoplasias Experimentales/terapia , ConejosRESUMEN
The haemagglutinin content of monovalent influenza whole virus and Tween-ether split vaccines derived therefrom, were assayed comparatively using single radial immunodiffusion (SRID, the only test recommended for influenza vaccines by the European Pharmacopoeia Commission), quantitative SDS-polyacrylamide gel electrophoresis and immunization of guinea pigs. If SRID was performed with split vaccines, reduced haemagglutinin values were consistently recorded which were 50-25% of values obtained before disruption of virions. If, however, disruption was conducted in the presence of excess detergent thus preventing aggregate formation of solubilized haemagglutinin, test values comparable to those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and the corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. From SDS-polyacrylamide gel electrophoresis and densitometer tracings obtained by scanning the gels after staining with either Coomassie Blue or fluorescein isothiocyanate-labelled concanavalin A it was calculated that about 90% of whole virus HA2 was recovered in Tween-ether split vaccines. From our experiments we conclude that precise quantification of solubilized haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.
Asunto(s)
Hemaglutininas Virales/análisis , Vacunas contra la Influenza/análisis , Animales , Anticuerpos Antivirales/biosíntesis , Electroforesis en Gel de Poliacrilamida , Éter , Cobayas , Inmunización , Inmunodifusión , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Polisorbatos , Dodecil Sulfato de SodioRESUMEN
Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.
Asunto(s)
Hemaglutininas/análisis , Vacunas contra la Influenza/inmunología , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Éteres , Cobayas , Pruebas de Hemaglutinación , Inmunodifusión , Orthomyxoviridae/efectos de los fármacos , Polisorbatos , Proteínas/análisisRESUMEN
Vero cells were grown in microcarrier culture and subsequently infected with herpes simplex virus (type 1). Amino acid analyses were conducted on culture supernatants using a rapid reverse-phase high performance liquid chromatography method. The utilization of amino acids by the cells during cell growth and virus production phases was examined. During cell growth, glutamine and histidine were rapidly depleted (80-90%) by Vero cells while arginine and tryptophan-menthionine (peaks not resolved) were only moderately reduced (ca. 40%). In contrast, a marked increase in the concentrations of alanine and serine in the culture medium was observed. During virus production, the rapid depletion of glutamine, as well as the release of alanine and serine, was again observed, while the concentrations of isoleucine, lysine, threonine and valine increased only moderately.
Asunto(s)
Aminoácidos/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Microesferas , Simplexvirus , Cultivo de VirusAsunto(s)
Fibroblastos/análisis , Interferones/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Amino Azúcares/análisis , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Concanavalina A , Dextranos , Electroforesis en Gel de Poliacrilamida , Humanos , Interferones/metabolismo , Leucocitos/análisis , Resinas de Plantas , Sefarosa/análogos & derivadosRESUMEN
The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met1-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-X-Gln-Lys.
Asunto(s)
Interferones , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Fibroblastos , Humanos , Interferones/aislamiento & purificaciónRESUMEN
Two methods are described for the preparation of NalphaB1,Nepsilon29-Boc2-insulin from Nalpha A1-trifluoroacetyl-insulin and Nalpha A1-citraconyl-insulin in 80 - 90% and 65% yields, respectively. Removal of the Boc protections afforded the fully active insulin. Application of this derivative was demonstrated by the preparations of des-GlyA1-insulin and [A1-guanidinoacetyl]insulin. The former compount exhibited 2% activity in the in vitro free fat cell assay and the latter 88 +/- 5% while NalphaB1-NepsilonB29-Boc2-insulin showed 45 +/- 3% activity only.
Asunto(s)
Insulina/análogos & derivados , Aminoácidos , Animales , Azidas/farmacología , Bovinos , Cromatografía por Intercambio Iónico , Compuestos de Dansilo , Electroforesis en Acetato de Celulosa , Electroforesis en Papel , Insulina/aislamiento & purificación , Ácido Trifluoroacético/metabolismoRESUMEN
The preparation of affinity columns that contain insulin attached to Sepharose in a targeted manner by way of biotin-avidin noncovalent bonds is described. Insulin was acylated selectively at the amino terminus of the B chain with the N-hydroxysuccinimido ester of biotin to form N(alpha,B1)-biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean +/- SD) 94 +/- 9.6% (P, 0.05) that of the control insulin. N(alpha,B1)-Biotinylinsulin displaced 4-hydroxyazobenzene-2'-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of the N(alpha,B1)-biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4. N(alpha,B1)-Biotinylinsulin combined with avidin attached to Sepharose to form affinity columns in which the hormone was attached to the support by strong noncovalent bonds. The determination of the loading of avidin-Sepharose columns with biotinylinsulin was greatly facilitated by the attached biotin which provided a marker whose concentration could be assessed accurately by titration with avidin. Biotinylinsulin attached to avidin-Sepharose beads retained the ability to stimulate rat epididymal adipocytes. The activity of several samples of these beads was about 15% that of free biotinylinsulin, based on the amount of biotinylinsulin anchored to the support. The advantages of biotinylated hormones for the targeted attachment of hormones to solid supports are discussed.