Incorporation Of 125I Research Articles

Iodination of Antibodies with Immobilized Iodogen.

Iodination, a chemical or enzymatic incorporation of 125I to specific amino acid side chains, is a commonly used method for labeling antibodies with radioisotopes. Commercially available products make iodination of antibodies a simple and quick process. One example, used here and available at Pierce, is the "Iodination bead," or N-chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads.

Radioiodination of the stable metabolic fragment of bradykinin, RPPGF

Arg-Pro-Pro-Gly-Phe (RPPGF, BK[1–5]), is a stable metabolite of the peptide hormone bradykinin. Considering the short half-life of bradykinin (BK, ∼15 secs), RPPGF has been used as a marker for BK’s endogenous generation. A lack of a radioiodinated RPPGF has precluded the development of a radioimmunoassay for this peptide. The present study describes a two-step reaction that allows for the incorporation of 125I into the aromatic ring of the phenylalanine of RPPGF. This radioiodinated analog is recognized by an antibody to RPPGF, demonstrating its utility for the development of a radioimmunoassay for measurements of RPPGF, a stable metabolic product of bradykinin.

Radioiodination of human interferon-α2 interferes with binding of C-terminal specific antibodies

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-α2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-α2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-α2 with Na 125I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30–53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues ∼120–145∼). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-α2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

P-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide−Protein Interaction Application to Substance P Receptor

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.

Labeling of insulin with non-radioactive 127I and application to incorporation of radioactive 125I for use in receptor-binding experiments by high-performance liquid chromatography

Conditions for the labeling of insulin with radioactive iodine isotopes were investigated by means of incorporation of non-radioactive 127I into the peptide. Either the chloramine-T (CT) or lactoperoxidase-hydrogen peroxide (LPO) technique was applied and reversed-phase high-performance liquid chromatography (RP-HPLC) was used for analysis of the reaction products. The LPO method provided the 127I-labeled peptide within 15–30 min, whereas the CT alternative yielded the labeled substrate even within 15 s. However, the latter reaction can only be controlled in a reproducible manner with difficulty and undesirad side-reactions became increasingly prominent when t a few seconds. In another experiment, the LPO technique was applied for radiolabeling insulin with 125I. The product was first purified by size-exclusion chromatography (SEC) and then subjected to RP-HPLC. SEC yielded two peaks. The smaller one, which eluted at a slightly higher K d value (accounting for about 14% of total radioactivity) predominantly consisted of material eluting at the column's void volume under the conditions of RP-HPLC, whereas the main SEC fraction (accounting for about 86% of total radioactivity) yielded a single peak, as shown by HPLC. The radioactive material attributable to the main SEC fraction revealed the expected receptor-binding properties, as evidenced by displacement experiments with non-radioactive insulin, as well as the action of tetradecanoyl phorbol acetate on the binding characteristics and thus indicating formation of a labeled hormone retaining biological activity.

Truncated Forms of the Human Prion Protein in Normal Brain and in Prion Diseases

The cellular form of the prion protein (PrPc) is a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety. An aberrant form of PrPc that is partially resistant to proteases, PrPres, is a hallmark of prion diseases, which in humans include Cruetzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. We have characterized the major forms of PrP in normal and pathological human brains. A COOH-terminal fragment of PrPc, designated C1, is abundant in normal and CJD brains as well as in human neuroblastoma cells. Sequence analysis revealed that C1 contains alternative NH2 termini starting at His-111 or Met-112. Like PrPc, C1 is glycosylated, anchored to the cell membrane, and is heat-stable. Consistent with the lack of the NH2-terminal region of PrPc, C1 is more acidic than PrPc and does not bind heparin. An additional fragment longer than C1, designated C2, is present in substantial amounts in CJD brains. Like PrPres, C2 is resistant to proteases and is detergent-insoluble. Our data indicate that C1 is a major product of normal PrPc metabolism, generated by a cleavage that disrupts the neurotoxic and amyloidogenic region of PrP comprising residues 106-126. This region remains intact in C2, suggesting a role for C2 in prion diseases.

Trifunctional Reagents for Derivatizing Sulfhydryl Groups

The syntheses of four trifunctional reagents for alkylating sulfhydryl groups in proteins are described: N-γ-maleimidobutyrylbiocytinyl-tyramine (compound I) and its sulfone, N-γ-maleimidobutyrylbiocytinyltyrosine (compound II), and N-α-4-maleimidobutyrylbiocytinamido-2 ′-[ p-(hydroxyphenyl)-propionamido] ethane (compound III). Each reagent contains a maleimide function capable of reacting with SH groups, a p-hydroxyphenyl group that can be iodinated, and a "biotin handle" to facilitate purification of the derivatized proteins or peptides derived from them by biotin-avidin affinity chromatography. Detailed conditions for obtaining the pure di-iododerivatives of the compounds have been developed. The biotin is attached to all the reagents via the ϵ-amino group of lysine (biocytin) to provide sufficient space for optimum binding to avidin. The half-times ( t 1 2 ) for dissociation of compound I from succinoyl avidin (36.7 days), its monoiodo (26.1 days) and di-iodo derivatives (21.4 days), and compound I sulfone (29.8 days), demonstrate that iodination does not significantly interfere with binding of the biotin residue to succinoyl avidin and that these reagents can be used effectively as affinity ligands. Remarkably, all the reagents can be iodinated without loss of their sulfhydryl alkylating capacity. Alkylation of highly purified human placental insulin receptor with the di-iodo derivatives of the reagents resulted in significant incorporation of 125I into the β-subunit of the receptor and the alkylation was prevented by prior exposure of the receptor to NEM. The advantages of these reagents over those previously available are that the parent molecules (1) are inexpensive to prepare, (2) are solids that can be stored indefiniteIy without degradation, (3) and can be radiolabeled to specific activity levels over seventy times higher with 125I than the specific activity available for 3H derivatives.

Development of an 125I-postlabeling assay as a simple, rapid, and sensitive index of DNA-protein cross-links.

A rapid, simple, and sensitive 125I-postlabeling technique has been developed to allow detection of DNA-protein cross-links induced by environmental contaminants and carcinogens. This method is based on specific incorporation of 125I into tyrosine residues associated with DNA. Cultured Chinese hamster ovary cells were exposed to various crosslinking agents, e.g., UV light, K2CrO4, or NiCl2. DNA was isolated by proteinase K/phenol/chloroform. The residual peptides cross-linked to DNA were radioiodinated with Na125I and chloramine T. After repeated precipitation with ethanol, the radioactivity was determined. The 125I method was compared with a 3[H]-tyrosine prelabeling method and found to be of similar sensitivity.

Variability of integrin alpha 2 beta 1 activity on human platelets

The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.

Synthesis and properties of sulfhydryl-group-specific reagents containing 125I

125I-containing compounds that react specifically with sulfhydryl groups were prepared in yields of 30 to 40% on the basis of starting 125I quantity. The synthetic precursors were commercially available heterobifunctional crosslinkers and the peptide l-arginyl- l-tyrosine. Two types of sulfhydryl specific reagents were prepared: 3-(2-pyridyldithio)propionylarginyl-[ 125I]-monoiodotyrosine, which permits reversible incorporation of 125I at sulfhydryl sites, and 3-maleimidopropionylarginyl-[ 125I]monoiodotyrosine, an irreversible labeling reagent. These products were isolated in a highly radiochemically pure form by C18 HPLC. The second-order rate constants for the reaction of 3-(2-pyridyldithio)propionylarginylmonoiodotyrosine and 3-maleimidopropionylarginylmonoiodotyrosine with N-acetylcysteine were 28 ± 3 m −1 s −1 and 154 ± 4 m −1 s −1, respectively, at pH 7.3. Storage of carrier-free 3-(2-pyridyldithio)propionylarginyl-[ 125I]monoiodotyrosine and 3-maleimidopropionylarginyl-[ 125I]monoiodotyrosine at −80°C at a radioactive concentration of 0.4 mCi/ml resulted in conversion of 125I to species that did not react covalently with sulfhydryl groups. This process occurred with first-order kinetics and a t 1 2 of 5.7 days for the pyridyldithio compound and 7.5 days for the maleimido compound. No conversion was observed during storage at −80°C at radioactive concentrations of 0.02 mCi/ml or less. The labeling properties of these compounds were examined using red blood cell proteins as a test system. 3-(2-Pyridyldithio)propionylarginyl-[ 125I]monoiodotyrosine and maleimidopropionylarginyl-[ 125I]monoiodotyrosine reacted preferentially with membrane - associated sulfhydryl groups when incubated with intact red blood cells.

Preparation of radioiodinated secretin for radioimmtmoassay

Radioiodination of synthetic human secretin on its N-terminal histidyl residue was not difficult when a greater amount of Chloramine T and a longer reaction time were employed to achieve better incorporation of 125I. The radioiodinated tracer for an optimal radioimmunoassay required purification. The combination of Sep-pak C18 Cartridge and high performance liquid chromatography for the purification of 125I-secretin in our study revealed that the Sep-pak cartridge was a preliminary step in removing unlabeled radioactive iodide, the reactant, and labeled materials unadsorbed to the cartridge. The eluate eluted from the Sep-pak containing high radioactivity and high immunoreactivity to the antibody were selected for further purification by HPLC which eliminated undesirable radiolabeled substances with lower immunoreactivity. The purified radiolabeled secretin was used in developing a sensitive radioimmunoassay.

Acceptance of murine thyroid allografts by pretreatment of anti-Ia antibody or anti-dendritic cell antibody in vitro.

A new method for thyroid allografts in mice was established. Thyroids of C67BL/6J mice were treated with collagenase, and the follicles were isolated using a Percoll density gradient technique. These follicles were treated with anti-Ia antibody (Ab) or anti-DC Ab plus complement in order to eliminate DCs. The follicles were then mixed with agarose and transplanted under the left renal capsule of BALB/c mice. One hundred days after transplantation, acceptance of the grafts was verified by both histological study and the incorporation of 125I into the grafts. Allografts treated with C were rejected, whereas allografts treated with Ab plus C were accepted. When nontreated thyroids of C57BL/6J mice were grafted under the right renal capsule of BALB/c mice that had accepted DC-depleted thyroids of C57BL/6J mice, the nontreated thyroids were rejected. These findings indicate that DCs play a crucial role in the rejection of mouse thyroid allografts, and that the depletion of DCs permits allografts to be accepted without inducing donor-specific tolerance. Our method presented here may be developed as a viable strategy for the treatment of patients with congenital or acquired hypothyroidism.

The lack of long-term suppressive effect of ciprofloxacin on murine bone marrow.

In order to investigate the long-term effect of therapy with ciprofloxacin on haemopoietic cells, we administered ciprofloxacin orally in a daily dosage of 150 mg/kg/day in three divided doses for 11 days to syngeneic mice. A control group of mice was treated with saline. On the second, sixth, tenth and sixteenth day of ciprofloxacin therapy complete blood counts (cbc) and determination of haemopoietic progenitor cells (cfu-gm) from the tibial and femoral bone marrow were performed and followed by the transplantation of the bone marrow into lethally irradiated syngeneic mice. Bone marrow engraftment was evaluated ten days following transplantation by counting the visible colonies formed on the spleen surface (cfu-s) and by measuring the incorporation of 125I by the spleen and by the femurs of the transplanted mice. The results revealed that there was no statistical significance between the control and the ciprofloxacin treated mice in cbc, cfu-gm and cfu-s parameters. It is concluded that the suppressive effect of ciprofloxacin on haemopoietic cells appears only with very high concentrations of ciprofloxacin and is short-term and reversible.

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Isolated thyroid follicles of C57BL/6J mice were treated with anti-dendritic cell antibody plus complement in order to eliminate dendritic cells. The follicles were transplanted under the renal capsule of BALB/c mice. Acceptance of the grafts was verified by both histological study and the incorporation of 125I into the grafts 100 days after transplantation. Allografts treated with complement were rejected, whereas allografts treated with the antibody plus complement were accepted. These findings indicate that dendritic cells, but not other Ia positive cells, play a crucial role in the induction of rejection in mouse thyroid allografts, and that the deletion of dendritic cells permits allografts to be accepted.

Effects of toxic doses of a novel histamine (H 2) antagonist on the rat thyroid gland

The oral administration of high doses of a histamine H 2 antagonist SK&F 93479 (up to 1000 mg/kg/day) to male rats for up to 21 days resulted in alterations in thyroid morphology indicative of increased activity of the thyroid gland. Measurement of thyroidal 125I incorporation substantiated these findings. Treatment with SK&F 93479 resulted in a dose-dependent increase in thyroidal iodide incorporation. This was apparent after a single dose of the compound and was reversible after dosing for 7 days. The increased incorporation of 125I into the thyroid gland was apparently dependent on thyroid-stimulating hormone (TSH) since both hypophysectomy and pretreatment with thyroxine (T 4) markedly reduced thyroidal 125I uptake. Hypothalamic thyrotropin-releasing hormone (TRH) and pituitary TSH concentrations were not altered by SK&F 93479 treatment, and in TRH challenge experiments circulating TSH concentrations showed no change from control levels. These data suggest that hypothalamic pituitary sensitivity was unaltered by treatment with SK&F 93479. Pharmacological ablation of thyroidal mast-cell function did not alter the thyroid response to 125I accumulation after SK&F 93479 dosing, indicating that the action of the compound is probably not dependent on changes in thyroid mast-cell histamine. Circulating T 4 and TSH levels were altered in SK&F 93479-treated rats. Generally, T 4 levels were reduced 6 hr after dosing and TSH levels were elevated 24 hr after dosing. Triiodothyronine (T 3) levels were unaffected by SK&F 93479 treatment. The effect of SK&F 93479 treatment on T 4 clearance was measured by examining the elimination of radioactivity from the circulation of rats previously injected with 125I-labelled T 4. One oral dose of 1000 mg SK&F 93479/kg markedly increased T 4 clearance. These results suggest that SK&F 93479 affects thyroid activity indirectly by a primary effect on T 4 clearance. Reductions in circulating T 4 lead to increased TSH levels and subsequent stimulation of thyroid activity.

Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor.

Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.

Effects of different batches of 125iodine on properties of 125I-hFSH and characteristics of radioligand-receptor assays

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCi carrier free Na 125I (Amersham Corp., 15 mCi/μg I 2) in the presence of lactoperoxidase. Incorporation of 125I into hFSH was determined by the method of R. C. Greenwood, W. M. Hunter, and J. S. Grover (1963) Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20°C) using different calf testis membrane preparations having similar receptor characteristics. Each 125I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of K a and R t. Incorporation of 125I into FSH was relatively constant over the large number of experiments (62.4 ± 6.4 μCi/μg; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of 125I-hFSH was related to the lot of 125I utilized, and was significantly ( P ≤ 0.01) lower and more variable (28.7 ± 10.5 μCi/μg). Maximum bindability of 125I-hFSH was not correlated to specific activity ( r = 0.06) but was negatively correlated to hFSH 125I incorporation ( r = −0.47; P ≤ 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na 125I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.

The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics.

A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.

Phagocytosis of latex microspheres by bovine meshwork cells in culture.

The ability of cultured bovine trabecular meshwork cells to phagocytose latex microspheres was studied by electron microscopy. In addition, the incorporation of 125I gamma-globulin-coated microspheres by the meshwork cells was monitored. The cultured cells, on the basis of quantitation by scanning electron microscopy, did not show significant preference between biotic particles (gamma-globulin-coated microspheres) and abiotic particles (uncoated microspheres). Surface microvilli appeared to be involved in the initial stages of phagocytosis; indeed a significant negative correlation was demonstrated between the incidence of microvilli and the commitment to phagocytosis of preconfluent cells. Two weeks postconfluent cells were shown to be significantly less phagocytic than preconfluent cells. The importance of these findings in terms of the behaviour of meshwork cells in vivo is discussed.

Electrophoretic purification of radioiodinated follicle-stimulating hormone for radioligand receptor assay and radioimmunoassay.

A method is described for electrophoretic purification of [125I]human (h) FSH after radioiodination that improves radioligand binding to FSH membrane receptors. Lactoperoxidase-iodinated hFSH was separated from reaction products by electrophoresis on 7.5% polyacrylamide tube gels (PAGE). Material eluted from 3-mm gel slices was analyzed for incorporation of 125I and binding to antibody (RIA) or receptor (RRA), and by sodium dodecyl sulfate-PAGE for protein composition. Sodium dodecyl sulfate-PAGE analysis of individual PAGE fractions demonstrated that iodinated proteins, both higher and lower in apparent mol wt than intact FSH, were separated by PAGE, but not by gel filtration chromatography (Sephadex G-25). PAGE purification of radioligand resulted in significantly greater (compared to gel filtration) RRA sensitivity and specificity. Maximum binding of PAGE-purified [125I]hFSH to excess calf tests membrane receptors was 45%, with a specific activity of approximately 26 microCi/micrograms, as determined by the method of self-displacement. Maximum binding to excess hFSH antisera (NIH anti-hFSH 4) was 80-85%. This allowed a useful final dilution of 1:120,000, thereby facilitating development of a sensitive and specific RIA with this antiserum. These data indicate that PAGE separation of intact [125I]hFSH from other iodinated proteins results in improved radioligand binding, assay sensitivity, and assay specificity. In addition, PAGE-purified lactoperoxidase-iodinated hFSH is suitable for use in both RIA and RRA.