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Despite improvements in health and longevity, many workers in the United States retire young. By age 62, only 44 percent of men and 24 percent of women are still working full-time. The combination of younger retirement and increasing longevity means that Americans are spending more years in retirement than at any time in history. The widespread availability of post-retirement benefits is an important aspect of this national trend. Eligibility for employer-provided retirement benefits can begin as young as age 50 and occurs quite frequently at age 55. Eligibility for Social Security benefits begins at age 62. Eligibility for Medicare begins at age 65. As the population ages, the implementation of cost-saving reforms in retirement programs has become an increasing policy concern. To sustain the major public entitlement programs, proposals have been made to raise the age of eligibility for Social Security and Medicare, or to reduce benefit levels, or to target benefits to those most in need. Other cost-saving changes have been considered, and in many cases implemented, in employer-provided retirement benefits. These policy changes will have implications for the retirement decisions of working Americans in the future. This report, drawing on research sponsored by the National Institute on Aging, reviews the trend in the United States toward earlier retirement as well as some recent research findings on how retirement decisions relate to public and private retirement policies. With the changing age demographics of the population, the implementation of cost-saving reforms to retirement policies and other changes in the economic circumstances of individuals as they age, the work and retirement decisions of older workers will continue to evolve over the coming decades.

Surface plasmon resonance (SPR) biosensors were constructed on miniature integrated sensors. Recognition elements were attached to the sensor surface using a gold-binding repeating polypeptide. Biosensors with fluorescyl groups attached to their surfaces were functional for at least 1 month of daily use with little decrease in response to the binding of an anti-fluorescyl monoclonal antibody. The coupling of protein A to the gold-binding polypeptide on the sensor surface enabled the biosensor to detect the binding of antibodies to the protein A and provided a sensor with convertible specificity. The system described herein provides a simple and rapid approach for the fabrication of highly specific, durable, portable and low cost SPR-based biosensors.

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed for mouse intestinal mast cell proteinase (IMCP). Specificity was demonstrated by the absence of immunoreactivity with extracts of isolated serosal mast cells (SMC), or with high concentrations (50 micrograms/ml) of the antigenically similar rat mast cell proteinases I or II. The small and large intestines in normal mice were the major sources of IMCP, there being little or no IMCP in non-mucosal tissues. Concentrations of IMCP in normal (non-parasitized) mice were low, but were increased 100-1000-fold intestines of mice infected 10 days earlier with Trichinella spiralis. The kinetic response of secreted IMCP into the blood of mice following infection with T. spiralis was also studied. Systemic release of IMCP coincided with the immune expulsion of adult worms from the intestine, and peak concentrations (9.45 micrograms/ml IMCP) occurred 9 days after infection. The tissue distribution of IMCP, its secretion into blood, and its enteric accumulation during parasite infection, are consistent with a mucosal mast cell (MMC) source for IMCP. The results are discussed in the context of similar findings for rat mast cell proteinase II.

BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.

An intracellular serine protease, which is believed to initiate the degradation of several intracellular pyridoxal phosphate-dependent enzymes, was localized by immunofluorescence in atypical mast cells of the lamina propria and in intraepithelial cells of the rat small intestine. Some mucus-secreting goblet cells also contained the protease antigen. Atypical mast cells containing the enzyme were present in large numbers beneath the epithelium of bronchioles. All atypical mast cells also contained low levels of the chymotrypsin-like protease of normal mast cells. Both enzymes were consistently present in normal connective tissue mast cells. Amino acid content, molecular weight, and lack of immunologic crossreactivity indicate that the two enzymes are similar but not identical. The cell-specific localization of the intestinal serine protease makes it unlikely that the enzyme has any general role in the degradation of pyridoxal phosphate-dependent enzymes. The function of the enzyme in mast cells, atypical mast cells, and intestinal goblet cells is not known.

The distribution of the predominant chymotrypsin-like enzyme of mucosal mast cells (rat mast cell protease II: RMCP II) was examined in naive and Nippostrongylus-primed rats both before and after the induction of systemic anaphylaxis. Anaphylactic secretion of RMCP II following i.v. challenge of primed rats with worm antigen was accompanied by significant depletion of this enzyme from the jejunal and gastric mucosae; the concentrations were not altered in the ileum and colon. Despite significant increases in the levels of RMCP II in lung and mesenteric lymph node following infection with N. brasiliensis there was no anaphylactic depletion of this enzyme from these sites. No RMCP II was detected in liver, spleen, kidney or bone marrow either before or after systemic anaphylaxis. Mucosal mast cells were depleted from the jejunal, gastric and colonic mucosae following antigen challenge of primed rats. These data provide further evidence that gastrointestinal mucosal mast cells are the major source of secreted RMCP II following systemic anaphylaxis in the rat.

We have used immunoprecipitation to test tumor biopsies and normal adult and fetal human tissues for p97, a tumor-associated protein. Five of nine melanoma biopsies contained p97 in low to very high levels. Three of seven non-melanoma tumors contained p97, but in smaller amounts. No p97 was detected in any of the normal adult tissues examined. The protein was, however, observed in samples of fetal colon and umbilical cord, and in one sample of fetal lung. One of two benign nevi contained high levels of p97, whole on benign angiofibroma was negative. We conclude that the presence of p97, in levels detectable by our method, appears to be characteristic of certain neoplastic and fetal tissues.

Factor VII is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it participates in blood coagulation by activating factor X and/or factor IX in the presence of tissue factor and calcium. Clones coding for factor VII were obtained from two cDNA libraries prepared from poly(A) RNA from human liver and Hep G2 cells. The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature protein that circulates in plasma is a single-chain polypeptide composed of 406 amino acids. The amino acid sequence analysis of the protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VIIa by the cleavage of a single internal bond between arginine and isoleucine. This results in the formation of a light chain (152 amino acids) and a heavy chain (254 amino acids) that are held together by a disulfide bond. The light chain contains a gamma-carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule. Factor VII shows a high degree of amino acid sequence homology with the other vitamin K-dependent plasma proteins.

"Group-specific" protease (GSP) is a serine protease, obtained from rat small intestine, which preferentially inactivates the apo forms of certain pyridoxal phosphate requiring enzymes. The enzyme contains 224 amino acid residues in a single polypeptide chain and three disulfide bonds. In the present work the covalent structure has been determined and its homologous relationship to those of chymotrypsin, trypsin, and elastase has been established (approximately 33% identity with each). The residues forming the "charge-relay" system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in GSP, whereas an alanyl residue at position 176 of GSP corresponds to a residue which participates in the primary substrate binding site in serine proteases (Asp-177 in trypsin; Ser-189 in chymotrypsin). Three disulfide bonds in GSP occur in similar positions in chymotrypsin, trypsin, and elastase. However, GSP lacks a disulfide bond which is present in all known serine proteases (linking Cys-191 to Cys-220 in chymotrypsin). In view of the close proximity of this bond to both the primary and the antiparallel binding sites of various serine proteases, it is likely that its absence in GSP is related to the substrate specificity of this enzyme. It is concluded that GSP diverged from a common ancestor preceding chymotrypsin but following trypsin.

Rats primed by infection with the intestinal nematode Nippostrongylus brasiliensis and challenged intravenously with soluble whole-worm antigen undergo systemic anaphylactic shock. The primary lesions are in the gut and include increased permeability of the mucosa together with release, into enteric secretions, of a mucosal mast cell (MMC)-specific serine proteinase, rat mast cell protease II (RMCP-II). This enzyme is also released into the blood of shocked rats. These manifestations of anaphylaxis were abolished in rats previously treated with corticosteroids (methylprednisolone acetate, 25 mg per kg of body weight, 48 and 24 hr before i.v. challenge with antigen). Suppression of the response was associated with depletion of RMCP-II and of MMC from the intestinal mucosa. Depletion occurred 4-24 hr after treatment with as little as 1 mg of methylprednisolone per kg. By contrast, neither connective tissue mast cells nor serum levels of parasite-specific IgE were depleted in rats given 2 X 25 mg of methylprednisolone per kg. The capacity of unprimed treated rats to mount passive cutaneous anaphylaxis was, however, impaired.

Spleen cells from a mouse immunized with human melanoma cells were fused with mouse myeloma cells, and somatic cell hybrids were grown in selective medium. Eight hybrids, which secreted antibodies to protein antigens of the melanoma cell line, were identified by immunoprecipitation of a 125I-labeled melanoma cell lysate followed by sodium dodecyl sulfate-gel electrophoresis of the immunoprecipitates and autoradiography. Seven of the eight melanoma proteins identified in this way were present at the cell surface. Two of the cell surface proteins, p80 and p97, were not detected in autologous fibroblasts.

Secretory granules of the rat basophilic leukemia (RBL-1) cell, a chemically generated tumor cell line maintained in tissue culture, were shown to stain with alcian blue but not with safranin counterstain and to have sparse, small, electron-dense granules. A Mr 25,000 protein was the major [3H]diisopropyl fluorophosphate-binding protein in extracts of RBL-1 cells. Double-immunodiffusion analysis of extracts revealed immunoreactivity for rat mast cell protease (RMCP)-II, a Mr 25,000 neutral protease present in the secretory granules of rat mucosal mast cells and cultured rat bone marrow-derived mast cells, but no immunoreactivity for RMCP-I, the predominant neutral protease of rat connective tissue mast cells. By radial immunodiffusion, there was 66.8 ng of RMCP-II per 10(6) cells. Whereas rat connective tissue mast cells stain with alcian blue and safranin and contain heparin proteoglycan, rat mucosal and rat bone marrow-derived mast cells stain with alcian blue only and contain a non-heparin proteoglycan and lesser amounts of histamine. Proliferation of rat mucosal mast cells in vivo and rat bone marrow-derived mast cells in vitro requires T-cell factors, whereas no comparable requirement has been observed for connective tissue mast cells. The transformed RBL-1 tumor cells, whose growth is independent of factors other than those present in standard tissue culture medium, has previously been shown to contain predominantly chondroitin sulfate di-B proteoglycans and low amounts of histamine. The similar histology and secretory granule biochemistry of the rat mucosal mast cells, rat culture-derived mast cell, and RBL-1 cell suggest that they comprise a single mast cell subclass distinct from the rat connective tissue mast cell.

Infestation of the gastrointestinal tract by parasitic nematodes is invariably associated with mucosal mastocytosis, which is a thymus-dependent phenomenon in parasitized rats, and is adoptively transferable with a T cell-enriched population of thoracic duct lymphocytes. When derived by in vitro culture, mucosal mast cells (MMC) arise from a bone marrow precursor after stimulation by T cell-derived factors. In rats infected with the nematode Trichinella spiralis, mucosal mastocytosis is temporally associated with the immune expulsion of the adult worms whereas in the case of Nippostrongylus brasiliensis, mastocytosis is frequently observed to occur after worm expulsion has been completed. Consequently, there has been doubt as to whether MMC are active and serve a functional role in the expulsion of rat intestinal nematodes. MMC contain and secrete a neutral proteinase, rat mast cell protease II (RMCP II); detection and assay of secreted RMCP II therefore provides a direct measurement of MMC activity. Here we describe the release of this enzyme into the blood of rats infected with N. brasiliensis or T. spiralis. Our results show that the systemic secretion of RMCP II coincides with the immune expulsion of these nematodes, demonstrating clearly for the first time that rat MMC are functionally active during the immune elimination of primary nematode infections.

BALB/c mice were immunized with human melanoma cells and their spleen cells hybridized with NS-1 myeloma cells. The hybrids were screened for the production of antibodies that bound to melanoma cells. Two hybridomas of interesting specificity were identified and cloned. Hybridoma 5.1 produce an IgG1 antibody that binds to about half of the melanomas and carcinomas tested. The target is a polypeptide with an apparent molecular weight of 210 kilodaltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The antigen, denoted p210, is also expressed in normal adult brain and in certain fetal tissues. Hybridoma 6.1 produces an IgM antibody that binds to about 50% of the melanomas, and 80% of the kidney carcinomas tested. The antigen defined by this antibody in melanomas has an apparent molecular weight of 155 kilodaltons and is denoted p155. It has not been observed on any normal adult or fetal tissues. The antigen present in the kidney carcinomas was not p155, but rather consisted of two proteins of approximately 60,000 and 250,000-300,000 daltons. This observation suggests the possibility that the antigenic determinant recognized by antibody 6.1 may be present on several distinct protein molecules.

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.

The systemic secretion of a serine protease, rat mast-cell protease II (RMCPII), a major product of rat mucosal mast cells (MMC), was measured by a sensitive enzyme-linked immunosorbent assay (ELISA) in the sera of naive and primed rats challenged with the nematode N. brasiliensis. The systemic secretion of RMCPII was both time- and dose-dependent in primed rats (1-3 micrograms RMCPII/ml serum, 1 hr after challenge). Systemic release of RMCPII was slower and less pronounced in naive rats following intraduodenal challenge with 5-day-old worms. No RMCPII was detected in the sera of naive rats challenged with 4-day-old N. brasiliensis. Soluble worm antigen had no effect in naive rats, but when it was given intraduodenally or intravenously to primed rats, the serum levels of RMCPII 1 hr later were 10.5 micrograms/ml and 122 micrograms/ml, respectively. Few morphological changes were detected in MMC following worm challenge and the jejunal content of RMCPII was unaltered. A substantial reduction in the number of MMC occurred following intravenous injection of worm antigen, and the remaining cells were vacuolated and pale-staining, although granule exocytosis was not observed. Significant reduction in the jejunal content of RMCPII was also evident. These results demonstrate, unequivocally, that MMC are activated in response to N. brasiliensis challenge infection or to parasite antigens. In addition, the ability to detect secreted RMCPII in the sera of test animals provides a highly sensitive and uniquely selective assay to determine the participation of MMC in pathological reactions at mucosal surfaces.

The physical, chemical, and immunologic properties of a protease from rat skeletal muscle, proposed to function in the degradation of certain intracellular enzymes, are identical to those of a chymotrypsin-like serine protease isolated from peritoneal mast cells. The results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea indicate that the two rat proteases have identical mobilities corresponding to a molecular weight of 26,000. The relative amino acid compositions of the proteases are nearly identical. Immunodiffusion tests for crossreaction between the muscle protease and antisera directed toward mast cell protease indicate that the former is immunologically identical to mast cell protease. The first 35 amino-terminal residues of the two enzymes are identical and indicate homology of these proteins to other mammalian serine proteases. The sequence analysis of the protease from muscle was extended for an additional 16 positions, and comparison of this amino-terminal sequence with that of a similar enzyme from small intestine showed approximately 75% sequence identity. In contrast, only 40% of the residues in this region of bovine chymotrypsin A were found at corresponding loci in rat muscle protease. It is concluded that the protease from muscle or mast cells is closely related to the enzyme from small intestine which recently was localized in the "atypical" mast cells of gut mucosa [Woodbury, R. G., Gruzenski, G. M. & Lagunoff, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2785-2789].

Culture supernatants from mitogen-stimulated splenocytes were found to stimulate protease production in P815Y mastocytoma cells. Such supernatants increased cell-associated plasminogen activator levels in a dose-dependent fashion, and under serum-free conditions. In contrast to peritoneal exudate cells, tumor-cell plasminogen activator was not enhanced by the mitogen ConA alone. The tumor cell line P815Y may, thus, be used as a homogeneous cell source for the quantitation of lymphocyte factors which activate or inhibit plasminogen activator activity.

Following infection with the intestinal nematode Nippostrongylus brasiliensis, mucosal mast cell (MMC) proliferated in the jejunum and the peak of this response was associated with a nine-fold increase in the level of mucosal mast cell protease (RMCPII) in the mucosa. At this stage the protease constituted 10%-15% of the soluble protein from gut homogenates. Concomitant immunoperoxidase studies showed that during the early proliferation of the MMC, only a proportion of the cells in lamina propria and none of the MMC within the epithelium contained detectable RMCPII. Fourteen and 20 days post infection and following a secondary challenge, staining for RMCPII in lamina propria MMC was much stronger and a few intraepithelial mast cells also contained RMCPII. With time after infection an increasing proportion of intestinal goblet cells were specifically labelled, indicating an accumulation either of RMCPII or of an antigenically similar enzyme within mucous glycoproteins. The significance of the high levels of protease in parasitized gut and of its apparent cellular distribution is discussed in relation to the protective response against the parasite.

The substrate specificity of rat mast cell protease I (RMCP I), a chymotrypsin-like serine protease localized in the secretory granules of mast cells, was compared to that of bovine alpha-chymotrypsin by using several peptide and protein substrates of known amino acid sequences. Although the overall specificities of the two proteases appeared similar, subtle but significant differences were observed. RMCP I was more prone than chymotrypsin to hydrolyze peptide bonds consisting of Leu-Xaa or two hydrophobic residues--e.g., Phe-Phe. Additionally, the hydrolysis of angiotensin I catalyzed by chymotrypsin, but not by RMCP I, resulted in the generation of angiotensin II as an intermediate product. In contrast to the solubilized enzyme, the RMCP I activity within the insoluble granules was completely stable for at least 2 months in suitable buffers at pH 8.0 or pH 7.2, at 4 degrees C. Carboxypeptidase A activity associated with isolated mast cell granules was completely inhibited by 10 mM o-phenanthroline. Polypeptides smaller than apomyoglobin (17,199 Da) were rapidly hydrolyzed by granule-bound RMCP I, whereas apomyoglobin and other larger proteins were not hydrolyzed. In contrast, the free protease readily hydrolyzed the larger proteins. Neither normal rat serum nor alpha 1-antitrypsin, both of which inhibited the activity of free RMCP I, was effective in inhibiting granule-associated RMCP I. The results indicate that granule-bound RMCP I is not released into solution from isolated secretory granules under physiological conditions of ionic strength and pH and that the granule structure limits the size of proteins that can be hydrolyzed by the protease.

We have used two highly sensitive assays to quantitate p97, a protein associated with human melanoma, in cultured cells and normal adult, fetal, and neoplastic tissues. To measure p97 at the surface of intact cells, radiolabeled Fab fragments of a monoclonal antibody specific for p97 were used in a binding assay. To measure p97 in detergent-solubilized membrane preparations, we used a novel double-determinant immunoassay that uses two monoclonal antibodies to two distinct antigenic determinants of p97. These assays revealed that although p97 is present in small amounts in normal adult tissues, it is present in much larger amounts in most melanomas, in some other tumors (both benign and malignant), and in certain fetal tissues. We conclude that monoclonal antibodies to p97 may prove to be of value for the diagnosis and therapy of melanoma.