Purification of a Human Prostate Specific Antigen.

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

Compromised osseous healing of dental extraction sites in zoledronic acid-treated dogs.

UNLABELLED: The goal of this study was to document how treatment with high doses of zoledronic acid affects dental extraction healing. Our results, showing significantly compromised osseous healing within the socket as well as presence of exposed bone and development of a sequestrum in one animal, provide a building block toward understanding osteonecrosis of the jaw. PURPOSE: The goal of this study was to document how treatment with a bisphosphonate affects the bone tissue following dental extraction. METHODS: Skeletally mature female beagle dogs were either untreated controls (CON) or treated with intravenous zoledronic acid (ZOL). Following the extraction of the fourth premolars, healing was allowed for 4 or 8 weeks. Properties of the extraction site were assessed using microcomputed tomography (micro-CT) and dynamic histomorphometry. RESULTS: The initial infilling of the extraction socket with bone was not affected by ZOL, but subsequent removal of this bone was significantly suppressed compared to CON. After 8 weeks of healing, the alveolar cortical bone adjacent to the extraction socket had a remodeling rate of ∼50% per year in CON animals while ZOL-treated animals had a rate of <1% per year. One ZOL-treated animal developed exposed bone post-extraction which eventually led to the formation of a sequestrum. Assessment of the sequestrum with micro-CT and histology showed that it had features consistent with those reported in humans with osteonecrosis of the jaw. CONCLUSIONS: These results, showing significantly compromised post-extraction osseous healing as well as presence of exposed bone and development of a sequestrum in one ZOL animal, provide a building block toward understanding the pathophysiology of osteonecrosis of the jaw.

In vivo effects of zoledronic acid on oral mucosal epithelial cells.

OBJECTIVE: Osteonecrosis of the jaw is a serious complication of bisphosphonate treatment for which the pathophysiology is unknown. The purpose of this study was to investigate whether in vivo zoledronic acid (ZA) induces alterations in cell proliferation, apoptosis, and matrix metalloproteinases (MMPs) expression in oral mucosal epithelial cells. METHODS: One-year-old dogs were either untreated (control group) or given high doses of intravenous ZA (ZA group) for 3 months. The doses of ZA were equivalent to those given to cancer patients, yet were administered two times more frequently (every 2 weeks). Mucosal tissues were assessed immunohistochemically for cell proliferation (proliferating cell nuclear antigen, PCNA), matrix metalloproteinase (MMP) expression, and apoptosis (caspase 3 and TUNEL). RESULTS: There were no significant differences between the groups with respect to PCNA, MMP-2, MMP-14, and TUNEL positive cells. However, the expression of MMP-9 was significantly higher in the control group than in the ZA group (P < 0.05), whereas the expression of caspase 3 was significantly lower in the control group than in the ZA group (P < 0.05). CONCLUSION: These results suggest that high doses of ZA resulted in higher levels of apoptosis and lower levels of MMP-9 in the oral epithelial cells supporting the idea of bisphosphonate treatment affects the oral mucosa.

Consistency of predictive signature genes and classifiers generated using different microarray platforms.

Microarray-based classifiers and associated signature genes generated from various platforms are abundantly reported in the literature; however, the utility of the classifiers and signature genes in cross-platform prediction applications remains largely uncertain. As part of the MicroArray Quality Control Phase II (MAQC-II) project, we show in this study 80-90% cross-platform prediction consistency using a large toxicogenomics data set by illustrating that: (1) the signature genes of a classifier generated from one platform can be directly applied to another platform to develop a predictive classifier; (2) a classifier developed using data generated from one platform can accurately predict samples that were profiled using a different platform. The results suggest the potential utility of using published signature genes in cross-platform applications and the possible adoption of the published classifiers for a variety of applications. The study reveals an opportunity for possible translation of biomarkers identified using microarrays to clinically validated non-array gene expression assays.

Genomic indicators in the blood predict drug-induced liver injury.

Genomic biomarkers for the detection of drug-induced liver injury (DILI) from blood are urgently needed for monitoring drug safety. We used a unique data set as part of the Food and Drug Administration led MicroArray Quality Control Phase-II (MAQC-II) project consisting of gene expression data from the two tissues (blood and liver) to test cross-tissue predictability of genomic indicators to a form of chemically induced liver injury. We then use the genomic indicators from the blood as biomarkers for prediction of acetaminophen-induced liver injury and show that the cross-tissue predictability of a response to the pharmaceutical agent (accuracy as high as 92.1%) is better than, or at least comparable to, that of non-therapeutic compounds. We provide a database of gene expression for the highly informative predictors, which brings biological context to the possible mechanisms involved in DILI. Pathway-based predictors were associated with inflammation, angiogenesis, Toll-like receptor signaling, apoptosis, and mitochondrial damage. The results show for the first time and support the hypothesis that genomic indicators in the blood can serve as potential diagnostic biomarkers predictive of DILI.

A fast-degrading thiol-acrylate based hydrogel for cranial regeneration.

Successful regeneration of the cranium in patients suffering from cranial bone defects is an integral step to restore craniofacial function. However, restoration of craniofacial structure has been challenging due to its complex geometry, limited donor site availability, and poor graft integration. To address these problems, we investigated the use of a thiol-acrylate hydrogel as a cell carrier to facilitate cranial regeneration. Thiol-acrylate hydrogels were formulated with 5-15 wt% poly(ethylene glycol)-diacrylate (PEGDA) and 1-9 mm dithiothreitol (DTT). The degradation rate, swelling ratio, and shear modulus of the resulting hydrogel were first characterized. Then, pre-osteoblast-like cells (MC3T3-E1) were encapsulated in the hydrogel and cultured for up to 21 d. Our results demonstrate that compared to samples formulated from 15 wt% PEGDA, 5 wt% PEGDA samples showed lower storage modulus at day 10 (0.7 kPa versus 8.3 kPa), 62.7% higher in weight change after soaking for 10 d. While the 5 wt% PEGDA group showed an 85% weight loss between day 10 and 21, the 15 wt% PEGDA group showed a 5% weight gain in the same time period. Cell viability with 15 wt% PEGDA and 5 mm DTT hydrogel decreased by 41.3% compared to 5 wt% PEGDA and 5mM DTT gel at day 7. However, histological analysis of cells after 21 d in culture revealed that they had pericellular mineral deposition indicating that the cells were differentiating into osteoblasts lineage in all experimental groups. This study shows that thiol-acrylate hydrogels can be tailored to achieve different degradation rates, in order to enhance cell viability and differentiation. Thus, the findings of this study provide a fundamental understanding for the application of thiol-acrylate hydrogels in cranial bone regeneration.

Carcinoembryonic antigen-binding immunoglobulin isolated from normal human serum by affinity chromatography.

A human immunoglobulin that binds carcinoembryonic antigen (CEA) was isolated from four individual normal human sera by affinity chromatography with the use of a CEA-Sepharose solid adsorbent. The yield of isolated protein, termed human CEA-binding protein (HCBP), ranged from 1.8 to 10 microgram/ml serum. HCBP is a gamma-globulin of restricted electrophoretic heterogeneity as shown by immunoelectrophoresis. HCBP was shown to bind radioiodine-labeled CEA both by a radioimmune precipitation assay and by a radioimmunoelectrophoresis assay. This protein was of practical interest because of its potential usefulness as a carrier of radioactivity or therapeutic agents to a CEA-producing tumor for therapeutic or diagnostic purposes.

Relationship between antigen density and immunotherapeutic response elicited by monoclonal antibodies against solid tumors.

The relationship between the level of cell surface antigen expression and solid tumor immunotherapy with monoclonal antibody (MoAb) was evaluated. Two MoAb's that were shown effective in the passive therapy of breast carcinomas of human origin, established and growing in female Swiss nude mice, were used for these studies. Several groups of tumors were produced from cell cultures of different passages; each cell culture possessed a distinct target antigen level. Results from immunotherapy experiments demonstrated that the amount of tumor reduction response after MoAb therapy was proportional to the antigen density at the cell surface. Analysis of these data indicated a theoretical improbability of a single MoAb treatment being able to completely eradicate solid tumors and may necessitate the use of multiple MoAb's to circumvent this problem.

Differential localization of human pancreas cancer-associated antigen and carcinoembryonic antigen in homologous pancreatic tumoral xenograft.

Tissue localization of a human pancreas cancer-associated antigen (PCAA) and carcinoembryonic antigen (CEA) was studied in a homologous pancreatic tumoral xenograft, a human pancreatic cancer line established from ascites (AsPC-1), with the use of the indirect immunofluorescence technique with specific anti-PCAA and anti-CEA antisera. Histologically, AsPC-1 xenograft was composed of mucinous adenocarcinoma of variable size and degree of glandular differentiation. PCAA was selectively associated with the columnar cytoplasm, was involved primarily in the epithelial proliferation, and originated at the basal aspect of the glands. In contrast, CEA appeared diffuse and was found predominantly in the cuboidal cells, with higher intensity at the luminal border and in mucin. Furthermore, growth comparison of the AsPC-1 adenocarcinoma revealed that, except in the juvenile gland that was morphologically consistent with grade 1 (G1) carcinoma in situ, the number of PCAA-positively stained cells decreased from G1 to grade 4 epithelial differentiation. In well-differentiated adenocarcinoma and degenerating adenocarcinoma, cells positive for PCAA were released into the lumen of the glands, whereas in poorly differentiated adenocarcinoma these cells were often scattered singly and/or in focal clusters and disappeared in very poorly differentiated adenocarcinoma. This study showed that the PCAA was different from CEA in its immunologic reactivity and distribution in its homologous tumoral tissues, in which PCAA was predominantly associated with the proliferative phase of the malignant epithelium. These results also indicated the potential applicability of this antigenic expression in the etiology, disease grading, and staging of human pancreatic cancer.

Human tissue polypeptide antigen in breast cancer.

Serum tissue polypeptide antigen (TPA) and plasma carcinoembryonic antigen (CEA) were simultaneously measured in 108 patients with breast cancer, in 40 healthy women, and in 26 women with benign breast disease. TPA levels were elevated (0.09 microgram/ml or higher) in 53% of 19 patients with primary breast cancer, and CEA levels were elevated (2.5 ng/ml) in 21%. Among 67 patients with metastatic breast cancer, TPA and CEA levels were increased in 70% and 61%, respectively. TPA was positive in 13% and CEA in 8% of the healthy women. CEA levels were not elevated in patients with benign breast disease, but levels of TPA were elevated in 27% of those studied. Elevation of TPA levels was more frequent in patients with visceral metastasis having higher values of the test results. Among 22 women with breast cancer who had no apparent cancer recurrence, TPA levels were elevated in 12 and CEA levels in 6. In another group of 39 patients with metastatic breast cancer who received palliative therapy, a limited correlation was noted between the clinical course of the disease and changes in TPA and CEA values measured in linear fashion. Thus TPA appeared to be equal to CEA as a tumor marker in most areas analyzed.

Identification of a macromolecule containing an anticarcinoembryonic antigen-reactive substance and immunoglobulin M in human pancreatic cancer.

Ascitic fluid from a patient with carcinoma of the pancreas was fractionated by ammonium sulfate precipitation. The fraction precipitated between 25 and 50% saturation of ammonium sulfate was sequentially chromatographed on Sephadex G-200 and Sepharose 6B. A macromolecular fraction (greater than 10(6) daltons) obtained was found to react with both antihuman IgM and antiserum to carcinoembryonic antigen (CEA). This fraction was further purified by adsorption with protein A-Sepharose CL-4B and chromatography on DEAE-Sephacel. The purified macromolecular fraction had a sedimentation value of 28S as determined by ultracentrifugation. Upon dissociation of the purified macromolecule at pH 2.3 and purification of the dissociated components on Sepharose CL-2B and BioGel A 1.5M, a 19S protein and a 5S protein were recovered. The 19S protein showed a complete line of identity with a reference human IgM when reacted with antihuman IgM in gel diffusion, whereas the 5S protein showed a partial immunologic identity with colon CEA against anti-CEA. These results indicated the existence of an IgM-containing macromolecular complex with an anti-CEA cross-reactive substance in the extracellular fluid of human pancreatic cancer.

Relative reliability of five serially measured markers for prognosis of progression in prostate cancer.

During an 8-year period, 1,065 serum specimens were collected from 79 patients with prostate cancer of stages B2 to D1 (group I) and 51 patients with newly diagnosed stage D2 prostate cancer (group II) to evaluate statistically the relative reliability of elevated tumor-associated markers for progressive disease in prostate cancer. Forty of the group I patients and 21 of the group II patients presented a clinical progression of disease during follow-up. With the use of Gail's modification of Cox's regression model, serial acid phosphatase (AcP), total alkaline phosphatase (TAP), bone alkaline phosphatase (BAP), prostatic acid phosphatase (PAP), and prostate-specific antigen (PA) were analyzed. Results from group I patients revealed that only PA (P = .0002) and PAP (P = .0684) were prognostically important markers for detection of imminent disease progression. However, all markers were prognostically important in group II patients. Comparative studies indicated that PA (P = .0052) and PAP (P = .0359) were the more reliable markers for group I patients, whereas PA (P less than .0001), BAP (P = .0007), and PAP (P = .0206) were the more reliable markers for group II patients. Multivariate analyses revealed that, after adjustment for the effect of PA, no other marker was significantly related to the risk of progression. Elevated PA levels were predictive of increased risk 6 months before disease progression in group I patients only (P less than .0001). Overall, the apparent order of prognostic reliability for disease progression was found to be PA greater than PAP greater than BAP greater than AcP greater than TAP.

Prostate antigen: a marker for human prostate epithelial cells.

Specificity of a previously reported prostate antigen (PA) was assessed by several immunologic procedures. This antigen, restricted in distribution to the prostate gland, was detected within ductal epithelial cells. Continuous established cell lines LNCaP and PC-3 of malignant prostate origin retained the expression of PA. Tumor cells released the antigen in vitro into the culture fluid and also in vivo into the circulation of nude mice preinoculated with LNCaP cells. Prostate cells in culture also specifically accreted immunoglobulin fragments of PA antiserum.

Multiple marker evaluation in human prostate cancer with the use of tissue-specific antigens.

Serum prostate-specific antigen and prostatic acid phosphatase were simultaneously evaluated in 22 healthy males, 29 patients with benign prostatic hypertrophy, and 192 patients with prostate cancers at various stages as well as in 30 patients with cancers other than prostate cancer. Both markers were quantitated by specific sandwich-type, enzyme-linked, immunosorbent assays with the use of specific antiserum reagents. Serum assays revealed a discordance between these two markers; thus expressions of these two biochemically and immunologically distinct prostate-specific proteins may reflect different aspects in the biology of prostate cancer. A combination test with the use of 7.5 ng of prostate antigen and 15.5 ng of prostatic acid phosphatase/ml of serum, respectively, as cutoff values resulted in a positive detection rate of 58% for prostate cancers of stages A (7/12) and B (21/36) each, 68% for prostate cancer of stage C (19/28), 92% for prostate cancer of stage D (106/116), and only 10% for benign prostatic hypertrophy (3/29). None of 52 other cancers or healthy controls was registered as positive. This study demonstrates that a multiple marker test of tissue-specific antigens can be of an additive value in the immunodiagnosis of cancer and may be a logical and effective approach at this time, in light of the unavailability of human tumor-specific markers.

Enhancement of murine lymphokine-activated killer cell activity by retinoic acid.

Since retinoids have been suggested to be capable of potentiating immunity, the present study was undertaken to determine the effect, if any, on lymphokine-activated killer (LAK) cell activity by retinoic acid, an active metabolite of vitamin A and a differentiation enhancer. Retinoic acid alone was shown to induce no cytotoxicity generated from nylon wool-treated nonadherent murine (BALB/c) splenocytes against natural killer-resistant, LAK-sensitive syngeneic target tumor cells. When combined with human recombinant interleukin-2 (IL-2), retinoic acid augmented LAK cell activity in both a dose- and time-dependent manner. The augmentation was detected at 10(-10) M retinoic acid and reached the maximum at 10(-7) M, a greater than 200% increase in lytic activity. Kinetic study revealed that retinoic acid augmented significantly LAK cell activity when incubated in IL-2-containing culture as short as for 6 h before cytotoxicity was measured. The removal of retinoic acid from culture resulted in the loss of the augmentation. Retinoic acid was found to augment LAK cell activity in a wide range of IL-2 concentrations (750-12,000 IU/ml), even at 6,000 IU/ml where the maximal induction of LAK cell activity had been reached. No phenotype or proliferation of LAK cells was altered by the addition of retinoic acid to IL-2-containing culture. However, cellular serine protease activity, measured as N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl-esterase, in LAK cells was increased by retinoic acid also in a dose- and time-dependent manner. The increase in LAK cellular serine protease activity was significantly correlated with that of augmented LAK cell activity. Overall these results demonstrated that IL-2-induced LAK cell activity was enhanced by retinoic acid and that the augmentation may be mediated by means of enhanced expression of cellular serine protease activity. This study also suggests that, in addition to its use in chemoprevention of cancer, retinoic acid is of potential in adoptive immunotherapy.

Demonstration of two molecular variants of carcinoembryonic antigen by concanavalin A sepharose affinity chromatography.

The carcinoembryonic antigen (CEA) active glycoproteins from perchloric acid extract of liver-metastasized primary colon tumor have been separated by concanavalin A Sepharose (Con A Sepharose) chromatography. The CEA activities separated by Con A Sepharose chromatography were designated as loosely bound and tightly bound which, respectively, eluted on the Con A Sepharose column between 0.12 and 0.15 M and 0.3 M alpha-methylmannose in a linear gradient of alpha-methylmannose. Further purification of these activities by Sephadex G-200, Bio-Gels A-1.5m and P-300 yielded two variants of glycoproteins (B1 and C2) with CEA activity. Both purified preparations of CEA had similar immunochemical properties. Their A280/A260 ratios were 1.30 and 1.56, respectively. The purified loosely bound CEA (B1) had immunological, chromatographic, and electrophoretic properties similar to those of 125I-CEA, whereas the tightly bound CEA (C2) had a lower molecular weight (120,000 to 140,000). Further, specificity to these two CEA's was established by their reactions in immunoelectrophoresis with preparations of specific goat anti-CEA anti-serum obtained from other investigators. The results indicate the practical use of Con A Sepharose affinity chromatography for the separation and characterization of glycoprotein tumor antigens.

Isolation of glycoprotein antigen from ascites fluid of pancreatic carcinoma.

A glycoprotein antigen from the ascites fluid of pancreatic carcinoma has been isolated with the use of perchloric acid extraction and chromatographies on Sepharose 4B, DEAE-Sephadex, and DE52, followed by Sephadex G-200. The purified glycoprotein was found to be homogenous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A molecular weight of 185,000 dattons was determined by gel filtration. The molecule contained 55% protein and 45% carbohydrate. Both the amino acid and carbohydrate compositions were different from those of carcinoembryonic antigen. This glycoprotein antigen of pancreatic cancer cross-reacted with anticarcinoembryonic antigen; however, the antiserum prepared from this antigen did not react with carcinoembryonic antigen. The biological significance of this glycoprotein in pancreatic cancer is being studied.

Prolongation of serum half-life of interleukin 2 and augmentation of lymphokine-activated killer cell activity by pepstatin in mice.

We reported previously using a murine model that the kidney is the organ involved in catabolism of exogenous human recombinant interleukin 2 (IL-2) and that cathepsin D, a major renal acid protease, is responsible for the degradation of IL-2. In the present report also using BALB/c mice we have investigated the effect of in vivo pepstatin, an acid protease inhibitor, treatment on serum half-life of IL-2, and generation of lymphokine-activated killer (LAK) cell activity. The in vivo pepstatin treatment by i.p. injection resulted in a significant reduction in the accumulation of 125I-IL-2 by the kidney in a reverse dose-response manner. Pepstatin treatment prolonged the serum half-life of 125I-IL-2, and the increase in serum half-life of 125I-IL-2 was pepstatin dose dependent. A significant reduction in renal cathepsin D activity, as monitored by the degradation of 125I-IL-2, was detected. In vivo pepstatin (0.6 mg/kg) treatment along with IL-2 (300,000 IU/mouse) daily for 3 or 6 days resulted in an augmentation of natural killer activity exhibited by freshly prepared and uncultured splenocytes against YAC-1 cells. An additional culturing of the splenocytes with IL-2 (3,000 IU/ml) in vitro for 1 day significantly enhanced the effect of in vivo pepstatin treatment; i.e., LAK cell activity generated from the splenocytes of animals treated with IL-2 plus pepstatin was greatly augmented in comparison with that treated with IL-2 alone. Phenotypic assessment by cell surface markers (Thy-1.2, Lyt-2, L3T4, and asialo-GM1) on the fresh splenocytes prepared from animals treated in vivo with pepstatin plus IL-2 revealed a decrease in the percentage of cells expressing Thy-1.2 and Lyt-2 and an increase in those carrying asialo-GM1. These results demonstrated that, as a result of in vivo pepstatin treatment, renal cathepsin D activity was greatly inhibited, which in turn reduced the degradation of circulating IL-2, then prolonged serum half-life of IL-2, and subsequently augmented natural killer and LAK cell activity. The in vivo pepstatin and IL-2 treatment decreased the T-cells and increased the natural killer-like LAK precursor cells, possibly also with an increase in its activity, which were further induced by in vitro IL-2 culture to generate an augmented LAK cell activity. This study also suggests the clinical potential of pepstatin in IL-2-related immunotherapy.

Purification and characterization of acid phosphatase from Dunning R3327H prostatic adenocarcinoma.

Acid phosphatase (phosphoric monoester hydrolase) was isolated from the Dunning R3327H prostatic adenocarcinoma, a slow-growing and hormone-sensitive rat prostate tumor histologically similar to well-differentiated human prostatic cancer. The enzyme was purified to homogeneity and characterized. In comparison with the acid phosphatase isolated from human malignant prostate, the acid phosphatase from the Dunning rat tumor was similar in molecular weight [100,000 +/- 10% (S.D.)]. However, it possessed a single isoelectric point of 7.6 (human prostatic acid phosphatase showed multiple isoenzymes at 4.4 to 5.3); an electrophoretic mobility of 0.5 in reference to human prostatic acid phosphatase on 7.5% polyacrylamide gel, pH 8.5; an optimal pH of 5.0 with alpha-naphthyl phosphate as the substrate in 0.1 M citrate buffer (human prostatic acid phosphatase, 5.5); and a Km (alpha-naphthyl phosphate) of 6.9 X 10(-4) M (human prostatic acid phosphatase, 4.4 X 10(-5) M). Furthermore, it did not cross-react with antiserum raised against human prostatic acid phosphatase. These results show that the acid phosphatase of the Dunning R3327H prostatic adenocarcinoma is biochemically and immunologically distinct from human prostatic acid phosphatase and may be unique for this animal model of prostatic cancer.

Detection of a breast tissue-associated antigen by antiserum to Raji cell-bound circulating immune complexes of human breast cancer.

Rabbits tolerant to human immunoglobulin G were used to raise antisera against the Raji cell-bound circulating immune complexes from human breast cancer sera. After solid-phase adsorption treatment with glutaraldehyde-cross-linked normal human plasma, acetone-extracted normal liver tissue powder, and glutaraldehyde-fixed Raji cells, one antiserum reacted specifically with breast tissue extracts but not with extracts of other tissues, as examined by a counterimmunoelectrophoresis technique. Immunological reactivity of the treated antiserum was removed by incubation with normal, primary, or metastatic breast tumor tissue extracts. Incubation with normal human serum or extracts derived from tissues other than the breast showed no neutralizing effect on the antibodies. This specific antiserum reagent was used in a modification of the Raji cell radioimmunoassay. Raji cells were incubated with sera from cancer patients or normal controls and then reacted with 125I-labeled F(ab')2 fraction of the treated antiserum reagent. The amount of 125I-F(ab')2 bound was then determined. Although all sera exhibited elevated circulating immune complexes by the conventional Raji cell radioimmunoassay, 14 of 18 breast carcinoma sera demonstrated a significant uptake when compared with the normal population group as opposed to five (three lung and two colon) of 29 other cancer sera examined (p less than 0.001). An immunologically reactive breast tissue-associated antigen, purified from malignant breast tumor or normal breast tissue extracts with the use of antiserum reagent, exhibited an apparent molecular weight of 85,000 by sodium dodecyl sulfate; polyacrylamide gel electrophoresis and a pI value of 4.9 +/- 0.2. These results demonstrated that a breast tissue-associated antigen rather than a breast tumor-associated neoantigen, was involved in circulating immune complexes of breast cancer patients as detected by Raji cell immunoassay. It also implied the occurrence of disease-related autoimmunity in human breast cancer.