Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays.

Prostate-specific antigen (PSA) in serum is primarily complexed with alpha 1-antichymotrypsin (alpha 1-ACT). However, 12-15% of prostate cancer (PCa) patients present with the predominant form being uncomplexed (free) PSA (Lilja et al., Clin Chem 1991;37:1618-24). We report that commercial immunoassays demonstrate variations in reactivity, especially to the uncomplexed form. We fractionated and analyzed commercial controls, PSA complexes prepared in vitro, and sera from patients with PCa or benign prostatic hyperplasia, using molecular sieve chromatography and Hybritech Tandem-R, Abbott IMx, and Ciba Corning ACS PSA assays. Peak integration of PCa samples demonstrated ACS:Tandem-R ratios of 1-1.3 for PSA/alpha 1-ACT complex. In contrast, ratios of uncomplexed peaks ranged from 2 to 4, suggesting a greater reactivity of the uncomplexed form in the ACS PSA assay. Discrepancies between assays, when PSA was measured in unfractionated sera, correlated directly with the percentage of the uncomplexed form. In controls, fractionation revealed the presence of uncomplexed PSA only, with ratios of ACS:Tandem-R and IMx:Tandem-R of 3:1 and 1.8:1, respectively. Immunoblots of PCa sera detected uncomplexed PSA (approximately 30 kDa) and PSA complexes of approximately 95 kDa (PSA/alpha 1-ACT) and > 200 kDa, indicative of alpha 2-macroglobulin. Maximal recognition of all forms of PSA may be important for early detection of disease progression.

Fiber optic evanescent wave immunosensors for medical diagnostics.

Immunodiagnostic sensors permit sensitive measurement of a wide variety of both high and low molecular mass analytes--serum analytes may be detected at picomolar concentrations. Due to the sensor monitoring antigen--antibody reactions directly, quantitative results are available within seconds to minutes of sample introduction. In recent years, the trend has been for immunodiagnostics to be used at sites away from centralized medical laboratories. Specific requirements associated with such use are being achieved by the development of optical devices incorporating evanescent wave sensors.

Measurement of ferritin-bearing peripheral mononuclear blood cells in cancer patients by radioimmunoassay.

A radioimmunoassay has been developed to measure ferritin bound to the surface of isolated human peripheral blood mononuclear white blood cells (PBMs) in order to investigate the possible relationship of this phenomenon to breast and other forms of cancer. The assay measures the specific binding (%SP) of affinity-purified 125I-labeled rabbit anti-Hodgkin's spleen ferritin antibody to isolated patient PBMs. A preliminary prospective, preclinical trial on 300 patients was run which included: (a) normals, benign breast disease, and medical/surgical patients as non-cancer controls; (b) postoperative primary cancer and advanced cancer in clinical remission as post cancer controls; and (c) both early preoperative breast cancer patients and cancer patients with localized recurrences or active disseminated disease as test groups. The mean %SP for the non-cancer control groups was in the range of 4.3 to 5.1 (n = 187), which was identical to that for inactive cancer or postoperative cancer, which was no evidence of recurrence. Using a %SP normal cutoff level of 6.5, which resulted in a false-positive rate of approximately 10% for both non-cancer and post-cancer control groups, only 27% of early preoperative cancers (n = 22) gave elevated %SP values. These results suggest that measurement of ferritin-PBM is inappropriate for early disease diagnosis. In contrast, 91% of patients with advanced active breast cancer and 73% of those patients with other types of advanced cancers, including tumors of ovarian, lung, colon or esophageal origin, showed elevated %SP values more than double those of post-cancer controls. The mean %SP value in active advanced cancer was 10.8 for breast (n = 12) and 10.6 for all other solid tumors investigated (n = 34). Paired patient comparisons of ferritin-PBM and plasma carcinoembryonic antigen in breast cancer showed elevations in 91% of the patients for ferritin-PBM and 67% for carcinoembryonic antigen. Overall, these results suggest that patients with advanced cancer display elevated levels of ferritin on the surface of their PBMs and that this measurement may be a useful adjunct in monitoring and evaluating the clinical status of cancer patients.

Characteristics of a specific radioimmunoassay for measurement of ferritin on the surface of peripheral mononuclear white blood cells in cancer patients.

Using 125I-labeled rabbit anti-Hodgkin's spleen ferritin antibody (RHF), a simple radioimmunoassay has been developed for quantitation of ferritin on the surface of peripheral blood mononuclear white blood cells (PBM). This method makes use of a % specific binding determination (%SP) by measuring the amount of 125I-labeled RHF bound to 1 X 10(6) PBM in the presence and absence of soluble ferritin. To standardize this procedure, artificial ferritin positive control cells were prepared by covalently coupling ferritin to cultured acute lymphoblastic leukemia cells. These cells were tested on a daily basis in parallel with patient PBM's to ensure inter and intra-assay precision and remained stable for over two years. Characteristics of 125I-labeled RHF binding to control and patient PBM's were evaluated to determine the specificity of interaction and optimum binding parameters. %SP was linear in the range of 1 X 10(5) - 1 X 10(6) PBM's and was progressively inhibited by graded concentrations of soluble ferritin. F(ab')2 preparations of RHF were equally as effective as intact RHF in blocking 125I-labeled RHF binding confirming that 125I-labeled RHF was not binding non-specifically to PBM Fc receptors. Additional experiments describing kinetics and methods of standardization of new lots of 125I-labeled RHF are also described.

The effect of ionic environment in specific FSH binding to plasma membrane receptor.

Studies were undertaken to define the effects of ionic strength and ion selectivity on specific binding of 125I-hFSH to both its particulate and Triton X-100 solubilized calf testis plasma membrane receptor. Plasma membrane vesicles migrated with a negative charge on free flow electrophoresis. There was no evidence of formation of inside-out vesicles. Specific FSH binding to the plasma membrane vesicles with both monovalent and divalent cations, used to reduce charge repulsion between the negatively charged FSH molecule and membrane vesicles, revealed similar binding maxima at an ionic strength range of 0.03 to 0.05. However, Mg2+ consistently resulted in 25-30% greater specific binding than with any other cation. That increased FSH binding suggested unmasking of specific FSH plasma membrane binding sites. However, specific FSH binding to solubilized plasma membrane vesicles was unaffected by ionic strength or ion selectivity. Enzymatic removal of sialic acid from plasma membrane vesicles significantly enhanced specific FSH binding. In previous studies, we had observed that sialic acid was not exposed within the FSH binding site. Consequently, enhanced FSH binding associated with desialylation of plasma membrane vesicles probably reflected modification of parareceptor sites which affected the conformation of the specific FSH receptor.

Hemoglobin electrophoresis at alkaline pH on agarose gels.

We describe a modified thin agarose-gel system for use in primary hemoglobin screening by electrophoresis. The system makes use of a Tris-EDTA-borate-buffered agarose gel (pH 8.8) and a sodium barbital electrophoresis buffer (pH 8.6). Separation is effected in 20 min at a constant potential of 250 V. Eight samples can be simultaneously separated and the patterns made visible in 47 min. There are fewer operative steps needed in running and staining. Normal and the more common abnormal hemoglobins are separated into easily visualized and differentiated bands, with the separation of HbA and F significantly improved over that attainable by present methods.