Human autologous mixed lymphocyte reactivity is primarily specific for xenoprotein determinants adsorbed to antigen-presenting cells during rosette formation with sheep erythrocytes.

We present evidence that most T cells proliferating in response to autologous sheep erythrocyte (SRBC)-separated non-T cells (NT) cells are not specific for autoantigens but for antigens derived from xenogeneic sources. The conclusion was based on the following three observations. First, we found that NT cells isolated in the absence of xenoproteins by means of density gradient centrifugation on Percoll only weakly stimulated autologous T cells. Because this weak proliferation could not be expanded in restimulation experiments, its significance as an immune recognitive event remains questionable. NT cells isolated by the above method in the absence of xenogeneic determinants readily acquired stimulatory capacity after brief exposure to either SRBC or fetal calf serum. Second, restimulation of T memory cells generated in 1 degree autologous mixed lymphocyte reaction (AMLR) against SRBC-separated autologous NT cells was exclusively seen when NT cells exposed to or separated with xenoproteins were used for restimulation. Third, T memory cells generated against SRBC-separated autologous NT cells were specifically restimulated by autologous Percoll-separated NT cells that had been pulsed with a variety of xenogeneic mammalian sera. These xenogeneic determinants were preferentially recognized in context with autologous HLA-DR+ cells. From these findings and from our previous results that indicated an absolute requirement of HLA-DR+-adherent NT cells (8), we conclude that human AMLR primarily does not represent an autoantigen but a xenoantigen response that is genetically restricted by the HLA-DR type of the antigen-presenting cell.

In vitro lymphocyte dysfunction in Hodgkin's disease.

In vitro lymphocyte function and delayed cutaneous hypersensitivity (DCH) response in vivo were assessed in 27 patients with Hodgkin's disease. Lymphocyte responsiveness to phytohemagglutinin (PHA) and to a standard mitomycin C-treated lymphoblastoid cell line in a one-way mixed lymphocyte culture (MLC) was measured simultaneously and compared to that of controls. Seventeen patients, including 6 of 11 untreated patients, had some defect either of DCH or of an in vitro lymphocyte function. In patients lacking a DCH response, the PHA and MLC responses were significantly depressed as compared to either those with intact DCH or normal controls. In patients with intact DCH, the PHA but not the MLC response was significantly depressed. PHA stimulation and MLC may define different subpopulations of responding thymus-derived lymphocytes in patients with Hodgkin's disease. Measurement of lymphocyte response to PHA and to a standard lymphoblastoid cell line in the one-way MLC may provide complementary in vitro means of assessing cell-mediated immunity in patients with Hodgkin's disease.

Alterations in cell surface phenotype of T- and B-cell chronic lymphocytic leukemia cells following in vitro differentiation by phorbol ester.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was used for the induction of in vitro differentiation in primary cultures of chronic lymphocytic leukemia cells to study its effects on B-cell antigens [surface IgG, HLA-DR, and the mouse erythrocyte receptor (MR)] and on T-cell antigens [T65 and Lyt-3 (sheep erythrocyte receptor)] found on these cells. Three distinct phenotypes were studied: 1) the common phenotype (slg+, HLA-DR+, MR+, T65+, Lyt-3-); 2) the T-cell phenotype (slg-, HLA-DR-, MR-, T65+, Lyt-3+); and 3) a unique phenotype (slg-, HLA-DR+, MR+, T65+, Lyt-3-). In both the common and unique phenotypes, TPA increased the expression of T65 and HLA-DR, decreased the formation of MR, and induced cytoplasmic immunoglobulin, but it did not induce Lyt-3. In the unique phenotype, TPA also induced surface immunoglobulin. These data suggest that both the common and unique phenotypes are derived from the same lineage, probably B-cell. In the T-cell phenotype, TPA increased the expression of T65 and Lyt-3, but it did not induce any B-cell antigens. These data suggest that the T-cell phenotype is derived from a T-cell lineage distinct from the two B-cell phenotypes.

Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody.

Acetone-fixed frozen tissue sections from 56 cases of human lung carcinoma were tested for reactivity by an indirect immunoperoxidase technique with a monoclonal antibody (MAb 528) specific for the external domain of the epidermal growth factor receptor (EGFR). MAb 528 reacted with all epidermoid (22/22) and large-cell (4/4) lung carcinomas evaluated. The antibody was also positive with a subset of lung adenocarcinomas (13/21) and did not react with small-cell lung cancers (SCLCs) (0/9). MAb 528 also stained normal bronchial epithelium identified within the tumor sections of 5 cases. Thus EGFR was expressed by all epidermoid and large-cell lung carcinomas examined, a subset of lung adenocarcinomas, and normal bronchial epithelium. EGFR expression was not identified in any of the SCLCs tested. These data imply that immunohistochemical detection of EGFR expression may find future application in distinguishing epidermoid, large-cell, and some adenocarcinomas of the lung from SCLCs.

Morphologic and cytochemical comparison of human lymphoblastoid T-cell and B-cell lines: light and electron microscopy.

Human lymphoblastoid cell lines characterized as T- or B-cells by various markers were compared morphologically and cytochemically by light and electron microscopy. Distinct differences in nuclear morphology, amount of cytoplasm, pyroninophilia, and periodic acid-Schiff (PAS) staining enabled us to discriminate between T- and B-cell lines. T-cells had nuclei with an irregular configuration, stippled heterochromatin, and small or absent nucleoli. The scanty cytoplasm of T-cells contained intensely stained, PAS-positive globules and was less pyroninophilic than the cytoplasm of B-cells. B-cells had more rounded, uniform, vesicular nuclei with prominent nucleoli and peripheral heterochromatin. The cytoplasm of B-cells was abundant and strongly pyroninophilic. Transmission electron microscopy generally confirmed these morphologic differences. These findings supported our contention that consistent cytologic features concordant with immunologic markers make it possible to identify certain lymphomas as being of B- or T-cell origin on purely morphologic grounds.

Establishment and characterization of an Epstein-Barr virus-negative lymphoma B-cell from a patient with a diffuse large cell lymphoma.

Cell line (LNPL) was established from a tumor biopsy of a nasopharyngeal "histiocytic" lymphoma composed of large noncleaved B-cells. LNPL expressed immunoglobulin M kappa surface immunoglobulin, as did the original tumor. LNPL also expressed Ia antigen but lacked Epstein-Barr virus nuclear antigen. It exhibited a translocation between chromosomes 8 and 14 which has been described in certain other malignant lymphoproliferative disorders. An autologous T-cell line lacked the translocation between chromosomes 8 and 14. LNPL is being used as an immunogen to produce monoclonal antibodies in our laboratory. Such cell lines may facilitate identification of lymphoma-associated surface antigens.

Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates.

Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared. Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain. In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis. Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity. Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar. Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain. Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.

Establishment of a human B-cell tumor in athymic mice.

Human B-cell tumors have been established in athymic, BALB/c mice using the EBV-positive Burkitt lymphoma cell line Namalwa. One-hundred-one of 104 animals (97%) developed tumors 10-14 days following s.c. injection of a mixture of 20 x 10(6) Namalwa and 5 X 10(6) irradiated human fibrosarcoma (HT-1080) cells. Tumors developed at the site of injection and reached approximately 300 mm2 (product of cross-sectional diameters) after 21 days; no metastases were found. Histological analysis showed that tumors consisted solely of lymphoid cells. Immunofluorescence assays demonstrated that while 85% of the tumor cells retained reactivity with the monoclonal B-cell antibody BA-1, 96% retained reactivity with antibody BA-2 and 43% with BA-3. A similar reactivity profile was observed with cultured Namalwa cells. Tumors were passaged serially 10 times without significant change in BA-1, BA-2, or BA-3 reactivity. Indirect immunofluorescence demonstrated that antibody BA-2 reached tumor cells within 2 h following i.p. injection; antigen modulation was not observed. These results demonstrate the suitability of this B-cell model for testing the in vivo efficacy and stability of anti-B-cell immunoconjugates.

Induction of in vitro and in vivo antigenic modulation by the anti-human T-cell monoclonal antibody T101.

Because of its implications for the therapeutic application of monoclonal antibodies, we have studied antigenic modulation in vitro and in vivo in patients receiving T101 monoclonal antibody. Incubation of normal peripheral blood T-cells, chronic lymphocytic leukemia cells, and cutaneous T-cell lymphoma cells with an excess of T101 at 37 degrees induced modulation of the T65 antigen. When assayed by indirect immunofluorescence, a change in cellular reactivity with T101 was seen after 1 hr. After 24 hr, normal T-cells showed a 94 +/- 4% (S.D.) decrease in fluorescence, compared to an 82 +/- 6% decrease for chronic lymphocytic leukemia cells and a 56 +/- 4% decrease for cutaneous T-cell lymphoma cells. When T101 was removed from the culture, the cells reexpressed T65. Modulation was inhibited by cold temperatures, suggesting that it is energy dependent. Patients with chronic lymphocytic leukemia, cutaneous T-cell lymphoma, or T-cell lymphoma have received 24-hr infusions of 3 to 500 mg T101 in therapeutic trials. After infusion, in vivo binding of T101 was observed in 39 of 43 treatments not associated with endogenous host anti-T101 antibodies. T65-target cells were seen in all 39 treatments associated with in vivo bound T101, suggesting that modulation had occurred. When cultured in vitro for 24 hr, these cells reexpressed T65. In vivo, reexpression of T65 occurred following disappearance of the serum T101 titer. The extent and duration of in vivo modulation were related to both the T101 dose and the tumor burden. These data suggest that the rapid rate of antigenic modulation may prevent potential target cell destruction by antibody-mediated cytotoxicity. However, if the process of modulation involves internalization of the antibody:antigen complex, it would be an advantage for the use of cytotoxic immunoconjugates.

Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells.

Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

Radioimmunotherapy of human B-cell lymphoma with 90Y-conjugated antiidiotype monoclonal antibody.

We report the first case of 90Y-conjugated monoclonal antibody (MoAb) administration for human radioimmunotherapy. Ten mCi 90Y-labeled antiidiotype (anti-Id) MoAb were administered to a patient with B-cell lymphoma whose tumor successfully imaged with 111In-labeled anti-Id MoAb. No significant toxicities were observed. More than 2 g of unlabeled anti-Id MoAb were administered while clearing the circulating IgM idiotype prior to administration of the 90Y-MoAb. Transient partial regression of disease was observed. Serial fine needle aspirations of a malignant lymph node documented in vivo anti-Id penetration into a site that did not image by radioimmunoscintigraphy. The radiosensitivity of B-cell lymphoma, the tumor specificity of anti-Id, the antitumor activity of anti-Id alone, and the safe administration of 10 mCi 90Y-labeled anti-Id MoAb in this report suggest further investigation of this radioimmunoconjugate for therapy of B-cell lymphoma is warranted.

Therapy of chronic lymphocytic leukemia and cutaneous T-cell lymphoma with T101 monoclonal antibody.

The findings accompanying the administration of 50 intravenous courses of monoclonal antibody to human T-cell (T101) in eight patients, four with chronic lymphocytic leukemia and four with cutaneous T-cell lymphoma are reported. Infusion rates of 0.7 to 1 mg/min were associated with unacceptable toxicity in the presence of circulating target cells, but slower rates were well-tolerated. Immunofluorescence techniques confirmed that circulating cells did bind the antibody in vivo and were subsequently removed from the circulation. Modulation of the antigen on target cells in the bone marrow and skin has important implications for the schedule of administration of such antibodies, and points out the possible limitation of effector cell-mediated cytotoxicity at the tissue level. Production of anti-mouse antibodies resulted in neutralization of therapy in two patients with cutaneous T-cell lymphoma, and suggests that whether such an anti-mouse response is produced may be secondary to the underlying immune status of the patient or the amount of mouse protein to which immunocompetent cells are exposed. The relative specificity and efficacy of monoclonal antibody therapy is encouraging, but the limited clinical benefit and problems of modulation and anti-mouse antibody production underscore the need for continued research into passive therapy and suggest that cytotoxic conjugates may be of more clinical value.

Chronic lymphocytic leukemia and other chronic lymphoid proliferations: surface marker phenotypes and clinical correlations.

A diagnosis of chronic lymphocytic leukemia (CLL) was made in 81 patients referred for peripheral blood lymphocyte typing (PBL). A retrospective review was undertaken to see if correlations existed between surface marker phenotype-determined subclasses and clinical features. Surface markers utilized were surface immunoglobulin (sIg), sheep erythrocyte receptor (E), 65,000-dalton human T lymphocyte antigen (T65), Ia antigen, and for sIg+ cells, heavy and light chains. All patients were Ia+. Cells of 70% of patients were sIg+ E- T65+ Ia+, and the clinical heterogeneity was that of classical CLL. Eight of the nine patients with sIg+ E- T65- Ia+ cells had a paraprotein. The sIg- E+ T65+ Ia+ phenotype represented classical T cell CLL. Three of the five patients in the sEg- E- T65+ Ia+ group had significant albuminuria, and two had nephrotic-range proteinuria. Use of additional monoclonal antibodies to B cell surface antigens should further subclassify CLL and other lymphoproliferative disorders. Interesting clinical correlations with certain phenotypic subclasses do exist, and further subclassification and long-term follow-up may yield correlations between phenotypes and prognosis.

The effects of postinduction intensification treatment with cytarabine and daunorubicin in adult acute lymphocytic leukemia: a prospective randomized clinical trial by Cancer and Leukemia Group B.

Cancer and Leukemia Group B undertook a randomized trial of intensification treatment in adults aged 15 to 79 years with acute lymphocytic leukemia (ALL) in complete remission (CR). Daunorubicin (DNR), prednisone, vincristine (VCR), intrathecal (IT) methotrexate (MTX), and asparaginase produced 177 CRs in 277 patients. One hundred fifty-one patients were randomly assigned to receive treatment as follows: 74 received intensive cytarabine and DNR, and 77 received cycles of mercaptopurine (6-MP) and MTX, followed by 6MP, MTX, VCR, and prednisone for 3 years in all. One hundred twelve patients received CNS prophylaxis. Intensification produced major myelosuppression but did not improve remission duration (median, 21 months). Of the 151 patients with CRs who entered the intensification phase, 29% remain in continuous CR (43 to 117 months); in 19 patients, CRs have lasted for longer than 7 years. No relapses occurred after 60 months. Median survival from the time of randomization was 30 months. Those under 30 years of age responded more frequently, with longer CR and survival. While 53% of those aged 15 to 19 years remain in continuous CR, 92% of patients over 59 years have relapsed. The presence of a myeloid antigen on the leukemic cells was adversely prognostic for CR achievement and for survival. Pretreatment WBC and platelet levels independently affected CR duration and survival. Early M1 marrow development presaged longer remissions. CNS relapse occurred in 47 of 256 patients with normal CSF before treatment, in 29 before CNS prophylaxis. CNS disease occurred after CNS prophylaxis in 18 patients: 13 of 61 who had received standard premaintenance and five of 51 who received intensification. No advantage in CR duration or survival resulted from intensive treatment with DNR and cytarabine following induction of CR.

IDEC-C2B8: results of a phase I multiple-dose trial in patients with relapsed non-Hodgkin's lymphoma.

PURPOSE: To evaluate the safety, pharmacokinetics, and biologic effect of multiple doses of the chimeric anti-CD20 monoclonal antibody (mAb) IDEC-C2B8 in patients with relapsed B-cell lymphoma. PATIENTS AND METHODS: Twenty patients with relapsed low-grade (n = 15) or intermediate-/high-grade (n = 5) lymphoma received weekly infusions times four of 125 mg/m2 (n = 3), 250 mg/m2 (n = 7), or 375 mg/m2 (n = 10) of IDEC-C2B8. RESULTS: Infusional side effects during the initial infusion were mainly grade I/II fever, asthenia, chills, nausea, rash, and urticaria. More serious events were rare. Peripheral-blood B cells were rapidly depleted and slowly recovered over 3 to 6 months. There was no change in mean immunoglobulin (Ig) levels. Antibody serum half-life (and maximum concentration [Cmax]) generally increased between the first and fourth infusions (33.2 hours v 76.6 hours, respectively) following the 375-mg/m2 doses. Six of 18 assessable patients had a partial remission (PR), with a median time to disease progression of 6.4 months (range, 3 to 21.7). Minor responses (MRs) were observed in five patients and progressive disease (PD) in seven. Tumor responses occurred in peripheral blood, bone marrow (BM), spleen, bulky lymph nodes, and extranodal sites, and in patients who had relapsed following high-dose myeloablative chemotherapy. Six of 14 patients (40%) with a low-grade histology responded. Four of six with bulky disease had a PR. CONCLUSION: IDEC-C2B8 chimeric anti-CD20 mAb therapy is well tolerated and has clinical activity in patients with relapsed B-cell lymphoma. The 375-mg/m2 dose has been selected for a phase II trial in patients with relapsed low-grade or follicular B-cell lymphoma.

Morphologic and cytochemical characterization of adult lymphoid leukemias which express myeloid antigen.

Cancer and Leukemia Group B demonstrated that adults with acute lymphoid leukemia (ALL) possessing blast cells with myeloid antigens (My+ALL), as identified by monoclonal antibodies against CD13 and CD33, have a worse prognosis than those lacking myeloid antigens (My-ALL). Consequently, we further studied this group of adults with ALL to determine if these immunological groups could be distinguished by morphological and cytochemical criteria. Bone marrow films were classified according to French-American-British Co-operative Group Criteria, assessed for myelodysplasia, and examined for blasts with azurophilic granules. More cases of My+ALL had L2 morphology than did My-ALL (68% vs. 49%, p = 0.04), and more cases of My+ALL were positive for acid alpha-naphthyl acetate esterase (61% vs. 31%, p = 0.03). The presence of myelodysplastic changes was not significantly different in My+ALL (13%) as compared to My-ALL (5%), but more cases of My+ALL had unusual blasts (monocytoid features and cytoplasmic buds) than did My-ALL (19% vs. 0%, p less than 0.01). In addition, more cases of My+ALL had greater than 5% of the blasts with azurophilic granules (42% vs. 13%, p = 0.01). In the My+ALL group the presence of azurophilic granules was associated with a longer median survival (13.5 months vs. 1.5 months, p less than 0.01). We conclude that My+ALL can be suspected when cases possess L2 morphology, unusual blasts, positive staining for acid alpha-naphthyl acetate esterase, and greater than 5% azurophilic granules. In addition, the poor risk group (My+ALL) can be further subdivided into better and poorer risk subgroups based on the presence of azurophilic granules.

Interleukin 2 gene therapy of colorectal carcinoma with autologous irradiated tumor cells and genetically engineered fibroblasts: a Phase I study.

The purpose of this study was to determine the safety, toxicity, and antitumor immune response following S.C. immunizations with a mixture of irradiated, autologous tumor cells and autologous fibroblasts that were genetically modified to express the gene for interleukin 2 (IL-2) in patients with colorectal carcinoma. Ten patients were treated with a fixed dose of tumor cells (10(7)) and escalating doses of fibroblasts secreting IL-2 (per 24 h): 100 units (three patients), 200 units (three patients), 400 units (three patients), and 800 units (one patient). Pre- and posttreatment peripheral blood mononuclear cells were evaluated for evidence of antitumor immune responses. Fatigue and/or flu-like symptoms were experienced by seven patients and delayed-type hypersensitivity-like skin reactions were observed at the sites of the second or subsequent vaccinations in five patients. Low frequencies of tumor cytotoxic T-cell precursors (range, 1/190,000-1/1,320,000 peripheral blood mononuclear cells) were detected prior to therapy in four of seven patients. There was a 5-fold increase following treatment in the frequency of tumor cytotoxic T-cell precursors in two of six evaluable patients. Some patients with colorectal cancer have low frequencies of tumor cytotoxic T-cell precursors that may be increased by this well-tolerated form of IL-2 gene therapy, which warrants continued clinical evaluation.

Cytokine gene therapy with interleukin-2-transduced fibroblasts: effects of IL-2 dose on anti-tumor immunity.

We evaluated the effects of different doses of interleukin-2 (IL-2)-transduced fibroblasts in the treatment of colorectal carcinoma in the CT-26 murine tumor model. Immunization with a mixture of irradiated tumor cells and IL-2-transduced fibroblasts (100 units of IL-2/24 hr) induced significantly greater protection against a live tumor challenge compared to irradiated tumor cells alone (22/35, 65% vs. 10/30, 33%, p < 0.02). Protective effects were observed with doses of IL-2-transduced fibroblasts secreting from 5 to 100 units of IL-2/24 hr. Parallel experiments in nude mice produced no protection, indicating that the effects of immunization were mediated by a T-cell-dependent mechanism. In animals with established tumors, complete tumor remissions were observed following immunization with a mixture of irradiated tumor cells and IL-2-transduced fibroblasts secreting 100 units of IL-2/24 hr, but not after immunization with irradiated tumor cells alone (7/16 vs. 0/11 complete remissions, p < 0.02). Fibroblasts secreting higher doses of IL-2 were ineffective in generating systemic immunity, but were required to prevent tumor implantation. A statistically significant difference in the prevention of tumor implantation was observed between groups inoculated with a mixture of live tumor cells and IL-2-transduced fibroblasts (1,750 units of IL-2/24 hr) compared to control fibroblasts (6/8 vs. 0/12, p < 0.001). Similar results were observed in nude mice, suggesting that the implantation rejection response is mediated in part by cells other than thymus-derived T cells. Our data support the utility of IL-2-transduced fibroblasts and indicate that the level of IL-2 expression is an important variable in activating different effector components of antitumor immune responses in IL-2 gene therapy.

Plasma calcitonin as a marker of disease activity in patients with small cell carcinoma of the lung.

To evaluate the usefulness of plasma calcitonin as an index of tumor burden and disease activity, we undertook a prospective study of serial calcitonin measurements in a group of patients with small cell carcinoma of the lung from diagnosis throughout a period of intensive therapy. Plasma calcitonin was significantly elevated in 84% of patients with extensive small cell carcinoma of the lung and was not elevated in patients with limited disease at the time of diagnosis. The elevated values fell significantly in response to chemotherapy and radiation therapy, and reflected regression of followable disease and improvement in clinical status. A significant correlation existed between plasma calcitonin and extent of disease. Relapse was generally associated with an increase in elevated plasma calcitonin levels, and calcitonin appeared to reflect tumor burden. The serial measurement of plasma calcitonin is useful in the management of the patient with small cell carcinoma of the lung.

An enzyme immunofiltration assay useful for detecting human monoclonal antibody.

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.