Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the second TD-10 workshop.

Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Elimination of malignant clonogenic breast cancer cells from human bone marrow.

Autologous bone marrow transplantation is a promising approach to the treatment of breast cancer but is at present limited to patients without bone marrow metastases. To eliminate malignant clonogenic breast cancer cells from normal human bone marrow, immunomagnetic separation has been combined with chemoseparation using 4-hydroperoxycyclophosphamide. Breast cancer cell lines have been mixed with a 10-fold excess of irradiated human bone marrow from normal donors. Mixtures have been incubated with a combination of five different monoclonal antibodies which bind to epithelial cell surface antigens of Mr 42,000, 55,000, 72,000, 200,000, and greater than 200,000. Antiglobulin coated microspheres which contained magnetite were added, and tumor cells were trapped in a magnetic field. Elimination of tumor cells from the decanted marrow was measured in a limiting dilution assay. Two treatments with antibody and microspheres permitted elimination of 2-4 logs of clonogenic breast cancer cells, depending upon the cell line studied. Similar treatment of nonirradiated normal marrow failed to affect levels of colony forming units-granulocyte-macrophage significantly. Use of immunomagnetic purging in combination with 4-hydroperoxycyclophosphamide eliminated up to 5 logs of tumor cells but reduced the recovery of colony forming units-granulocyte-macrophage. If prompt engraftment is observed following reinfusion of similarly treated marrow in phase I trials, these techniques should permit extension of autologous bone marrow transplantation to a larger population of breast cancer patients.

Selection of monoclonal antibodies for use in an immunometric assay for carcinoembryonic antigen.

Six high-affinity mouse monoclonal antibodies against carcinoembryonic antigen produced in our laboratory were evaluated for use in a solid-phase immunoradiometric assay. The antibodies all recognized single peptide epitopes, one being highly conformation dependent. Cross-inhibition studies demonstrated that three antibodies bound to different epitopes, while the remaining three bound to the same or to very closely related epitodes. All antibodies strongly stained colorectal cancer tissue. The three antibodies binding to the same epitode also stained normal granulocytes, indicating a reactivity with the non-specific cross-reacting antigen. On the basis of the results of the characterization, two antibodies were selected for use in a sandwich immunometric assay. One of these was coupled to 2.8 microns magnetizable polymer particles, while the other one was radiolabelled. The assay required 2 h incubation to reach equilibrium, having a working range up to 135 micrograms/l and a sensitivity (Zero + 2 SD) of 0.1 micrograms/l. A comparison of the new assay with two commercial CEA kits that also use monoclonal antibodies was carried out on a small panel of serum samples from colorectal cancer patients and revealed satisfactory correlations but also discrepancies that must be attributed to differences in epitope specificity.

A rapid and sensitive immunoassay for tumor necrosis factor using magnetic monodisperse polymer particles.

A sensitive and rapid immunoassay for the detection of tumor necrosis factor (TNF) has been developed. Magnetic monodisperse polymer particles (M-280 Dynabeads) used as solid phase material, were coated with a neutralizing mouse monoclonal antibody to TNF. The coated Dynabeads were shown to have a more rapid binding capacity for recombinant (r) TNF as compared to standard immunowells coated with antibodies to TNF. The amount of TNF bound to the Dynabeads was quantified using either a polyclonal antibody to TNF or a mouse monoclonal antibody to TNF. The antibodies used for detection were either labelled with 125I or peroxidase. The linear assay range for the TNF standard curve was form 62 to 4000 pg/ml, and the assay time was less than 60 min. The sensitivity could be increased 5-8-fold by increasing the sample volume from 0.1 to 2 ml.

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

A sequential binding assay with a working range extending beyond seven orders of magnitude.

A new immunometric sequential binding assay has been developed in which the sample is first reacted with a solid phase binding partner in low concentration, and subsequently with a second binding partner at a higher concentration. The amounts of analyte bound to the two solid phase binding partners are separately measured, thus establishing a double standard curve. There is a shift between the two standard curves along the concentration axis. Thus an unambiguous determination of analyte concentration is obtained, even in the descending region of the curves where the 'hook' effect causes decreasing signal with increasing analyte concentration. A two-particle immunofluorometric assay for AFP based on this principle measured by flow cytometry, resulted in an assay with rapid binding (approximately 2 h), a detection limit of 0.1 kIU/l and a working range (0.3 to > 3 x 10(6) kIU/l) in excess of 7 log10 orders. Assay results compared well with those of an immunoradiometric assay.

In vivo evaluation of radiolabelled antibodies with antigen-coated polymer particles in diffusion chambers.

A method for the evaluation of in vivo immunolocalization of labelled monoclonal antibodies (MoAb) is presented. The technique is an alternative to the nude mouse xenograft system. The antigen reservoir is an intraperitoneal diffusion chamber (DC) filled with a suspension of antigen-coated polymer particles. Intravenously injected 125I-labelled MoAb are allowed to specifically bind to this artificial abdominal 'tumour', which can be removed and measured for radioactivity after animal killing. The model can be used as a preclinical in vivo method for the evaluation of labelled MoAbs prepared for immunodiagnostics or therapy. The DC system permits the amounts of both antibody and antigen to be controlled and antibody access to the antigen within the DC is presumably the same in every animal. The model permits a systematic comparison of different antibodies and antibody fragments, labels and labelling procedures, as well as routes of administration in immunocompetent animals.

Dual analyte assay based on particle types of different size measured by flow cytometry.

Simultaneous flow cytometric assays have been developed for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), with internal determination of sample related non-specific binding (NSB). The assays use particles of 7.5, 6.5 and 5.5 microns diameter coated with, respectively, monoclonal antibodies specific for AFP, hCG or an epitope normally not present in serum. The different particle types were identified simultaneously by light-scatter measurements as their specific immunofluorometric responses were determined. The NSB in the simultaneous assay of AFP and hCG was increased by approximately 30% compared to corresponding single analyte assays. The working range of the dual analyte assays was 0.6-2000 kIU/l for AFP and 6-10,000 IU/l for hCG. No significant interference from the presence of the other analyte was observed in the measurement of either AFP or hCG. The 95% confidence interval for the ratio of dual over single analyte assay results was [0.81, 1.11] for AFP and [0.88, 1.16] for hCG.

Excess antibody immunoassays for rat glandular kallikreins. Measurement of kallikrein from different organs in the presence of cross-reacting antigens.

An immunoradiometric assay has previously been developed for measurement of rat glandular kallikrein. In the present paper, further studies on the specificity and sensitivity of the method are described. Problems of interference of immunologically cross-reacting antigens were overcome by proper preabsorption of the antibody. A method was thus established in which enzymatic activity of the immunoreactive kallikrein could be measured even in the presence of enzymes sharing immunological determinants and substrate specificity with kallikrein. Two variants of the immunoradiometric assay have been evaluated. A simplified version with simultaneous addition of all reagents gave results equal to those obtained in the original assay. A further modification with delayed addition of the solid-phase antibody, gave considerable improvement in assay sensitivity.

Excess antibody immunoassay for rat glandular kallikrein. Measurement of kallikrein complexed with inhibitors and in plasma.

We have recently developed an immunoradiometric assay (IRMA) for specific measurement of immunoreactive kallikrein which allows a simultaneous determination of the enzymatic activity of kallikrein. This paper describes the application of this method for measurements of glandular kallikrein complexed with inhibitors. Interference by low molecular weight inhibitors such as benzamidine and Trasylol was easily overcome by increasing the amount of immobilized anti-kallikrein antibody added in the assay, and by prolonging the incubation time of the antigen-binding step. The recovery of kallikrein in complex with plasma inhibitors was complete only when the anti-kallikrein antibody was immunoadsorbed onto a solid-phase sheep anti-rabbit immunoglobulin. The dose-response curve of glandular kallikrein in plasma paralleled that of purified kallikrein in both the immunoradiometric and the immunoenzymometric assays. The concentration of immunoreactive glandular kallikrein in normal rat plasma was 12.8 +/- 4.3 nU/ml. The enzymatic activity of this immunoreactive kallikrein was 86% inhibited.

Excess antibody immunoassay for the measurement of tonin in rat tissues and plasma.

Tonin, a proteolytic enzyme isolated from the rat submandibular gland, can generate angiotensin II directly from angiotensinogen. To date a method for the measurement of tonin in plasma has not been available and the present paper describes a sensitive and specific excess antibody immunoassay for determination of tonin in tissue homogenates and plasma. Interference from immunologically cross-reacting proteins was evaluated and the assay was found to be specific for tonin. Tonin measured in various tissue homogenates was directly proportional to the amount of sample added, giving a linear dose-response curve. The slope of this curve was determined by the recovery of tonin, which was better than 55% for urine and all tissues tested. The highest concentration of tonin was seen in the submandibular and sublingual gland (69 and 0.7 microgram/mg protein, respectively). The parotid gland, the exorbital lacrimal gland, liver, kidney, pancreas, and lung contained only negligible amounts (less than 4 ng/mg protein). Tonin in plasma was bound to one major inhibitor with a molecular weight of about 650,000-750,000. A partial splitting of the tonin-inhibitor complex was obtained by preincubating plasma with guanidine, allowing tonin to be measured with a recovery of 38 +/- 13% (n = 16) and with a linear dose-response curve. The concentration of immunoreactive tonin in normal arterial plasma from adult male rats was 0.90 +/- 0.53 ng/ml (n = 16). The concentration decreased after removal of the submandibular glands and increased after sympathetic stimulation.

Excess antibody immunoassay for rat glandular kallikrein. Monosized polymer particles as the preferred solid phase material.

The development of an excess antibody assay for rat glandular kallikrein is described. This assay permits immunological determination of kallikrein as well as a simultaneous specific measurement of kallikrein enzymatic activity. The assay is based on coupling of immunopurified anti-kallikrein immunoglobulin to a solid phase. In a first incubation step, kallikrein was bound to the immobilized antibody. Determination of kallikrein was subsequently done in a second incubation step; immunologically by addition of iodinated anti-kallikrein antibody, or enzymatically by a kallikrein substrate. Enzymatic quantification could also be followed by immunological measurements on the same sample. Comparison of Sepharose, cellulose, and acrylate based polymer particles proved the latter to be the best matrix in this assay. The main advantage of the polymer particles was the low non-specific binding of labelled antibody.

Immunomagnetic DNA aptamer assay.

DNA aptamers, oligonucleotides with antibody-like binding properties, are easy to manufacture and modify. As a class of molecules, they represent the biggest revolution to immunodiagnostics since the discovery of monoclonal antibodies. To demonstrate that DNA aptamers are versatile reagents for use as in vitro diagnostic tools, we developed a hybrid immunobead assay based on a 5'-biotinylated DNA thrombin aptamer (5'-GGTTGGTGTGGTTGG-3') and an anti-thrombin antibody (EST-7). Our results show that the thrombin DNA aptamer is capable of binding to its target molecule under stringent in vitro assay conditions and at physiological concentrations. These findings also support the view that DNA aptamers have potential value as complementary reagents in diagnostic assays.

Immunohistochemical localization of kallikrein in human pancreas and salivary glands.

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.

Localization of kininogenase in the rat kidney.

1. The rat kidney kininogenase (KGA) activity was located mainly in the kidney cortex.2. Differential centrifugation of kidney cortex homogenate revealed that the microsomal fraction contained more KGA activity per unit of protein than the other subcellular fractions.3. Subfractionation of the microsomal fraction showed that the KGA activity was recovered in a subfraction also containing high specific activity of both alkaline phosphate and glucose-6-phosphatase. It is suggested that the KGA activity is localized in the plasma membrane and/or endoplasmic reticulum membranes of kidney cortical cells.4. Hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester at pH 8.5 paralleled the KGA activity.

The relationship between kidney and urinary kininogenase.

1. Rat kidneys which were perfused with saline contained both kininogenase (KGA) and kininase activity. These activities were separated by gel filtration on a Sephadex G-100 column. The kininase activity was excluded from the column whereas the KGA activity was retained. Kidney KGA activity was primarily found in the sedimentable fraction of the homogenate.2. The kidney KGA activity was compared with the urinary KGA activity, and the following properties were found to be the same: molecular dimension, pH optimum, effect of inhibitors, and ability to liberate kinins from kininogens.3. A urinary sample collected over 24 h contained about 8 times the KGA activity found in the corresponding kidneys at the end of the collection period. The urine: kidney ratio for alkaline phosphatase was about 0.01.4. The ability of kidney and urinary samples to hydrolyse N-alpha-benzoyl-L-arginine ethyl ester (BAEE) at pH 8.5 paralleled the KGA activity.

Subcellular localization of renin and kininogenase in the rat kidney.

1. The distribution of enzymatic activities was determined in subcellular fractions of rat kidney cortex homogenates after various homogenization procedures. The specific activities of kininogenase (KGA), BAEE esterase (pH 8.5), alkaline phosphatase and glucose-6-phosphatase were, on average, 3.4 times higher in the microsomal fraction than in the whole homogenate. The total amount of these activities in the microsomal fraction after gentle, ordinary and forced homogenization were about 15, 40 and 65% of total recovered activities, respectively. These results confirmed the localization of KGA in the microsomal fraction.2. Renin activity was primarily recovered in the heavy mitochondrial fraction. When the force of the homogenization was increased some renin activity was shifted to the soluble fraction.3. When a mixture of renin and purified urinary KGA was given intravenously to an anaesthetized rat, a hypotensive response due to the KGA was followed by a hypertensive renin response. Over a certain range of concentrations KGA and renin could be measured simultaneously. In fractions of kidney homogenates, however, KGA activity was too low to be measured by this method.

Synthesis of kallikreins by rat kidney slices.

1 Four radioactive akllikreins were isolated from rat kidney slices incubated with (3H)-L-leucine. 2 The kallikreins were purified by procedures previously used for the isolation of rat urinary kallikreins (Nustad & Pierce, 1974), and by affinity chromatography on a column of insolubilized anti-rat urinary kallikrein. 3 The kidney kallikreins resembled the urinary kallikreins in their relative amounts, isoelectric points and electrophoretic mobilities on polyacrylamide disc gels. 4 The data indicate that the kidney synthesizes four kallikreins which are released into urine. The kallikreins are not changed by passage through the lower urinary tract.

Rat submandibular gland kallikreins: purification and cellular localization.

1 Four submandibular gland kallikreins (E.C.3.4.21.8) were isolated by chromatography on DEAE-Sephadex A-50 and hydroxyapatite, followed by gel filtration and electrofocusing. The pI values were 3.87, 3.96, 4.07 and 4.16, and a common molecular weight of 34,000 was found. 2 The kallikreins were localized by direct immunofluorescence with an antibody to rat urinary kallikrein, to the granular tubules, striated duct cells and some main duct cells in the submandibular gland, and to striated duct cells in the sublingual gland. Kallikrein was not found in acini and stroma. 3 Several non-kallikrein esterases present in the submandibular gland reacted with the antibody to rat urinary kallikrein. The antibody was made monospecific for kallikrein by absorption with the crossreacting esterases. 4 We suggest that kallikrein is produced in striated duct cells. Granular tubules, which are differentiated from striated duct cells, have preserved the ability to produce kallikrein. These cells also store large quantities of kallikrein.