Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Hybrid antibodies in cancer diagnosis and therapy

Monoclonal Antibodies (Mabs) represent a promising tool for cancer diagnosis and therapy. Administration of MAbs alone or conjugated to cytotoxic agents has been attempted but has significant limitations. Another potentially effective approach is the use of bispecific or bifunctional antibodies where the capacity to recognize the tumor cell and the toxic agent or lymphocyte activation molecule are united in one MAb. The hybrid molecule can be produced by chemical linkage between the two parental antibodies, or alternatively by a biological approach that consists in the fusion of the two selected hybridomas. In the resulting quadroma cell the hybridoma immunoglobulin chains recombine randomly to form the bifunctional MAb. In different in vitro and in vivo models, bifunctional MAbs against tumor and CD3 at nanomolar concentration has been shown to promote tumor cell killing by cytotoxic T cells. Specific localization of chemotherapeutic drugs in xenografted tumors has been demonstrated in mice pretreated with hybrid MAbs. The advantages of the hybrid MAb approach are that it should reduce the MAb biodistribution problem and that it involves no chemical manipulation between the functional agent and the MAb molecules.     

Therapeutic Use of Antimelanoma Monoclonal Antibodies

Monoclonal antibodies (MAb) to tumor-associated antigens are attracting much attention for tumor therapy. Melanomas belong to the tumors most studied in this respect, and several melanoma-associated antigens have been studied in great detail. These include the melanoma-associated glycoprotein p97, the melanoma-associated proteoglycan, and glycolipid antigens. Although none of the antigens is absolutely specific for tumor, the degree of relative specificity appears to be sufficient to use several of the melanoma antigens as therapeutic “targets”. Antimelanoma MAb can be applied therapeutically in several ways. The most straightforward approach is use of MAb without further modification. MAb which kill melanoma cells in the presence of human serum as the source of complement or mediate antibody-dependent cellular cytotoxicity with human natural killer (NK) cells or macrophages as effectors are logical choices for this. Some cases of partial or even complete regression of metastatic melanoma have been observed in patients treated with such MAb. Combinations of such MAb with interleukin 2 (IL-2) or other immunological response modifiers are of great interest. Alternatively, one may use antimelanoma MAb (or fragments prepared from MAb) as carriers of antitumor agents, including radioactive isotopes, toxins, or chemotherapeutic drugs. Although it is premature to make any conclusions about the efficacy of such conjugates, we are optimistic that it will be feasible by using the right combination of MAb and antitumor agent to achieve therapeutic benefit. Another approach is to develop therapeutic “vaccines” for active immunization, once an antigen characterized by using a MAb has proven to have a relatively high level of tumor selectively. Anti-idiotypic antibodies and live recombinant viruses inducing tumor antigen expression in infected cells provide alternative strategies to this approach.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

Complementation of expression of carcinoembryonic antigen and tumor associated glycoprotein-72 (TAG-72) in human colon adenocarcinomas.

Enzyme-labeled monoclonal antibodies (MAbs) were used in an immunohistochemical, dual-staining study of 10 colon adenocarcinomas. MAbs B72.3 and COL-4, reactive with the high molecular weight tumor-associated glycoprotein-72 (TAG-72) antigen and carcinoembryonic antigen (CEA), respectively, were labeled with horseradish peroxidase or alkaline phosphatase. Dual staining using the two MAbs on a single tissue section (formalin-fixed, paraffin-embedded) showed that greater numbers of carcinoma cells could be detected by using the combination of the two MAbs than could be detected by use of either MAb alone. In many tumors, some carcinoma cells reacted with MAb B72.3, some reacted with MAb COL-4, and some cells reacted with both MAbs. Only 1 of 10 carcinomas showed greater than 75% reactive cells when stained with each MAb individually. In 9 of 10 cases, however, greater than 75% of cells reacted when the combination of MAbs was used. Cell surface and cytoplasmic patterns of reactivity were observed with both MAbs while some pools of extracellular mucin were composed of both TAG-72 and CEA. This study supports the rationale for the use of a combination of anti-TAG-72 and anti-CEA MAbs for in vitro immunologic detection and potential in vivo immunodiagnostic and immunotherapeutic applications for these MAbs in colon adenocarcinoma patients.     

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells

One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.     

Role of Myxococcus xanthus cell surface antigen 1604 in development.

The inhibition of development of Myxococcus xanthus by monoclonal antibody (MAb) 1604 has been further investigated with two MAbs produced against the affinity-purified cell surface antigen (CSA) 1604. Both of these second-generation MAbs, 4070 and 4054, reacted with the same band at 150 kilodaltons (kDa) on Western immunoblots of lysed and reduced cells. This band was also identified by MAb 1604. However, the affinity-purified CSA was a complex of the two proteins (51 and 23 kDa) and lipopolysaccharide (LPS) that the 150-kDa material comprised. One of the three MAbs, 4070, reacted with LPS on Western immunoblots. Another MAb, 4054, reacted with the 23-kDa protein, and MAb 1604 reacted with the 51-kDa protein found in the CSA complex. Competitive binding studies verified that MAbs 4054 and 1604 identified different epitopes, and MAb 4070 probably reacted with a third epitope of the CSA 1604 complex. MAb 4054 blocked development, although not as thoroughly as MAb 1604 did, when added at 60 micrograms/ml to cells undergoing submerged development. In contrast, MAb 4070 prevented sporulation in submerged development and induced the cells to reaggregate in rings around the initial aggregation centers. A mutant strain of M. xanthus that is deficient in the epitope for MAb 1604 retained the epitope for MAb 4054. The affinity-purified antigen 1604, when added to cells at greater than or equal to 550 ng/ml, altered the appearance of the fruiting bodies and at higher concentrations prevented fruiting body formation. The CSA 1604 moiety responsible for this inhibitory effect is apparently a peptide constituent and not the LPS.     

Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain

Despite the development of high‐titer bioprocesses capable of producing >10 g L^?1 of recombinant monoclonal antibody (MAb), some so called “difficult‐to‐express” (DTE) MAbs only reach much lower process titers. For widely utilized “platform” processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10‐day fed‐batch transient production process varied in titer 5.6‐fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)‐specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC‐HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:188–197, 2014     

Production and characterisation of monoclonal antibodies to ovine interleukin-5

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.     

Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.     

In vitro and in vivo regulation of tumor antigen expression by human recombinant interferons

In vitro treatment with either type I or type II Interferon (IFN) can selectively enhance the expession of several tumor antigens, such as the carcinoembryonic antigen (CEA) and the tumor-associated glycoprotein-72 (TAG-72) in different human carcinoma cell lines and result in enhanced level of monoclonal antibody (MAb) binding to the cell surface. In vivo animal studies demonstrated that treatment of athymic mice with a type I interferon [i.e. interferon-α(A)]significantly increased the expression of a 90 kDa tumor antigen which improved the targeting of a MAb to the carcinoma xenograft. More recent studies reported that in vitro IFN treatment of human adenocarcinoma cells isolated from human malignant serous effusions selectively increased the expression of TAG-72 and CEA. One can envision that the ability of these cytokines to upregulate the level of expression of human tumor antigens presents an important experimental model in which to study the regulation of markers often correlated with epithelial cell differentiation. In addition, the increase of selective MAb-defined antigens may also be exploited in an adjuvant setting to localize higher amounts of MAbs to the tumor cell surface and, thereby, improve the effectiveness of a MAb for tumor diagnosis and, possibly, therapy.     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Preclinical and phase I studies of monoclonal antibodies in melanoma: application to boron neutron capture therapy of melanoma

Monoclonal antibodies (MAbs) provide an attractive method of selectively localizing sufficient boron atoms around tumour cells to capture neutrons. Assuming that 10(8)-10(10) 10B atoms are needed for one capture event and that 10(3)-10(4) atoms can be coupled to each antibody molecule, then 10(5)-10(6) antibody molecules gathered on an individual cell will destroy that cell. Binding to normal tissues, on the other hand, would need to be at least 20-fold less than that to tumour tissues to avoid toxic effects of neutrons on surrounding tissues. Preclinical studies in animals show that several MAbs may bind to melanoma cells in sufficient quantities in vitro to localize the required amount of Boron per cell. Whether this will occur in vivo, however, may depend not only on antigen density but a variety of other properties of the tumour cells and MAbs. These include the Ig class and affinity of the antibody and whether the antibody is internalized into the tumour cell. The ratio of uptake between tumour and normal tissue is governed by such factors as the percentage of tumour cells within a tumour expressing the antigen and whether the MAb react with normal tissues. Use of Fab or F(ab)2 preparations of the MAb may increase the uptake ratio by preventing uptake of MAb by cells with Fc receptors. In contrast to preclinical animal studies, tumour/normal tissue uptake ratios in phase I studies in humans have been disappointingly low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies

We evaluated the capacity of freshly isolated blood monocytes to mediate antibody-dependent cellular-mediated cytotoxicity (ADCC) in cooperation with murine anti-tumor monoclonal antibodies (MAbs). Blood monocytes isolated from most donors by adherence selection to fibronectin-coated plastic surfaces and subsequently depleted of natural killer/killer (NK/K) cells exhibited significant ADCC activity against tumor cell lines in combination with an IgG3 antitumor MAb (BR55-2). However, significant variation in ADCC competence was observed among donors. Culture parameters influencing monocyte ADCC activity were evaluated and optimized. The influence of MAb isotype on ADCC capacity of anti-tumor MAbs was also evaluated using anti-tumor class-switch variant hybridoma proteins and a panel of anti-tumor MAbs. MAbs of the IgG2a and IgG3 subclasses exhibited high ADCC potential, whereas MAbs of the IgG2b subclass exhibited no ADCC activity. One of two IgG1 MAbs tested exhibited high ADCC activity with monocyte effectors. The role of monocytes or macrophages in tumor remission of cancer patients undergoing MAb immunotherapy is not known. However, correlative studies of monocyte ADCC capacity and responsiveness of cancer patients to MAb immunotherapy may help to establish the role of these effectors in MAb-mediated tumor remissions.     

Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440.

Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida (pWW0) released in unsterile lake mesocosms.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

Tumor localization and radioimaging with mixtures of radioiodinated monoclonal antibodies directed to different colon cancer associated antigens

After demonstrating enhanced tumor cell binding with a mixture of monoclonal antibodies (MAbs) in vitro, biodistribution and immunoscintigraphy studies with 3 radioiodinated anti-colon cancer MAbs and a non-specific control MAb (MOPC) were conducted in a human colon cancer (GW-39)-hamster model system. Each of the specific MAbs, but not MOPC, demonstrated extensive tumor binding and in scintigrams affected visualization of all large tumors (>0.85 g) over background. Using single MAbs, few small tumors (0.19–0.50 g) were defined above background (0–29%). However, with combinations of these specific MAbs small tumors were more frequently defined in scintigrams (43–67%). Radioimages using higher doses of MAbs and small, younger tumors more clearly demonstrated the superiority of a MAb mixture. These results confirmed that combinations of MAbs to different antigens can detect smaller tumors with better tumor localization when compared to component MAbs used singly. This study supports the concept that tumor targeting and detection may be enhanced with appropriate mixtures of MAbs.     

Preclinical studies of monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Eighty percent of bladder cancers present as superficial disease. Many are multifocal, and apparently successful treatment is frequently followed by recurrence. The use of monoclonal antibodies (MAbs) to target radiotherapy to these tumors offers great potential, especially since they can be administered directly into the bladder (intravesically) bypassing many of the side effects encountered to date with systemic MAb-based therapy. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. Tumor size can be monitored by X-ray analysis after administration of urograffin. Comparative studies of two murine MAbs, BLCA-8, IgG3, and C1-137, IgG1, against malignant human bladder cancer cells have been performed. Radio-immunoconjugates produced with¹²⁵Iodine (¹²⁵I) have been used for biodistribution studies following administration directly into rat bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intravesical administration of radio-immunoconjugates to tumor-bearing nude rats indicate better tumor uptake of C1-137 than BLCA-8. Further studies to test two-step intravesical administration of biotinylated MAb followed by radioiodinated streptavidin are in progress. Our studies indicate that the C1-137 MAb may have considerable potential for intravesical radioimmunotherapy of patients with superficial bladder tumors.     

Tritium Labeling of Hexachlorocyclohexane Isomers by Gas Exposure Method. II

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II) epitope mapping by monoclonal antibodies

PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94. J. Cell. Biochem. 65:186–197. © 1997 Wiley-Liss, Inc.