Linker-free antibody conjugation for sensitive hydrogel microparticle-based multiplex immunoassay.

Multiplex immunoassay, or the simultaneous detection of multiple proteins in a single sample, is expected to enable a new level of protein analysis across diverse disciplines, such as medical diagnostics and biomarker discovery. A bead-based assay using graphically encoded hydrogel microparticles synthesized using stop flow lithography has been a promising platform because of its high multiplex capacity and its superior sensitivity and dynamic range compared to the enzyme-linked immunosorbent assay (ELISA). The functionalization of these particles has been dependent on the use of a heterobifunctional linker to conjugate the capture antibodies on the hydrogel. However, the linker chemistry, which is based on linking the primary amine groups of antibodies with acrylate functional groups on the hydrogel monomer, is vulnerable to hydrolysis in aqueous conditions and can potentially damage the antigen binding region of the antibody. In this work, we introduce a new antibody conjugation method that avoids the use of the linker and further enhances the sensitivity of hydrogel microparticle-based immunoassays. Disulfide bonds in antibodies are reduced to liberate free thiols, which can directly bond with the double bonds remaining in the hydrogel after particle synthesis. We characterize the optimal reduction of antibodies for producing the highest detection signal and demonstrate an average two-fold improvement in sensitivity compared to the linker-dependent antibody conjugation method. Lastly, we validate the accuracy and specificity of the multiplex assays with particles conjugated with antibodies using the linker-free method.

Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and ß-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, ß-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.

Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry.

Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked ß subunit of hCG (hCGßn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGßn44/45 and hCGß47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGß peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGß, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGß-mAbs cannot discern between hCGßn44/45, hCGßn47/48, or intact hCGß as all 15 mAbs recognizing hCGß also recognized both nicked variants irrespective of which of the three major hCGß antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGßCTP. LC-MS/MS confirmed that their epitopes were not located on hCGß peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGßn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.

Vaccination with the repeat ß-hCG C-terminal peptide carried by heat shock protein-65 (HSP65) for inducing antitumor effects.

The ß-subunit of human chorionic gonadotropin (ß-hCG) is ectopically expressed in various types of cancer and has been utilized as an antigenic target in anti-cancer vaccines. In view of the low immunogenicity of this self-peptide, we designed a method based on the isocaudamer technique to generate 14 tandem repeats of the 10-residue sequence X of ß-hCG (109-118). These tandemly repeated copies were then combined with ß-hCG C-terminal 37 peptides (CTP37) and finally fused to mycobacterial heat-shock protein 65 (HSP65) to construct a fusion protein HSP65-X14-ßhCGCTP37 as an immunogen. In this study, BALB/c female mice were immunized via subcutaneous injection of the designed protein. Humoral immune and cellular immune responses were effectively elicited. A high titer of anti-ß-hCG antibody was detected in immunized mice sera by enzyme-linked immunosorbent assay and verified by Western blot analysis. The fusion protein, HSP65-X14-ß-hCGCTP37, effectively inhibited the growth of Ehrlich ascites carcinoma in mice. These results suggest that HSP65-X14-ßhCGCTP37 may be an effective tumor vaccine, and the use of multiple tandem repeats of a certain epitope is an effective method to overcome the low immunogenicity of self-peptide antigens.

TCR mimic monoclonal antibody targets a specific peptide/HLA class I complex and significantly impedes tumor growth in vivo using breast cancer models.

Our laboratory has developed a process for generating mAbs with selectivity to unique peptides in the context of MHC molecules. Recently, we reported that RL4B, an mAb that we have called a TCR mimic (TCRm) because it recognizes peptide in the context of MHC, has cytotoxic activity in vitro and prevented growth of tumor cells in a prophylactic setting. When presented in the context of HLA-A2, RL4B TCRm recognizes the peptide GVLPALPQV derived from human chorionic gonadotropin (hCG)-beta. In this study, we show that RL4B TCRm has strong binding affinity for the GVLPALPQV peptide/HLA-A2 epitope and fine binding specificity for cells that express endogenous hCGbeta Ag and HLA-A2. In addition, suppression of tumor growth with RL4B TCRm was observed in orthotopic models for breast cancer. Using two aggressive human tumor cell lines, MDA-MB-231 and MCF-7, we provide evidence that RL4B TCRm significantly retards tumor growth, supporting a possible role for TCRm agents in therapeutic settings. Moreover, tumors in mice responded to RL4B TCRm therapy in a dose-dependent manner, eliminating tumors at the highest dose. RL4B TCRm strongly detects the hCGbeta peptide/HLA-A2 epitope in human primary breast tumor tissue, but does not react or reacts weakly with normal breast tissue from the same patient. These results further illustrate the selective nature of TCRm Abs and the clinical relevance of the GVLPALPQV peptide/HLA-A2 epitope expression in tumor cells, because they provide the first evidence that Abs that mimic the TCR can be used to markedly reduce and suppress tumor growth.

Recognition based hormonal 95kDa monoclonal antibody on three human cancer cell lines for developing targeted radio-immuno-imaging and therapy.

The present study aimed to explore the possibility of developing a immuno-imaging/therapeutic agent for hCG-expressing tumors by using antibodies raised against them. Three human cancer cell lines were selected:lung adenocarcinoma (A549), glioblastoma (U87MG) and breast cancer (MCF7). Anti-ß-hCG monoclonal antibody, obtained from ascitic fluid, was purified by affinity chromatography followed by characterization and titration. Ectopic expression of hCG on these cell lines was tested by flow cytometry and in-vitro cytotoxicity with antibodies was tested by MTT assay on the cell line with the highest percentage binding. For positive and negative controls, immortalized trophoblast cells (SW71) and peripheral blood monocytes were used. Antibody was then radiolabeled with lutetium-177 ((177)Lu) and in vivo biodistribution studies were conducted in murine tumor model. Antibodies could be purified to homogeneity with a concentration 28mg/mL. Percentage receptor expression on A549, U87MG and MCF7 cells was 95%, 66% and 55% respectively. About 90% of A549 cells could be killed with antibody at 72h post-treatment. No cytotoxicity was observed on SW71 despite a high binding percentage (96%). Antibodies were radiolabeled with high efficiency (∼98%). In-vivo studies using radiolabeled antibodies showed hepato-biliary excretion route and significant uptake in A549 tumor. In conclusion, among the 3 cancer cell lines, lung adenocarcinoma significantly expresses ß-hCG and shows dose dependent cytotoxicity with anti-ß-hCG antibody. Radiolabeling of this antibody can aid in imaging and also has the potential of enhancing its therapeutic potential. This study provides a platform for further studies for targeted radio-immuno imaging and subsequent therapy of hCG-expressing cancers.

Development of a novel 7 immune-related genes prognostic model for oral cancer: A study based on TCGA database.

Oral squamous cell carcinoma (OSCC) is an aggressive tumor whose prognosis has little improvement in the last three decades. Various immune-related genes have been suggested as significant roles in the development and progression of malignant cancers. In this study, we acquired and integrated differentially expressed genes of OSCC patients, including immune-related genes and transcription factors (TFs), from The Cancer Genome Atlas (TCGA) database. TF-mediated network was established to exploring the regulatory mechanisms of prognostic immune-related genes. A 7 immune-related genes prognostic model for OSCC was obtained, including CGB8, CTLA4, TNFRSF19, CCL26, NRG1, TPM2 and PLAU, which was further proved to be an independent prognostic indicator after adjusting for other clinical factors. The immune-related genes prognostic index was significantly negatively correlated to the infiltration abundances of B cells (P < 0.05) and CD8+ T cells (P < 0.05). The novel proposed immune-based prognostic model not only provided a promising biomarker and a way to monitor the long-term treatment of OSCC, but also gave a new insight into a potential immunotherapy strategy.

Evaluation of the immunogenicity of a single chain chimeric peptide composed of hCGß and oLHα for inhibition of the growth of hCGß-expressing cancer cells.

Human chorionic gonadotropin (hCG) is a membrane-associated protein highly expressed in several types of human cancer cells. The expression in the cancer cells indicates that hCG may be a potential target molecule for cancer immunotherapy. The objective of this study was to develop a novel immunogenic molecule, which can efficiently induce the neutralizing antibody against hCG and which is also suitable for mass production. The immunogenicity of the recombinant single chain chimeric protein of hCGß-oLHα expressed by yeast was examined. Additionally, the inhibitory effects of the anti-hCGß-oLHα antibody on the growth of hCG-positive cancer cells were determined. It was found that hCGß-oLHα yielded high titers of anti-hCG rabbit antibody that could effectively neutralize the bioactivity of hCG. The rabbit anti-hCGß-oLHα IgG inhibited the proliferation of hCG-expressing human colorectal cancer cells (LS-174, HCT-116, HCT-15 and KM-12) in a dose-dependent manner. Furthermore, an intact anti-tumor vaccine was prepared by conjugating hCGß-oLHα with tetanus toxoid (TT) and this was used to immunize Balb/c mice bearing hCG-expressing SP2/0 tumor cells. The progression of tumors in these immunized mice was remarkably inhibited. These results suggest that hCGß-oLHα is a new promising immunogenic molecule for the development of an anti-hCG-based cancer vaccine.

Deciphering a conformation-specific epitope of hCG-beta through Immunokinetics.

Proteins and peptides are comprised of both sequence-specific and conformation-specific epitopes. Sequence-specific epitopes are delineated by a peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays, etc. Available methods for deciphering conformation-specific epitopes are cumbersome (X-ray crystallography, etc.), time-consuming, and require expensive equipment. Therefore, it is indispensable to develop a simple method for identification and mapping of conformation-specific epitopes. In the present investigation, the radiolabeled human chorionic gonadotropin-beta ((125)IhCGbeta) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G(1)G(10).1. The NC-G(1)G(10).1-(125)IhCGbeta complex (NC(com)) was prepared and the dissociation of radiolabeled hCGbeta was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k(-1)), association constants (k(+1)), and affinity constants (k(a)) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGbeta involved in interaction with the complementary paratope on MAb G(1)G(10).1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of a conformation-specific epitope of hCGbeta consists of Arg (94, 95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Therefore, the results of the present investigation suggested that the dissociation kinetics coupled with SS-SPRIA unequivocally assists in deciphering amino acid residues constituting a conformation-specific epitope of hCGbeta.

Kinetic analysis of a human chorionic gonadotropin-beta epitope-paratope interaction.

Kinetics of protein-protein or ligand-ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand-ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-beta ((125)IhCGbeta) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G(1)G(10).1. The NC-G(1)G(10).1-(125)IhCGbeta complex (NC(com)) was prepared and the dissociation of radiolabeled hCGbeta was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k(- 1)), association constants (k(+1)) and affinity constants (k(a)) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGbeta involved in interaction with the complementary paratope on MAb G(1)G(10).1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGbeta epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand-ligate interactions and epitope analysis.

Fusion of hC3d3 to hCGbeta enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge.

Contraceptive vaccines based on hCGbeta have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGbeta and pCI-gs-signal-6His-hCGbeta-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10 nM, 100 nM hCGbeta, hCGbeta-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100 nM hCGbeta-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGbeta. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100 nM hCGbeta-C3d3, but no anti-hCG antibody was produced in the challenge with hCGbeta. The hCGbeta-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGbeta (P<0.05). The hCGbeta-hC3d3 promoted human PBMC producing more IL-2 than hCGbeta. These findings indicate that the fusion of hC3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future.

Immune response to lytic peptides conjugated to a betaCG fragment in treated BALB/C mice.

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.

Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells.

Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGbeta). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGbeta-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

Inhibition of tumor growth in vitro and in vivo by a monoclonal antibody against human chorionic gonadotropin beta.

Human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) has been detected in a wide variety of tumors and implicated in tumor maintenance and progression. To better facilitate the investigation of the expression and biological roles of hCGbeta, we generated a set of monoclonal antibodies (mAbs) against hCGbeta by the approach of DNA immunization. All the generated mAbs worked well in detecting native hCGbeta antigen, while some of them were surprisingly found to exhibit potential cytotoxicity to tumor cells in our preliminary experiments. Here, one of those cytotoxic anti-hCGbeta mAb 6H1 was evaluated in detail for its anti-tumor efficacy in vitro and in vivo. 6H1 showed high binding specificity to hCGbeta, which was analyzed by Western blot and ELISA as well as indirect immunofluorescence assay. Treatment with 6H1 inhibited the growth of a panel of hCGbeta-expressing tumor cell lines (HeLa, HL-60, HepG2, SMMC-7721, PC-3) in vitro. Moreover, 6H1 significantly delayed the growth of HeLa-borne tumors in nude mice and prolonged the survival of tumor-bearing mice. The anti-tumor effect of 6H1 was associated with the induction of apoptosis, which was estimated by Hoechst 33258 staining, DNA ladder assay and flow cytometry. Collectively, 6H1 revealed potent anti-tumor activity in vitro and in vivo and therefore may be an effective therapeutic candidate for immuno-intervention of cancers that ectopically express hCGbeta.

The antigen binding sites of various hCG monoclonal antibodies show homology to different domains of LH receptor.

The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Therefore, it can be hypothesized that the paratope of antibodies may exhibit homology with distinct domains of the receptor. By locating the hormone epitopes and determining the structure of the paratopes, it should be possible to identify the contact points between the ligand and the receptor. This hypothesis has been tested using hCG monoclonal antibodies (MAbs) recognizing different epitopes and having different effects on hormone binding and response. The beta subunit and heterodimer specific antibodies inhibited both hormone binding and response, while the alpha subunit specific antibodies inhibited response without affecting binding. The single chain fragment variables (ScFvs) produced from these antibodies also retained the properties of the parent antibodies. The amino acid sequences of the ScFvs exhibited homology to different regions of the receptor; the beta subunit specific antibody being homologous to the concave surface of the leucine rich repeats (LRR) of the receptor, particularly the concave surface of the LRRs, while the heterodimer specific antibody showed homology to the hinge region. The alpha subunit specific antibody showed homology to the transmembrane domain of the receptor. The exact locations of the epitopes of the monoclonal antibodies in the hormone molecule have also been identified. The data presented here also support the model of glycoprotein hormone-receptor interaction in which the hormone binds to the extracellular domain through the beta subunit and then the alpha subunit is brought in contact with the transmembrane domain leading to signal transduction.

Designing a new generation of anti-hCG vaccines for cancer therapy.

The heterodimeric 'pregnancy-specific' hormone human chorionic gonadotropin (hCG) has been used as the basis for a contraceptive vaccine. More recently, the observation that hCG, particularly in the form of the beta-chain expressed in the absence of alpha-chain, is aberrantly expressed in a number of different tumors has opened up a second potential application for such vaccines. Drawbacks of the currently available vaccines are that they are either relatively weakly immunogenic or that they induce antibodies that cross-react with human leuteinizing hormone (hLH). We have explored the possibility of creating mutated versions of the hCG beta-chain with improved immunologic properties.

Time-dependent folding of immunological epitopes of the human chorionic gonadotropin beta-subunit.

We have explored the possibility to use 14 different monoclonal antibodies in order to follow the formation of the respective epitopes during the biosynthesis of hCG subunits and their association in JEG-3 choriocarcinoma cells using pulse (30s to 5 min)-chase (0-180 min) experiments. We found central cystine knot epitope structures (epitope beta1) to be formed immediately and simultaneously with epitopes on the protruding hCG-beta loops 1 and 3. We found also differences in the time-dependent folding of beta2 and beta4 epitopes, which are highly overlapping structures on the loops 1+3. These differences were reinforced by decreasing the temperature during the pulse-chase experiments to 25 degrees C. Moreover, we describe for the first time an intracellular intact hCG beta-subunit form that showed the transient expression of the hCG-beta-core fragment epitope beta11 in the course of the maturation of this subunit which casts new light on the presence of hCG-beta-core fragment in Down's syndrome, tumors and pregnancy.

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

A novel monoclonal antibody with catalytic activity against beta human chorionic gonadotropin.

In this study, we report for the first time, production of monoclonal antibody (MAb) against beta human chorionic gonadotropin (hCG) with proteolytic activity. MAb "7D9" was raised in Balb/C mice using purified human chorionic gonadotropin. Immunoblot analysis and enzyme-linked immunoabsorbent assay (ELISA) showed that this MAb reacts with beta hCG. The epitope for this antibody appears to be located in the C-terminal of beta chain as suggested by the absence of cross-reaction with other glycoprotein hormones such as FSH, TSH and LH. Our data reveal that this MAb is very unstable and has autodegradation characteristics. Zymogram analyses also show that 7D9 MAb has a high level of hydrolytic activity against different substrates such as casein and gelatin. This proteolytic activity can be inhibited by EDTA. These findings demonstrate the proteolytic character of 7D9 MAb and consequently explain its instability.