Monoclonal antibodies against human platelet membrane glycoprotein IIIa and collagen-induced platelet activation

Two monoclonal antibodies against human platelet membrane glycoprotein IIIa (GPIIIa) were obtained. One monoclonal antibody, designated as 1B1, was found to inhibit both collagen-induced platelet aggregation and release reactions. This antibody also inhibited the binding of 125I-labeled collagen to human platelets. On the other hand, the other antibody, designated as B10, had no effect on platelet activation induced by a number of physiological stimulants including collagen. Direct binding studies involving 125I-labeled 1B1 or B10 demonstrated that the binding sites for these antibodies on unstimulated platelets have dissociation constants of 4.2 and 14.0 nM, respectively. The binding of 125I-labeled 1B1 or B10 to platelets was not inhibited by the other antibody. Purified 1B1 and B10 were covalently coupled to Affi-Gel and then proteolytic fragments of GPIIIa were applied to the Affi-Gel immunoadsorbent columns. Of the several proteolytic fragments, the 56 kilodaltons (kDa) fragment obtained on digestion with V8 protease bound to both of the columns. The 69 and 55 kDa fragments obtained with BrCN bound to only the 1B1 Affi-Gel column, while the 63 kDa fragment obtained with chymotrypsin only bound to the B10-Affi-Gel column. Based on the partial amino acid sequences of these fragments and the amino acid sequence of GPIIIa (C. A. Fitzgerald, B. Steiner, S. C. Rall, Jr., S. Lo and D. R. Phillips, J. Biol. Chem., 262, 3936 (1987), the epitopes for 1B1 and B10 were concluded to be located at amino acids 335 to 582 and 206 to 335, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions

The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG. Two new glycosynthase mutants (EndoS-D233A and D233Q) were generated by site-directed mutagenesis of EndoS (an endoglycosidase from Streptococcus pyogenes ) and were found to be capable of efficiently transferring predefined N-glycans from corresponding glycan oxazolines to the Fc-deglycosylated intact IgGs without product hydrolysis. As a model study, rituximab (a therapeutic monoclonal antibody) was successfully transformed from mixtures of G0F, G1F, and G2F glycoforms to well-defined homogeneous glycoforms, including a fully sialylated (S2G2F) glycoform that may gain anti-inflammatory activity, a nonfucosylated G2 glycoform that showed significantly enhanced FcγIIIa receptor-binding activity, and an azido-tagged glycoform that can be further transformed into other glycoforms. We also found that EndoS could selectively remove the Fc N-glycans in the presence of FAB glycosylation. This finding, coupled with the remarkable transglycosylation activity of the EndoS glycosynthase mutants, permitted a highly selective glycoengineering of the IVIG's Fc glycans into a fully sialylated Fc glycoform, which may possess significantly enhanced anti-inflammatory activity. The glycoengineering approach described here provides a general platform to modulate the effector functions of IgG antibodies, enabling the optimization of therapeutic efficacy and gain of new functions of monoclonal antibodies and IVIG.     

Antibody adsorption and orientation on hydrophobic surfaces

The orientation of a monoclonal, anti-streptavidin human IgG1 antibody on a model hydrophobic, CH(3)-terminated surface (1-dodecanethiol self-assembled monolayer on gold) was studied by monitoring the mechanical coupling between the adsorbed layer and the surface as well as the binding of molecular probes to the antibodies. In this study, the streptavidin antigen was used as a probe for the Fab portions of the antibody, while bacteria-derived Protein G' was used as a probe for the Fc region. Bovine serum albumin (BSA) acted as a blocking protein. Monolayer coverage occurred around 468 ng/cm(2). Below 100 ng/cm(2), antibodies were found to adsorb flat-on, tightly coupled to the surface and unable to capture their antigen, whereas the Fc region was able to bind Protein G'. At half-monolayer coverage, there was a transition in the mechanism of adsorption to allow for vertically oriented antibodies, as evidenced by the binding of both Protein G' and streptavidin as well as looser mechanical coupling with the surface. Monolayer coverage was characterized by a reduced level in probe binding per antibody and an even less rigid coupling to the surface.     

The use of PhastSystem crossed immunoelectrophoresis with immunoblotting to demonstrate a complex between glycoprotein Ib and the actin-binding protein (ABP) of human platelets

The study shows how a technique described in an accompanying paper can be applied to solve a biological problem. The technique makes use of the observation that a monoclonal antibody that has been coprecipitated with its antigen during crossed immunoelectrophoresis can be transferred to a nitrocellulose membrane and visualized. Previous studies using crossed immunoelectrophoresis of Triton X-100 extracts of platelets have indicated that a particular immunoprecipitate (peak III) of the membrane receptor glycoprotein Ib (GP Ib) might contain a complex between the receptor and the actin-binding protein (filamin). When a monoclonal antibody (PM6/317) directed towards the actin-binding protein was added to a platelet extract prior to immunoelectrophoresis and blotting, this was visualized on the blot as a replica of the peak III immunoprecipitate. This demonstrates a colocalization of GP Ib and the actin-binding protein in the precipitate, and thus the existence of a complex between the membrane receptor and the cytoskeletal protein.     

Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies.

The majority of methods used to prepare immunosorbents immobilize antibodies through their reactive amino acid residues. The bound antibody activity of these immunosorbents is low. Hydrazide-based matrices couple antibodies through carbohydrate chains frequently located in the Fc region. This paper reports a comparative study of the performance of immunosorbents prepared by cyanogen bromide or hydrazide immobilization methods. The experiments utilized murine monoclonal antibodies to the human plasma proteins Factor IX or Protein C. The antibodies were immobilized at low densities to beaded agarose matrices which had similar properties. The hydrazide immunosorbents had binding efficiencies which were lower (anti-Factor IX) or up to 1.6-fold higher (anti-Protein C) than comparable cyanogen bromide coupled gels. However, there was no improvement in performance due to lower recoveries of bound protein from the hydrazide gels. Control experiments demonstrated that oxidation of antibody which is required for its coupling to hydrazide gels had no effect on antibody binding to antigen. Our results indicate that, as with cyanogen bromide coupling methods, site-directed immobilization through carbohydrate residues results in a restricted ability to bind to antigen. Both monoclonals were found to contain carbohydrate in their Fab' regions through which coupling may have occurred. The frequency of carbohydrate in the Fab region and the ability to control glycosylation at these sites are factors which may impact the utility of carbohydrate-directed immobilization of antibodies.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

A multifunctional micro-fluidic system for dielectrophoretic concentration coupled with immuno-capture of low numbers of Listeria monocytogenes

In this study, we demonstrated a micro-fluidic system with multiple functions, including concentration of bacteria using dielectrophoresis (DEP) and selective capture using antibody recognition, resulting in a high capture efficiency of bacterial cells. The device consisted of an array of oxide covered interdigitated electrodes on a flat silicon substrate and a approximately 16 microm high and approximately 260 microm wide micro-channel within a PDMS cover. For selective capture of Listeria monocytogenes from the samples, the channel surface was functionalized with a biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Positive DEP (at 20 V(pp) and 1 MHz) was used to concentrate bacterial cells from the fluid flow. DEP could collect approximately 90% of the cells in a continuous flow at a flow rate of 0.2 microl min(-1) into the micro-channel with concentration factors between 10(2)-10(3), in sample volumes of 5-20 microl. A high flow rate of 0.6 microl min(-1) reduced the DEP capture efficiency to approximately 65%. Positive DEP attracts cells to the edges of the electrodes where the field gradient is the highest. Cells concentrated by DEP were captured by the antibodies immobilized on the channel surface with efficiencies of 18 to 27% with bacterial cell numbers ranging from 10(1) to 10(3) cells. It was found that DEP operation in our experiments did not cause any irreversible damage to bacterial cells in terms of cell viability. In addition, increased antigen expression (antigens to C11E9 monoclonal antibody) on cell membranes was observed following the exposure to DEP.     

Cyclic Peptidomimetics Derived from the Apical Membrane Antigen I of Plasmodium falciparum and Their Use in Malaria Vaccine Design

A library of 35 cyclic peptidomimetics has been prepared, by a combination of solid‐ and solution‐phase methods, which, together, scan amino acid residues 444–489 in a protruding loop in the subdomain‐III of the apical membrane antigen‐I (AMA‐I), an integral membrane protein found on the surface of Plasmodium falciparum merozoites. The mimetics each contain twelve residues from AMA‐I linked through the N‐ and C‐termini to a β‐hairpin‐inducing template, comprising the dipeptide D ‐proline‐L ‐Apro (Apro=cis‐4‐amino‐L ‐proline). These peptidomimetics were each coupled via the 4‐amino group to a phospholipid, which allows their incorporation into reconstituted influenza virus‐like particles (virosomes) for immunization of mice, as well as their use in ELISA to characterize epitopes recognized by anti‐AMA‐I growth‐inhibitory monoclonal antibodies.     

A synthetic mimic of human Fc receptors: defined chemical modification of cell surfaces enables efficient endocytic uptake of human immunoglobulin-G

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.     

Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and epitope mapping of two monoclonal antibodies against human CD99

CD99 plays a critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 x 10(-7) and 7.08 x 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Selection of hapten structures for indirect immunosensor arrays

A multianalyte immunosensor array can be implemented by immobilization of different haptens in distinct areas of a single cavity or flow cell. In this case a mixture of different antibodies for different analytes is used in an indirect ELISA-format. The selection of the right hapten structures is very important to build up an array successfully. A system of independent hapten/antibody combinations is needed, with one immobilized hapten (coating antigen) reacting only with one antibody. If more than one antibody binds to a coating antigen no ideal calibration curves are obtained. This phenomenon is known as shared-reactivity and can lead to double-sigmoidal curves. To use monoclonal antibodies to 2,4,6-trinitrotoluene (TNT) and 2,4-dichlorophenoxyacetic acid (2,4-D), two different haptens had to be found, one only reacting with the TNT-antibody, the other only binding to the 2,4-D-antibody. 2,4-Dichlorophenoxybutyric acid was used for the 2,4-D antibody and 2,4,6-trinitrophenyl-8-aminooctanoic acid for the TNT antibody. Although 4-nitrotoluene, 2,4-dinitrotoluene and 4-amino-2,6-dinitrotoluene showed only very low cross-reactivities to the 2,4-D antibody the corresponding haptens 4-nitrophenylacetic acid, 2,4-dinitrophenyl-6-aminohexanonic acid, and 4-amino-2,6-dinitrotoluyl-(N)-glutarate are useful coating antigens for this antibody. The structure of the coating antigens had no significant influence on the midpoints (IC50) of the test for 2,4-D and even haptens with very low cross-reactivities could be used. With all haptens a test midpoint of about 0.2 μg/L for 2,4-D was achieved. For the direct assay format with immobilized antibodies the same test midpoint of 0.2 μg/L for 2,4-D was obtained. As a conclusion, the selectivity of a monoclonal antibody should not be influenced by the used tracer or coating antigen as well. It could be shown that the affinity constants of an antibody to the analytes are the main sensitivity and selectivity determining parameters for competitive immunoassays. A two-dimensional microtiter plate array was used to determine the analytes 2,4-D and TNT in parallel with a mixture of antibodies. Received: 29 July 1998 / Revised: 21 October 1998 / Accepted: 10 November 1998     

A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies

For the development of human antibody Fc (fraction crystallizable) region-containing therapeutic protein candidates, which can be either monoclonal antibodies (mAbs) or pharmacologically active proteins/peptides fused to the Fc region of human Immunoglobulin G (IgG), reliable quantification of these proteins in animal pharmacokinetic study plasma samples is critical. LC-MS/MS has emerged as a promising assay platform for this purpose. LC-MS/MS assays used for bioanalysis of human antibody Fc region-containing therapeutic protein candidates frequently rely upon quantification of a 'signature' surrogate peptide whose sequence is unique to the protein analyte of interest. One drawback of the signature peptide approach is that a new LC-MS/MS assay must be developed for each new human Fc region-containing therapeutic protein. To address this issue, we propose an alternative 'universal surrogate peptide' approach for the quantification of human antibody Fc region-containing therapeutic protein candidates in plasma samples from all nonclinical species. A single surrogate tryptic peptide was identified in the Fc region of most human antibody Fc-containing therapeutic protein candidates. An LC-MS-MS method based upon this peptide was shown to be capable of supporting bioanalysis of a diversity of human Fc region-containing therapeutic protein candidates in plasma samples of all commonly used animal species.     

Construction,Purification, and Characterization of Anti-BAFF scFv–Fc Fusion Antibody Expressed in CHO/dhfr<Superscript>−</Superscript> Cells

Elevated levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, we have constructed a vector for the expression of a novel compact antibody composed of anti-BAFF single-chain antibody fragment (scFv) and the Fc region (the hinge region, CH2, and CH3 domains) of human IgG1 in Chinese hamster ovary cells. The scFv–Fc fusion protein, showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges, was affinity-purified on protein A Sepharose from culture supernatant. The scFv–Fc antibody was demonstrated to retain high binding affinity to antigen and prolonged clearance time in blood and to possess some human IgG crystallizable fragment effector functions such as protein A binding and antibody-dependent cellular cytotoxicity. These results suggest that this recombinant antibody may have therapeutic applications in the therapy of autoimmune disorders mediated by BAFF.     

A high-throughput microchip-based glycan screening assay for antibody cell culture samples

A high-throughput screening assay was developed to quantify major glycan species in the crude mammalian cell culture samples for monoclonal antibodies (mAbs). This method utilizes high-speed microchip electrophoresis separation following a fast sample preparation procedure. Using a 96-well ultra-filtration membrane, interfering species in the cell culture media were efficiently removed as the samples were concentrated. A commercial microchip electrophoresis instrument was used for high-speed separation, allowing each sample to be analyzed in less than 1 min. This method is well suited for the purpose of high-throughput antibody glycan profiling during cell culture expression, including clone selection and cell culture process optimization. The relative levels of high mannose (HM), fucosylated and galactosylated glycan species in the Fc domain can be determined for hundreds of crude cell culture samples in a few hours.     

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Metallothionein (MT) is a low‐molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot‐ and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 μL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit‐anti‐chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3‐aminoethyl‐9‐carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 μL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry‐Brdicka reaction.     

Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces

This report presents the development of pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose antibody immobilization platforms for real-time QCM-D immunoassay of sepsis-related biomarkers. These platforms differ significantly from recent trends in QCM-based assays, a rapidly expanding field given the affordability and sensitivity of the transduction system, by providing ultrafast biointerface deposition through cross-linking of polysaccharides. Using rhIL-1ra (17 kDa), a known sepsis biomarker, for development, various immunoassay modifications to increase sensitivity were investigated, including the use of Protein A, Protein G, and anti-IgG Fc specific antibody capture ligands for oriented antibody immobilization, higher-frequency QCM-D crystals, and amplification using secondary antibodies. The optimized assay employs Protein A oriented immobilization on pre-cross-linked polymer and secondary antibodies to achieve a detection limit of 25 ng/mL on 5 MHz crystals. Assay repeatability using the optimized chemistry is robust, with no loss in 100 ng/mL antigen detection over 20 cycles of the 10 min sandwich assay. Nonspecific adsorption of human serum albumin, as characterized by ToF-SIMS, is minimal and negligible for the pre-cross-linked and in situ cross-linked compositions, respectively.     

Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10⁹ independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.     

Production and evaluation of monoclonal antibodies for alphafetoprotein

Human AFP was used as an antigen for the development of monoclonal antibodies by the hybridoma technique. Balb/c mice were immunized with highly purified AFP. The preparation of I¹²⁵-AFP was carried out by lactoperoxidase oxidation method, preparation of AFP standards was carried out from cord sera. The antibody titer of the serum was tested by RIA-AFP system. The spleen of the immunized Balb/c was fused with Sp₂0 mouse myeloma cells. The cells from the positive hybridomas were cloned twice using limiting dilution method. Eleven stable clones were thus established for secreting monoclonal antibodies to AFP. Cells in this growth phase were chosen for freezing.