Interferon-α conjugation to human osteogenic sarcoma monoclonal antibody 791T/36

Human lymphoblastoid interferon-alpha (IFN-alpha) has been coupled using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to a murine monoclonal antibody (791T/36) which reacts with antigens expressed on human osteogenic sarcomas. The purified conjugates retain antibody activity as defined by their capacity to compete with binding of fluorescein isothiocyanate-labelled 791T/36 antibody to 791T cells. IFN-alpha-791T/36 antibody conjugates synthesized with 125I-trace-labelled IFN-alpha and 131I-trace-labelled antibody also bound to 791T cells, but not to bladder carcinoma T24 cells. The conjugates also retain the capacity of free IFN to activate natural killer cells in human peripheral blood lymphocytes and show specific localization in human osteogenic sarcoma xenografts developing in immunodeprived mice. These findings establish that conjugates containing IFN linked to a monoclonal antibody reacting with osteogenic sarcoma-associated antigens have potential for targeted immunotherapy and in related investigations with antibody has been shown by gamma camera imaging of patients following infusion of 131I-labelled antibody to localize in primary osteogenic sarcomas.     

Localization of anti-osteogenic sarcoma monoclonal antibody 791T/36 in a primary human osteogenic sarcoma and its subsequent xenograft in immunodeprived mice

Summary A primary human osteogenic sarcoma was visualized in situ by external gamma-camera imaging following administration of ¹³¹I-labelled anti-osteogenic sarcoma monoclonal antibody 791T/36. A xenograft of the tumour established in immunodeprived mice also showed localization of ¹³¹I-791T/36 determined by both gamma-camera imaging and organ distribution studies. Radiolabelled normal immunoglobulin showed no tumour localization. Expression of the 791T/36-defined antigen on xenografted tissue was further confirmed by reaction of its in vitro-cultured cells with fluorescein-labelled 791T/36 antibody.     

Production of a human monoclonal antibody recognising a determinant on mouse IgG2b from a patient receiving mouse monoclonal antibody for diagnostic imaging

Summary The development of human antibodies recognising mouse immunoglobulins represents an obstacle to effective antibody therapy. This study shows that patients produce modest titres of antibodies (predominantly antimouse rather than anti-idiotypic) after a single low-dose injection for immunoscintigraphy, suggesting that repeated imaging with the same or a different antibody could be a problem. Fusion of the lymphocytes from a patient who had been imaged twice previously resulted in a monoclonal antibody that specifically binds to an IgG2b isotypic determinant. Anti-IgG2b antibodies predominated in this patient's serum. Production of human monoclonal antibodies from patients given mouse monoclonal antibodies not only allows a finer dissection of the immune repertoire but also provides possible reagents for controlling the human anti-(mouse Ig) response, for selection of class-switch variants of mouse monoclonal antibodies and enhancing tumour imaging.     

A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.     

Preparation and characterization of a human aurora-A kinase monoclonal antibody

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an Elisa (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.     

Adriamycin custom-tailored immunoconjugates in the treatment of human malignancies

Twenty-three patients with disseminated refractory malignancies each received a tailored combination of adriamycin-conjugated murine monoclonal antibodies. Tumors were typed using a panel of antibodies. Cocktails of up to six antibodies were selected based on binding greater than 80% of the malignant cells as tested by immunoperoxidase and flow cytometry. These monoclonal antibodies were then conjugated to Adriamycin and administered intravenously. Seventeen of 23 patients had reactions to the administration of immunoconjugates, but these were tolerable in all but two patients. Fever, chills, pruritis, and skin rash were by far the most common transitory reactions. All were well controlled with premedication. In several patients there was limited antigenic drift among various biopsies within the same patient over time. This observation confirms the necessity for the use of a cocktail of antibodies if one wishes to cover all tumor cells. Preliminary serologic evidence suggests that the development of an IgM antibody, which is specific against the mouse monoclonal antibody, has the specificity and sensitivity to predict clinical reactions. Selected patients were re-treated. One patient with chronic lymphocytic leukemia had re-treatment on three occasions and demonstrated regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions and two patients with tongue carcinoma had shrinkage of their lesions. In the course of the study free Adriamycin released from the monoclonal antibodies was discovered to be a limiting factor in the amount of antibody that could be administered. Up to 1 g of Adriamycin and up to 5 g of monoclonal antibody were administered. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody that unpredictably caused hemopoietic depression. This study demonstrates the feasibility and reviews technical considerations in preparing immunoconjugate cocktails for patients with refractory malignancies. The major technical hurdle appears to be the selection of an effective conjugation method that can be used to optimally bind Adriamycin to monoclonal antibodies for targeted cancer therapy.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Human antibody responses to components of the monoclonal antimelanoma antibody ricin A chain immunotoxin XomaZyme-MEL

Human antibody responses to immunotoxin components were evaluated in 21 melanoma patients who were treated with XomaZyme-MEL, a murine monoclonal antimelanoma antibody-ricin A chain conjugate. Twenty of the 21 melanoma patients produced antibodies against ricin A chain, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1 to 2 weeks following initiation of therapy, with peak levels generally attained 2 to 4 weeks posttherapy. Titers of the anti-ricin A chain antibodies were generally higher than those of the antimurine monoclonal antibodies for the dose range tested. There was no clear correlation between the dose of immunotoxin administered and the antibody titer. By use of a competitive flow cytometry assay, antiidiotype responses were demonstrated in eight of 10 melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. The development of antimmunotoxin antibodies can reduce the therapeutic potential of immunotoxins through several mechanisms. The development of antibodies in a significant number of patients suggests that optimally effective, repeated courses of therapy will require some procedure for suppressing or abrogating the response against the immunotoxin.     

Protection against Campylobacter jejuni infection in suckling mice by anti-flagellar antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.     

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

Summary Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15–30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody-coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow. mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment.Supported by the Dutch Cancer Society (grant NKI 84-14)     

Human immune response to multiple injections of murine monoclonal IgG

Murine monoclonal antibody infusions in humans should induce a human anti-mouse immunoglobulin (mIgG) immune response, especially if multiple infusions over an extended period of time are necessary for therapeutic efficacy. We have administered multiple infusions of the murine monoclonal antibody T101 to patients with cutaneous T cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL). Five of 10 CTCL patients, compared with zero of six CLL patients, developed antibodies to mIgG. In those CTCL patients who did not demonstrate anti-mIgG antibodies, we were unable to correlate the lack of response to any of a large number of clinical parameters. Anti-mIgG antibodies were of both the mu and gamma isotypes and were detectable 14 days after the first infusion. Multiple infusions were associated with elevated titers. The anti-idiotypic portion of the anti-mIgG titer steadily increased with each infusion until eventually, in one patient receiving eight weekly infusions, well over one-half the serum anti-mIgG recognized only T101 and not four other murine IgG2AK antibodies tested. To increase our confidence in these findings, four separate assay systems were used to make these determinations. The identification of anti-idiotype antibodies as the dominant species of the immune response to multiple infusions of murine monoclonal antibody has major implications for future work with monoclonal antibodies. Although it has been suggested that human monoclonal antibodies would obviate an immune response, our work implies that such antibodies might still induce anti-idiotype antibodies if multiple infusions are administered.     

Characterization of a mouse/human chimeric monoclonal antibody (17-1A) to a colon cancer tumor-associated antigen

Mouse monoclonal antibody 17-1A is specific for an antigen expressed on cells of human gastrointestinal malignancies and has been used in radioimmune imaging and therapy trials for patients with colon and pancreatic cancer. The cell line SG3/5 was generated by transfection of a nonproducing mouse myeloma line (SP2/0) with a chimeric gene construct composed of variable regions from the mouse 17-1A immunoglobulin (gamma 2a, kappa) and constant regions of human k and gamma 3 immunoglobulin genes. The secreted immunoglobulin was bound by mouse monoclonal antibodies to human IgG(Fc) and IgG3 but not by staphylococcal protein A. Gel filtration HPLC profiles of purified chimeric antibody were similar to normal human IgG3 but quite different from native 17-1A and normal human IgG1, 2, and 4. Native and chimeric 17-1A had similar patterns of reactivity with colon cancer, other adenocarcinoma, and leukemic cell lines. Competitive inhibition documented that native and chimeric 17-1A had identical capacities to inhibit radiolabeled native 17-1A binding to colon cancer cell lines. Thus, the chimeric 17-1A exhibits molecular characteristics of normal human IgG3 but retains the specificity and binding affinity of the native 17-1A murine monoclonal antibody. The native and chimeric 17-1A mediated similar modest degrees of human lymphocyte and monocyte ADCC in a 4-hr 51Cr release assay, and both failed to mediate complement lysis of colon carcinoma cell lines in the presence of human complement. This human/mouse chimeric monoclonal antibody may be a good candidate for use in clinical trials because it retains the tumor antigen specificity and human effector cell recognition of the native 17-1A, would presumably have a fivefold to 10-fold longer circulating half-life in man, and should be considerably less immunogenic as compared with native murine immunoglobulins.     

Trypanosoma cruzi specific antibody response in immunized C3H(He) mice during infection

C3H(He) mice previously immunized with live culture derived Corpus Christi strain T. cruzi are significantly protected (up to 100% survival) against challenge by Brazil strain blood trypanosomes. The antibody response, directed against the Brazil strain or the Corpus Christi strain, in these mice has been observed by comparing sera from mice immunized only, infected only, or immunized and infected. The anti- T. cruzi titers determined by both direct agglutination (DA) and indirect fluorescence (IFA) were routinely found to be highest for immunized and infected mice with immunized mice and infected mice following in decreasing order. The use of mercaptoethanol treatment of sera (DA) and isotope specific second antibody (IFA) showed that IgG is the major parasite specific immunoglobulin response through infection. Evidence of cross-reacting antigens on the two parasite strains was found. By both DA and IFA, 11 of 18 anti-Brazil strain monoclonal antibodies were found to react (IFA titers of 320 or greater) with both parasite strains. No evidence of localization of cross-reacting antigens (using mouse antisera) or antigenic determinants (using monoclonal antibodies) was found in that uniform fluorescence over the parasite was observed in all IFA tests.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

应用哺乳动物抗体检测阿维菌素抗性果蝇P糖蛋白的表达

【目的】随着杀虫剂阿维菌素的广泛应用,靶标生物对其抗性问题日益严重。以往的研究显示,细胞膜转运蛋白P糖蛋白可能与抗药性有关。但在昆虫对阿维菌素的抗性研究中,由于目前市场上缺少专门针对昆虫的商业化抗体,使得这一研究受限。为此,本研究尝试应用其他物种的抗体开展P糖蛋白的检测。【方法】以果蝇为测试昆虫,以阿维菌素作为测试药物,采用免疫印迹方法用鼠抗人P糖蛋白单克隆抗体检测阿维菌素敏感品系与阿维菌素抗性品系果蝇中的P糖蛋白的表达水平。【结果】检测出果蝇体内P糖蛋白的特异性表达,且无明显非特异性条带;与敏感品系果蝇相比,阿维菌素抗性品系果蝇P糖蛋白的表达水平明显升高。【结论】用针对人及其他脊椎动物的P糖蛋白单克隆抗体检测果蝇P糖蛋白的表达可行,且果蝇对阿维菌素的抗性可能与P糖蛋白的表达升高有关。     

Local and systemic antibody response in infants after oral administration of inactivated enteropathogenicE. coli serotype O111 and O55

In ten infants divided into two groups (up to one month of age and at 2–7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intenstine and in serum after orally administered inactivated enteropathogenicE. coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method. Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens. Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d. The infants aged 2–7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens. The serum immunoglobulin levels did not change after oral antigen administration.     

A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice

Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammal; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.