Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies. Persistent expression of DNA

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.     

The humoral immune response of mice to intra-mammary immunization with ovalbumin.

Groups of lactating mice were immunized intra-mammarily on the second day of lactation with 20 micrograms, 150 micrograms or 400 micrograms of ovalbumin (OVA). This resulted in the appearance of IgG in serum, and IgA and IgG in milk. In serum, no IgA antibodies were detected 16 days after immunization in any of the groups. The serum response of IgG was variable and not related directly to the immunizing dose. Both IgA and IgG antibodies were absent in milk 5 days after immunization and IgG antibody level in milk increased significantly throughout lactation as measured 10 and 15 days after inoculation. No IgA antibodies appeared in the milk of the 20 micrograms and 150 micrograms group; however, responses appeared in milk with the highest dose (400 micrograms), but the number of responders for IgG increased in milk but not in blood. The results suggest that intra-mammary immunization can provoke a local IgA response in milk, and that serum is not a major source of IgG in that fluid. Moreover, the kinetics of the IgA and IgG responses differ.     

双分沁功能IgM、IgG2a单克隆抗体杂交瘤细胞株的建立与鉴定

应用HB_sA_g采取两种不同的免疫方法免疫小鼠后。取免疫的脾细胞与小鼠骨髓瘤细胞Sp2/0融合,筛选出两株抗HB_s的单克隆抗体杂交瘤细胞株。常规免疫方法获得的细胞株为WHB_s1,分泌IgG1。脾内免疫方法获得的细胞株WHB_s2,分泌IgM和IgG_(2a)抗体。经过3个月的连续培养,能稳定分泌效价高,特异性强的单克隆抗体。     

The construction and use of a human-mouse myeloma analogue suitable for the routine production of hybridomas secreting human monoclonal antibodies

A human-mouse myeloma analogue termed HMMA2.11TG/O was constructed by fusion of the mouse myeloma cell line P3x63Ag8.653, a mutant derivative of MOPC21, with bone marrow mononuclear cells from a patients with IgA myeloma. The HMMA2.11TG/O cell line is resistant to 6-thioguanine and ouabain and sensitive to HAT. The cell line secretes no detectable immunoglobulin and has a hybrid karyotype and cell surface phenotype. An average fusion efficiency for growth of hybridomas of 1/17,000 fused cells was obtained in fusions with human peripheral blood mononuclear cells (PBM), Pokeweed Mitogen (PWM) stimulated PBM, and Epstein-Barr Virus (EBV) transformed polyclonal B cell lines. Over 75% of hybrids secrete detectable immunoglobulin and the cloning efficiency of the hybrids at 1 cell/well averages 25%. Antibody secreting cloned hybridoma cell lines were obtained by fusion directly with PBM from an immunized volunteer and by fusion with in vitro, secondarily immunized, EBV transformed polyclonal cell lines. Five hybridomas secreting human monoclonal IgM anti-tetanus antibodies and 2 secreting human monoclonal IgG anti-tetanus antibodies were selected and cloned from 6 fusions performed specifically for anti-tetanus antibody. Immunoglobulin and antibody secretion by cloned hybrids has been stable for 5-10 months at present. Immunoglobulin and antibody secretion in routine cultures passaged every 3-4 days has been 8-42 micrograms/ml. This human-mouse myeloma analogue should prove useful for the routine production of human monoclonal antibodies.     

Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein

DNA immunization, in theory, is of great interest as a source of specific antibodies against different antigens. In an attempt to produce polyclonal and monoclonal antibodies against cell surface molecules by using the DNA immunization strategy, intramuscular and intrasplenic routes of DNA injection were compared. Two to five, but not a single, intramuscular DNA immunizations induced anti-CD54 and anti-CD147 antibody production. In contrast, a single intrasplenic immunization of CD54-encoding DNA could induce anti-CD54 antibody production. To produce monoclonal antibody (mAb), spleen cells obtained from an intrasplenic CD54-encoding DNA immunized mouse were fused with myeloma cells using the standard hybridoma technique. A hybridoma secreting specific mAb to CD54 was established. The generated mAb reacted to CD54 protein expressed on transfected COS cells and various cell types, the same as using standard CD54 mAb MEM-111. Our results demonstrated that direct immunization of antigen-encoding DNA into spleen is an effective route for production of both polyclonal and monoclonal antibodies to cell surface molecules. This finding is very useful for the production of antibodies to cell surface molecules where the protein antigen is not available or difficult to prepare, but cDNA encoding the corresponding protein is available.     

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.     

Use of 'single shot' intrasplenic immunization for production of monoclonal antibodies specific for human IgM

We report here the use of 'single shot' intrasplenic injection of human IgM for immunization of mice to obtain splenocytes for use in the production of hybridomas secreting antibodies against human IgM. Fusion was performed 3 days after intrasplenic injection of 20 micrograms of myeloma IgM. IgM-specific antibodies were found in 12% of the fusion wells; only 1 well contained antibodies which cross-reacted with other immunoglobulin classes. Two monoclonal antibodies (McAbs) have been fully characterized as specific for different epitopes on Fc mu. These antibodies can be used to detect IgM on the surface of human B cells by immunofluorescence and in solution by solid-phase radiobinding assay or single radial immunodiffusion. Both McAbs can also detect IgM fragments by immunoblotting from non-reducing SDS-polyacrylamide gels.     

Species-specific immune response to immunization with human versus rodent A beta peptide

Amyloid beta (A beta) immunization of amyloid precursor protein (APP)-transgenic (tg) mice with human A beta induces humoral immunity, however, the immune response to endogenous rodent A beta is unknown. Fourteen-month J20 APP-tg mice and non-tg littermates were immunized subcutaneously followed by chronic intranasal boosting with human or rodent A beta peptide and adjuvant LT(R192G). Rodent A beta-immunized APP-tg mice had anti-rodent A beta antibody levels of 257.8 micrograms/ml and those immunized with human A beta had anti-human A beta antibodies of 120.8 micrograms/ml. Non-tg littermates had anti-rodent and anti-human A beta antibody concentrations of 98.8 and 231.1 microgram/ml, respectively. Inter-species cross-reactivity was minimal. Anti-human A beta antibodies were predominately IgG1 and IgG2b, while anti-rodent A beta antibodies were equally IgG1, IgG2a, and IgG2b. Anti-human A beta antibodies recognized an epitope within human A beta1-9. Anti-rodent A beta antibodies did not stain Alzheimer's disease (AD) plaques but bound some plaques in APP-tg mice. Splenocytes proliferated modestly to their respective antigen and secreted low levels of IL-2 and IFN-gamma. Therefore, immunizing APP-tg and non-tg mice with rodent A beta resulted in a species-specific humoral response with modest T cell reactivity.     

Comparison of resistance level and circulating IgG response in chickens experimentally inoculated with a multiple or single immunizing doses of Eimeria acervulina.

Two immunizing methods (Trickle or single immunizing doses) against E. acervulina were tested in chickens. The effects of immunization and challenge upon growth, oocyst output and circulating antibodies response (IgG) were compared. Neither immunization method produced pathogenic effects, similar numbers of oocysts were produced, and the levels of IgG in serum were similar and low in each case. After the challenge, immunized birds showed a high level of resistance but susceptible controls produced very large number of oocysts and showed a marked reduction in the growth. Birds immunized by a trickle infection produced oocysts on two days only and the total number of oocysts per bird was very low, whereas those immunized by a single infection produced oocysts over a period of nine days and the total number of oocysts was higher. Susceptible and birds immunized by a single inoculation showed similar IgG concentration and these were statistically higher than birds immunized by a trickle infection. In susceptible birds the kinetic of IgG was delayed about 4 days.     

Identification of the components of glomerular immune deposits using monoclonal antibodies

Monoclonal antibodies have been raised against components of glomerular immune deposits in experimental glomerulonephritis and idiopathic human glomerulonephritis. An accelerated model of chronic serum sickness in the rat using cationized human serum albumin was employed to obtain renal tissue with capillary loop and mesangial immune deposits. Mice were immunized with isolated rat glomeruli or a preparation of glomerular basement membrane and mouse spleen cells fused with myeloma cells. Anti-human serum albumin monoclonal antibodies were produced from all technically successful fusions irrespective of the size of the deposits in the immunizing tissue or whether whole glomeruli or glomerular basement membrane were used for immunization. Monoclonal antibodies were then produced following immunization with tissue from postmortem human kidneys with idiopathic membranous and mesangiocapillary glomerulonephritis. Sixteen monoclonal antibodies, apparently reactive with glomerular immune deposits, were cloned; most of these were reactive with components of the complement system including a previously undescribed complement-related protein. These studies demonstrate that monoclonal antibody technology may be useful in determining the identity of antigen and non-antigen components of glomerular immune deposits.     

Intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated Salmonella enterica serovar enteritidis antigen

Induction of intestinal mucosal immune responses against Salmonella enterica serovar enteritidis was studied by immunizing chickens with liposome-associated antigen. An ultrasonicated whole cell extract of the bacteria was used for immunizing antigen. Intraocular immunization induced serum IgA, IgG and IgM responses. Also, significant IgA and IgG antibodies were detected in the intestinal tract. Immunization with antigen alone induced only IgG response in the intestine. Salmonella enteritidis-specific antibody-secreting lymphocytes were detected in the spleen and lamina propria of the intestinal tract of immunized chickens. Immunoglobulin (Ig) fractions extracted from intestines of immunized chickens inhibited the adherence of S. enteritidis to cultured HeLa cells. These results indicate that intraocular immunization with liposome-associated S. enteritidis elicits specific antibody-producing lymphocytes in the intestinal tract, and that Ig secreted in the intestine inhibits adherence of the bacteria to intestinal epithelial cells, suppressing the spread of bacterial infection in the host.     

Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.     

The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Two novel immunization methods (intrasplenic and intra-inguinal lymph node) have been developed for the production of polyclonal and monoclonal antibodies in mice. Freund's complete adjuvant and antigen were mixed in the ratio of 1 : 2 (v/v). Various concentrations of human serum albumin (HSA) were used as antigen. No primary immune response was induced with 0.1 μg of HSA in either of the methods studied. Intrasplenic immunization resulted in the strongest primary immune responses using all other doses of HSA. The primary immune response induced by intrasplenic immunization with 0.5 μg of HSA was higher than any response induced by subcutaneous immunization with various doses of HSA. Inguinal lymph node immunization was less effective than intrasplenic immunization but better than subcutaneous immunization with 1–50 μg of HSA. Comparisons were also made of the efficacy of different adjuvants when inducing primary immune responses with 1 μg of HSA. Freund's complete adjuvant resulted in a much stronger response than Freund's incomplete adjuvant and alum. Both intrasplenic and inguinal lymph node immunization using 1–5 μg of HSA were able to induce strong primary immune responses. Secondary immunization with either method or intravenous injection 3 days before fusion resulted in a higher frequency of specific monoclonal antibodies.     

Variable regions of antibodies to synthetic polypeptides--I. Characterization of an idiotope expressed on antibodies and T-cell factors

Spleen cells from a Lewis rat immunized with affinity-purified B10 anti-(T,G)-A-L antibody were fused with the non-secreting murine hybridoma SP2/0. Cell lines secreting monoclonal antibodies specific for mu- and kappa-chains, as well as an idiotope on anti-(T,G)-A-L antibodies, were isolated and characterized. The anti-mu and -kappa antibodies, are true anti-isotypes, reacting with sera from all strains of mice tested. The anti-idiotope antibodies recognize a determinant on antibodies binding a GT-containing epitope. The proportion of anti-GAT antibody bearing the idiotope varies markedly in different murine strains. A 1000-fold higher level of antibody from Igha mice than from Ighb and Ighe mice is required to give an equivalent inhibition of the idiotope-anti-idiotope reaction. Analysis of monoclonal antibodies expressing the idiotope indicates that the affinity of binding between idiotope and anti-idiotope can vary by as much as two orders of magnitude. Immunoadsorbants prepared with anti-idiotope antibody bind suppressor factor secreted by a GAT-specific T-cell hybridoma.     

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin.

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.     

Use of passive immunization for the production of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 1,6 and 12.

Hyperimmune sera from BALB/c mice immunized intraperitoneally (IP) with Actinobacillus pleuropneumoniae serotype 2 were used for passive intraperitoneal (i.p.) immunization of BALB/c mice. The immunized mice were subsequently immunized i.p. with a mixture of A. pleuropneumoniae serotypes 1, 6 and 12. Numerous monoclonal antibodies specific for serotypes 1, 6 and 12 were obtained. Using this immunization scheme antibodies can be obtained against specific antigens from closely related bacteria.     

Presence of natural autoantibodies in hyperimmunized mice.

Mice were immunized with various antigens in complete Freund's adjuvant following various injection schedules. Hybridomas were produced from the spleens of these immunized mice and examined for production of antibodies directed against the antigen injected and against a panel of self (tubulin, actin, myosin, DNA) and non-self antigens (myoglobin, spectrin, peroxidase, trinitrobenzene). Two to five percent of the hybrids were found to secrete polyspecific antibodies able to react with two or more antigens of the panel. Several of these hybrids were subcloned and expanded into ascites. The monoclonal immunoglobulins they secreted were isolated and shown to be IgM (kappa) and to possess the polyspecific antibody function. Several hybrids were also found to secrete antibodies reacting with the immunizing antigen as well as one or more antigens of the panel. The antibody secreted by one subclone which reacts with both the immunizing antigen, prolactin and one of the panel antigens, TNP, has been isolated using a DNP-immunoadsorbent. The isolated antibody was found to be a monoclonal IgM (kappa) immunoglobulin and to react both with prolactin and TNP. The hypothesis is advanced that cells carrying polyspecific natural antibodies as receptors after a given antigenic stimulation proliferate into cells producing highly specific antibodies for epitopes of that given antigen; the cells with polyspecific receptors will be continuously replaced by new cells probably on bone-marrow origin.     

Recent insight into class-specific properties of murine immunoglobulins obtained with the help of monoclonal antibodies

BALB/c and C57BL/6 mice were immunized with monoclonal anti-DNP IgE, obtained by fusion of PX63AG8-6-5-3 cells with spleen cells from immunized C57BL/6 or BALB/c mice, respectively. With the antiallotype sera thus produced the allotype of IgE (i.e. 7) could be defined since BALB/c is of the Igh-a and C57BL/6 of the Igh-b allotype. The antiallotype 7b does not cross-react with other allotypes. Antiallotype 7a recognizes in addition to allotype 7a also allotypes 7j, 7d, and 7e as expected. Both the complement fixing and the sensitizing activities of murine anti-TNP and anti-DNP monoclonal antibodies were examined. The minimal amount/ml for complement fixation was 0.46 micrograms from one of the IgG1 antibodies, 0.46 from IgG2a, 12.5 ng from IgM, 0.7 micrograms from IgG3. Complement fixation by IgG1 was unexpected. The minimal amount/ml for mouse PCA was 0.5-0.7 micrograms from IgG1, 1.44-2.3 micrograms from IgG2a, 5.6 micrograms from purified IgG2b and about 15 ng from IgE. Sensitization by IgG2a and IgG2b were new findings. Minimal amount/ml for guinea pig PCA was 0.23 micrograms from IgG2a. Minimal amount/ml for rat PCA was 2 ng from IgE.     

Rat anti-T15 monoclonal antibodies with specificity for VH- and VH-VL epitopes

BALB/c mice immunized with phosphorylcholine (PC) produce antibodies which are predominantly of the T15 idiotype. Monoclonal anti-T15 antibodies have been generated in a number of laboratories by allogenic or syngenic immunization. Most of these mouse monoclonal antibodies react with idiotopes that are in or near the PC-binding site and require the presence of both T15 heavy and T15 light chain variable regions. By immunizing rats with T15 immunoglobulins, we have obtained monoclonal antibodies that recognize idiotopes that are not near the antigen-binding site. Four of these rat anti-T15 monoclonal antibodies react with free T15 heavy chains and with T15 heavy chains associated with irrelevant light chains while four other rat monoclonal antibodies require the presence of both T15 heavy and T15 light chains. This battery of rat anti-T15 monoclonal antibodies is useful in searching for heterogeneity within the T15 antibodies and in following the expression of the T15 heavy chain variable region in different strains of mice.     

抗乙肝病毒核心抗体抗独特型抗体的研究——Ⅱ.制备抗人HBcAb的抗独特型抗体的探讨

为了研究抗独特型抗体对乙型肝炎免疫反应的调节作用,我们用人的多克隆抗HBc作为免疫原,制备单克隆抗独特型抗体(抗-Id)。将人HBcAb用亲和层析纯化,并经SPA-Sepharose4B柱提取Fab片段。用4株抗正常人IgG的单克隆抗体与人HBcAb复合,制成免疫复合物,再用作免疫原,采用3种不同的免疫方案,除了采用国内外公认的抗原-抗体结合抑制试验检测外,还做了中和试验。在“独特型结合试验”中,在用人HBcAb包放的同时,还用正常人IgG作对照,以排除其抗异种免疫球蛋白的干扰。同时,还进行了交叉反应独特型的筛选工作。我们共对6300个克隆进行了检测,没有检出特异性的抗独特型抗体,却有1450个抗正常人IgG的阳性克隆。对出现上述结果的可能原因进行了探讨。