Antibody Production In Rabbits Research Articles

Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27–54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85–105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1–3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST RECOMBINANT VP6 ANTIGEN OF ROTAVIRUS

Rotavirus is the most common cause of diarrhea in infants and young children under five years old. The VP6 protein of rotavirus was considered as an important antigen in the development of novel vaccine generation and diagnostic tools. Polyclonal antibodies of VP6 antigen could be used to generate a rapid test based on the lateral flow immunoassay. In the present study, we showed the results of synthesis of polyclonal antibodies against recombinant VP6 protein of rotavirus. Recombinant VP6 protein was strongly produced by E. coli in suitable conditions. The properties of recombinant VP6 protein were characterized by Western blot and ELISA methods. Pure recombinant VP6 protein was used as an antigen to inject into the rabbits. The recombinant VP6 protein was highly immunogenic in rabbits. Anti-VP6 antibodies were purified from the rabbit serum using the Protein A affinity chromatography. The specificity of the antibody towards its antigen was characterized by Western blot and ELISA immunoassay. The purity of antibody was reached as 97 %. The efficiency of antibody production in rabbit was 3.94 mg/ml serum. The amount and quality of the obtained anti-rotavirus VP6 antibody was suitable for application in the development of the diagnostic kits.

An oral microjet vaccination system elicits antibody production in rabbits.

Noninvasive immunization technologies have the potential to revolutionize global health by providing easy-to-administer vaccines at low cost, enabling mass immunizations during pandemics. Existing technologies such as transdermal microneedles are costly, deliver drugs slowly, and cannot generate mucosal immunity, which is important for optimal immunity against pathogens. We present a needle-free microjet immunization device termed MucoJet, which is a three-dimensional microelectromechanical systems-based drug delivery technology. MucoJet is administered orally, placed adjacent to the buccal tissue within the oral cavity, and uses a self-contained gas-generating chemical reaction within its two-compartment plastic housing to produce a high-pressure liquid jet of vaccine. We show that the vaccine jet ejected from the MucoJet device is capable of penetrating the buccal mucosal layer in silico, in porcine buccal tissue ex vivo, and in rabbits in vivo. Rabbits treated with ovalbumin by MucoJet delivery have antibody titers of anti-ovalbumin immunoglobulins G and A in blood serum and buccal tissue, respectively, that are three orders of magnitude higher than rabbits receiving free ovalbumin delivered topically by a dropper in the buccal region. MucoJet has the potential to accelerate the development of noninvasive oral vaccines, given its ability to elicit antibody production that is detectable locally in the buccal tissue and systemically via the circulation.

Epitope spreading induced by immunization with synthetic SSB peptides.

Sjogren's syndrome type B (SSB)/La antibody is an autoantibody generally observed in connective tissue diseases whereas double-stranded deoxyribonucleic acid (dsDNA) antibodies are the most characteristic autoantibodies found in systemic lupus erythematosus (SLE) patients. The relationship of these autoantibodies remains unclear. The aim of the study was to determine the profile of antibody production in rabbits immunized with synthetic SSB peptides alone or with dsDNA. For this purpose, 214–225aa peptide of SSB antigen was synthesized based on the organic chemistry solid-phase peptide synthesis. Rabbits were immunized with the following antigens: i) synthetic SSB peptides linked with keyhole limpet hemocyanin (KLH); ii) dsDNA; iii) SSB plus dsDNA; iv) KLH; and v) phosphate-buffered saline. SSB peptide antibody was measured using the enzyme-linked immunosorbent assay while extractable nuclear antigens (ENA) antibody and dsDNA antibody were measured by immunoblotting and immunofluorescence, respectively. The results showed that a specific anti-SSB peptide antibody was produced following immunization with SSB epitope alone or with dsDNA. The SSB peptide antibody titer in the coimmunization group was higher than that of the SSB alone group. In addition, antibodies against ribonucleoprotein (RNP), Smith and/or dsDNA were detected in rabbits of the coimmunization group. The presence of anti-dsDNA antibodies in the rabbits immunized with SSB peptide suggested the induction of epitope spreading. In conclusions, SSB antibodies were produced in rabbits immunized with SSB peptide or SSB+dsDNA, whereas SSB antibody titers were higher in the coimmunization group. Furthermore, coimmunization was associated with epitope spreading.

Erratum: Antibody production in rabbits administered Freund's complete adjuvant and carprofen concurrently

Erratum: Antibody production in rabbits administered Freund's complete adjuvant and carprofen concurrently

Antibody production in rabbits administered Freund's complete adjuvant and carprofen concurrently.

Freund's complete adjuvant (FCA) is a commonly used immunopotentiator that can boost polyclonal antibody production in animal models such as rabbits, but FCA is also known to cause inflammation and pain. It is important to balance the welfare of animals with the goal of efficiently producing antibodies, but little is known about how common treatments for pain and inflammation, such as non-steroidal anti-inflammatory drugs (NSAIDs), affect the production of polyclonal antibodies. The purpose of this study was to measure polyclonal antibody production in rabbits that were administered FCA either with or without a concurrent treatment of a NSAID, carprofen. Rabbits were divided into two groups and were administered identical treatments of an antigen with adjuvant, and the treatment group also received carprofen injections at different stages of the study. Carprofen treatment did not significantly affect polyclonal antibody production, which suggests that carprofen and other NSAIDs can be used alongside FCA in rabbits to achieve desired levels of antibody production while minimizing pain and distress associated with the use of FCA.

Development of CpG-Oligodeoxynucleotides for Effective Activation of Rabbit TLR9 Mediated Immune Responses

CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18–24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund’s adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

P151 Ligand recognition profiles of TLR9 from different species

Bacterial and viral DNA are potent stimuli to immune cells. Synthetic CpG-oligodeoxynucleotides (CpG-ODN) mimic the stimulatory effect of these microbial DNA in activation of immune cells. The activity of a CpG-ODN is determined by its sequence context including its length, the number of CpG motifs, and the spacing, position, and surrounding bases of these motifs. Toll-like receptor 9 (TLR9) is the cellular receptor for these CpG-ODN. Previous studies have shown different ligand recognition profiles of mouse TLR9 and human TLR9. Rabbits are commonly used for production of antibodies. Although CpG-ODN have been shown to be a safety and effective adjuvant in boosting antibody production in rabbits, the interaction between CpG-ODN have not yet investigated. In the present study, we cloned and characterized rabbit TLR9 cDNA. We further established cell-based activations with this rabbit TLR9 cDNA, and cDNAs for mouse and human TLR9 to comparatively investigate activation of these TLR9s. The complete open reading frame of rabTLR9 encodes 1028 amino acid residues, which share 70.6% and 75.5% of the identities of mTLR9 and hTLR9, respectively. Similiar to that of human TLR9, rabbit TLR9 is preferentially expressed in immune cells riched tissues, and is localized in intracellular vesicles. While mTLR9 and hTLR9 displayed species-specific recognition of their optimized CpG-ODN, rabbit TLR9 was activated by these CpG-ODN without any preference. The results indicated a different ligand recognition profiles of these TLR9 from these species.

Cytoplasmic expression, antibody production, and characterization of the novel zinc finger protein 637

Zinc finger protein 637 (zfp637), belonging to the Kruppel-like protein family, comprises one atypical C(2)H(2) and six consecutive typical zinc finger motifs. Based on the structural characterization of zfp637 and its location in the cell nucleus, we predict that zfp637 might function as a DNA-binding protein to regulate gene transcription. However, the absence of both a purified zfp637 protein and any commercial antibody for detecting it in cells and tissues has limited functional studies of zfp637 to date. Here, we developed and optimized an expression system by fusing zfp637 with glutathione S-transferase (GST) to achieve a maximal yield of soluble GST-zfp637 fusion protein in Escherichia coli BL21(DE3) cells. The yield was about 10 mg/l of the original bacterial culture. The recombinant GST-zfp637 fusion protein was purified and used for polyclonal antibody production in rabbits. In addition, we developed a method to remove the anti-GST antibody component and obtained a highly purified anti-zfp637 antibody, as demonstrated by an enzyme-linked immunosorbent assay. Western blotting showed that the anti-zfp637 antibody recognized not only the recombinant zfp637 protein but also endogenous zfp637 in several cell lines. The protein was localized mainly in the cell nucleus by immunofluorescence and immunohistochemistry. The expression levels of zfp637 mRNA and protein were significantly increased in NIH3T3 cells treated with 200 μM of H(2)O(2) in a time-dependent manner. The recombinant GST-zfp637 fusion protein and our purified anti-zfp637 antibody will help in elucidating the function of zfp637.

Production of rabbit antibodies against purified Glucose oxidase

Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti-rVP28 antiserum to detect the presence of the virus in WSSV-infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR-positive WSSV-infected shrimp samples. Immunodot blot was found to be equivalent to first-step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 105 viral DNA copies.

Dendrigraft Poly-l-lysine: A Non-Immunogenic Synthetic Carrier for Antibody Production

An easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens.

Expression of Pisum sativum SAD polypeptides in production hosts and in planta: Tetrameric organization of the protein

In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members ( SAD-A, -B, and -C). Expression of two of these genes ( SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody production in rabbits. With these antibodies the recombinant SAD-C protein (which was most highly expressed of the two isoforms) was shown to be a tetramer consisting of a dimer of dimers. The SAD genes are transiently expressed in plants by short exposures to ultraviolet-B radiation (UV-B), as judged by northern blotting. In turn, mRNA accumulation leads to formation of SAD protein in leaf and stem tissue upon prolonged UV-B irradiation.

Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli

Tristetraprolin (TTP/ZFP36) family proteins are anti‐inflammatory. They bind and destabilize some AU‐rich element‐containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over‐expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. This antiserum, and an antiserum against recombinant mouse TTP, was used to detect ZFP36L1 and TTP in mouse 3T3‐L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed, but TTP was induced by insulin and cinnamon and not by lipopolysaccharide (LPS) in 3T3‐L1 adipocytes. In contrast, ZFP36L1 was undetectable but TTP was strongly induced in LPS‐stimulated RAW264.7 cells. Quantitative real‐time polymerase chain reaction (PCR) confirmed higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW264.7 cells. Low levels of ZFP36L1 expression were also confirmed by northern blotting in mouse embryonic fibroblasts. These results demonstrate that TTP and ZFP36L1 are differentially expressed at the mRNA and protein levels in mouse adipocytes and macrophages (supported in part by the Intramural Research Program of the NIH, NIEHS and USDA‐ARS Human Nutrition Research Program).

Production and Characterization of ZFP36L1 Antiserum against Recombinant Protein from Escherichia coli

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was overexpressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed with a size corresponding to the lower mass size of ZFP36L1 expressed in transfected human embryonic kidney 293 cells, but TTP was induced by cinnamon extract and not by lipopolysaccharide (LPS) in adipocytes. In contrast, ZFP36L1 was undetectable, but TTP was strongly induced in LPS-stimulated RAW cells. Quantitative real-time polymerase chain reaction confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW cells. Low levels of ZFP36L1 expression were also confirmed by Northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein and that TTP and ZFP36L1 are differentially expressed and regulated at the mRNA and protein levels in mouse adipocytes and macrophages.

SSB peptide and DNA co-immunization induces inhibition of anti-dsDNA antibody production in rabbits

Patients with systemic lupus erythematosus often have various autoantibodies. The relationship between these antibodies is still poorly understood. The aim of the present study was to observe the anti-SSB antibody and anti-dsDNA antibody production profiles following immunization with synthetic SSB peptide alone, DNA alone or co-immunization with these two antigens. SSB 214 - 225 aa peptide was synthesized by organic chemistry solid-phase peptide synthesis. Rabbits were immunized with the following antigens: synthetic SSB peptide linked with keyhole limpet hemocyanin (KLH), DNA, SSB plus dsDNA, KLH and PBS. Antibodies were measured by ELISA. Histopathology and direct immufluorescence assays were also applied. Anti-SSB and anti-dsDNA antibodies were produced following immunization with SSB peptide and DNA respectively. The level of SSB antibody in the co-immunization group was higher than that of the SSB peptide immunization group. The level of anti-dsDNA antibody in the co-immunization group was, however, lower than that in the DNA immunization group. Meanwhile, the level of anti-SSB antibody was higher than that of anti-DNA antibody in the co-immunization group. No morphological or immunological abnormalities were found in the heart, liver, kidney, spleen or skin tissues. Inhibition of anti-dsDNA-antibody was induced by co-immunization with synthesized SSB peptide and DNA, which might explain, at least partly, the mild disease in some LE subsets associated with SSB antibody.