Immunochemical assay for recognition of 2-S-glutathionyl acetate, a glutathione conjugate derived from 1,1-dichloroethylene-epoxide

Cytotoxicities induced by 1,1-dichloroethylene (DCE) are ascribed to cytochrome P450-dependent metabolism to an epoxide. Conjugation of the DCE-epoxide with glutathione (GSH) results in the formation of the conjugates 2-S-glutathionyl acetate (GTA) and 2-(S-glutathionyl) acetyl glutathione (GAG); GAG undergoes hydrolysis to form GTA, and thus GTA is a major metabolite of DCE metabolism. Our objective is to develop an antiserum against the chemically synthesized GTA, and for immunization, we have used a hapten that consists of GTA conjugated to bovine serum albumin (BSA) as the carrier protein and glutaraldehyde (GLUT) as a chemical cross-linker. The antisera were raised in rabbits and were characterized by using the following synthesized structural analogs: GTA, glycine-GLUT-BSA (GLY-GLUT-BSA), GTA-GLUT-ovalbumin (GTA-GLUT-OVB), GTA-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-BSA (GTA-EDC-BSA), TRIS-GLUT-BSA, glutathione-GLUT-BSA (GSH-GLUT-BSA). The enzyme-linked immunosorbent assay (ELISA) and slot immunoblotting were used to characterize the specificity of the antisera. Noncompetitive ELISA experiments showed that the reaction of the antiserum with the antigen was concentration-dependent. In the competitive ELISA, GTA-GLUT-BSA inhibited binding efficiently; in contrast, the unconjugated GTA did not inhibit binding to the antigen. Competitive studies with the other analogs indicated low or minimal reactivities with the antibodies, which were blocked by incubation with GLY-GLUT-BSA. However, there was residual reactivity with the antigen that was not competitively inhibited by either the GTA-EDC-BSA or the GSH-GLUT-BSA conjugates. Slot-blotting experiments confirmed the findings of the ELISA studies and revealed high specificity of the antiserum to detect the hapten. These results demonstrated the successful development of polyclonal antibodies to detect GTA and hence DCE-epoxide.     

Detection of benzoquinone adducts to rat liver protein sulfhydryl groups using specific antibodies

Benzoquinone is an electrophilic metabolite of bromobenzene and other simple aromatic compounds of toxicological interest including benzene, phenol, hydroquinone, and acetaminophen. In reacting with proteins benzoquinone shows great selectivity for Michael addition to sulfhydryl groups and formation of S-(2,5-dihydroxyphenyl) protein adducts. To facilitate the specific detection and eventual isolation and identification of such adducted proteins, we prepared an antiserum capable of recognizing hydroquinone moieties by immunizing rabbits with keyhole limpet hemocyanin modified with 3-[2,5-dihydroxyphenyl)thio]propanoyl groups as haptens. The antiserum had a high titer and showed high specificity for hapten in competitive ELISA with hapten analogues. In Western blot experiments the antiserum detected not only synthetically haptenized control proteins but also several proteins from rat liver microsomes that had been incubated in vitro with [14C]bromobenzene. This binding was completely blocked by free hapten, showing that it was hapten-specific. Each of the microsomal protein bands detected in the Western blots also contained radioactivity, but not all radioactive protein bands reacted with antibody. This antiserum should prove useful in exploring the role of protein arylation by benzoquinone in cytotoxic responses to its metabolic precursors.     

Use of the protective antigen of Erysipelothrix rhusiopathiae in the enzyme-linked immunosorbent assay and latex agglutination

To establish a safe and convenient serodiagnostic method for swine erysipelas, a purified protective protein antigen of Erysipelothrix rhusiopathiae, which included a large amount of protective protein (64 kDa protein), was used for enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) test. In the ELISA, the antisera to four different serovars (1a, 2, 5 and 20) of E. rhusiopathiae exhibit a positive reaction, while antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) exhibit a negative reaction. In the LA test, the antisera to three different serovars (1a, 2 and 5) of E. rhusiopathiae reacted with P64-sensitized latex beads, while the antiserum to serovar 20 (2553 strain) did not. Moreover, the antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) did not in this test. Comparing the results of the growth agglutination (GA), ELISA and LA tests of 284 swine sera, there was a high degree of correlation among the results. The detection of anti-E. rhusiopathiae antibodies in the GA, ELISA and LA tests were compared using sera from pigs immunized with P64, alkaline extract (AE) and live-cell vaccine (LV). In all three tests, anti-E. rhusiopathiae antibodies could be detected 1 week after immunization. The serum antibody titre as determined by the LA test increased moderately, as did that by the GA test, while that determined by ELISA increased rapidly. These results suggested that ELISA could be used to monitor changes in anti-E. rhusiopathiae antibody titre and the LA test could be used in the screening test for swine erysipelas.     

Baculovirus expression of the fusion protein gene of bovine respiratory syncytial virus and utility of the recombinant protein in a diagnostic enzyme immunoassay

The fusion (F) protein of bovine respiratory syncytial virus (BRSV) was expressed by using a baculovirus vector. Antigenicity was tested by immunofluorescence analysis with F-specific monoclonal and polyclonal antibodies. Antibodies to recombinant F protein raised in a rabbit neutralized BRSV and human respiratory syncytial virus infectivity when tested in a plaque reduction assay. The recombinant F protein was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA), and this ELISA was compared with the virus neutralization (VN) test for detecting BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA with the baculovirus-expressed F protein as an antigen compared favorably with the VN test and is a rapid, sensitive, and specific method for detecting serum antibodies to BRSV.     

Use of the recombinant 38-kDa antigen of Mycobacterium tuberculosis as an immunogen for specific antisera production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Characterization of variable immunodominant antigens of Cowdria ruminantium by ELISA and immunoblots

Cross-immunization experiments have revealed a significant antigenic diversity of the isolate of Cowdria ruminantium which needs to be characterized for the development of vaccines. We identified polymorphic immunodominant antigens by ELISA and immunoblot. Using serum from a goat immune to the Gardel stock of Cowdria (isolated in Guadeloupe) adsorbed on antigen of the Senegal stock of this pathogen, distinct serogroups were revealed by ELISA among six isolates from different geographical origins. Furthermore, a goat serum directed against the Senegal stock and adsorbed on Gardel antigens was shown to be specific for the Senegal stock, thus confirming the existence of serotypes in Cowdria. The Major Antigenic Protein 1 (MAP1) of Cowdria was shown to have variable antigenic determinants. Also in a group of variable proteins ranging from 23 to 29 kDa, one antigen of 26-27 kDa had a determinant specific for the Gardel isolate. These polymorphic antigens may be relevant components of Cowdria ruminantium for a vaccine as the sera revealing these antigens originated from a goal surviving a lethal challenge. However, the presence of T-cell epitopes and the ability of the these antigens to confer protection to ruminants remain to be investigated. The production of a rabbit antiserum against this group of polypeptides will be of great use for their purification and for the screening of expression libraries.     

Ischaemic preconditioning: present position and future directions

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over the time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the fallen antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54-95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA-peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA-peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54-95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH-deficient rats. The growth-promoting effect of bGH was significantly enhanced by mixing with bovine anti-peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54-95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.     

Development of an ELISA for pantothenic acid (vitamin B5) for application in the nutrition and biological fields

Immunological assays appear to be the only alternative to the microbiological method for analysis of pantothenic acid in foods and blood. In order to evaluate the influence of the linker on the immunogenicity of the hapten, we have tried to raise antisera against pantothenic acid in rabbits using different conjugates. The hapten was coupled to a carrier protein (BSA or thyroglobulin) using adipoyl dichloride (adipoyl conjugate) or bromoacetyl bromide (acetyl conjugate). Only the acetyl conjugate has induced the production of a specific antibody. With this antibody, an assay on microplate using the ELISA inhibition technique was developed to measure pantothenic acid. The use of pantothenic acid coupled to thyroglobulin with adipoyl dichloride as the capture antigen has improved the sensitivity of the ELISA. This assay was applied to food products and blood.     

Isolation, characterization and comparison of antipeptide and antiprotein rabbit antibodies to the pi-isoform of glutathione S-transferase

The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-pi-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-pi-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-pi-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of pi-GST.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus. This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis.     

Hepatitis B and C viruses among Egyptian dentists

The present study was conducted on 105 subjects, 70 dentists working or studying at the Faculty of Oral and Dental Medicine, Cairo University, and 35 non medical non dental subjects. There were 47 males and 48 females, their ages ranged from 20-50 years. Detailed history and the required information were collected from each subject and were recorded in a specially a prepared questionnaire study cards. 10 C.C. of venous blood were obtained from each candidate using Venoject, sera were separated from clotted blood by centrifuge; their sera were tested for Hepatitis B surface antigen (HBsAg), Antibody to hepatitis B surface antigen (anti HBs), Antibody to hepatitis B core antigen (anti HBc) and Antibody to hepatitis C virus antigen (anti HCV) using ELISA (enzyme linked immuno sorbent assay) techniques according to Abbott Laboratories (West Germany). The ALT level (Alanine aminotransferase) was determined only for HBsAg and anti HCV positive cases using commercial Biomérieux kits (France). After statistical analysis of the results, the exposure rate of HBV among dentists was found to be 27.1% with a carrier rate of 7.1% compared to 31.4% with a carrier rate of 17.1% in the control group. The exposure rate of HCV infection among dentists was 1.4% compared to 17.1% in the control group. The exposure rate of HBV and HCV infections were 2.9% compared to 5.7% in the control group; these results and other important conclusions were adequately discussed.     

Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.     

Shigellosis in children: a clinico-epidemiological comparison between Shigella dysenteriae type I and Shigella flexneri

A new method was developed to measure the content of a Lumbricus component in a traditional Chinese medicine (TCM). An antiserum specific to Lumbricus was elicited in a rabbit following immunization with a suspension of Lumbricus fragments. A characteristic antigen protein, 70 kDa, was found in Lumbricus and was purified almost to singleness using a column chromatography series of gel filtration and DEAE-Sepharose. A selected antibody enzyme immunoassay (SAEIA) was developed using the antiserum and the purified 70 kDa protein as a solid-phase antigen. The SAEIA was specific to Lumbricus species, and showed no cross-reaction with any crude drugs other than Lumbricus. This SAEIA detected 70 kDa protein in the amount of 10 ng/ml with excellent reproducibility (coefficient of variation=3.0%) and an EC50 of 0.24 microg/ml. Using this assay, Lumbricus levels were easily determined in a Lumbricus-based TCM Kazecoll, but not in the control Kazecoll (Kakkonto) prepared without Lumbricus. The SAEIA for 70 kDa protein was simple, accurate, reproducible and may provide a general analytical method for the quality control of Lumbricus-based TCMs.     

Characterization of binding site of uremic toxins on human serum albumin

The interaction of uremic toxins including indole-3-acetic acid (IA), indoxyl sulfate (IS), hippuric acid (HA) and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) with human serum albumin (HSA) has been investigated by three methods of fluorescent probe displacement, ultrafiltration and equilibrium dialysis. The binding parameter of CMPF was found to have the strongest affinity (10(7)M-1) among all the uremic toxins studied. Competitive experiment based on the method of Kragh-Hansen suggested that IA, IS and HA bind to site II, whereas CMPF binds to site I. The present limited data indicated that the four uremic toxins caused inhibition to any endo- or exogenous substances on HSA.     

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification

The preparation and characteristics of compound-specific and group-specific antibodies against 7-alkylguanines (7-alkGua) are described. A compound-specific antibody against 7-methylguanine was prepared using a hapten bound to carrier protein through the N2 position. In a competitive enzyme-linked immunosorbent assay (ELISA) 7-methylguanine (7-MeGua) showed 50% inhibition (I50%) at 10 pmol/well at room temperature, but the inhibition was found to be 40 times better at 4 degrees C (I50% at 250 fmol/well). When the antibody was bound to protein A-Sepharose CL4B 7-MeGua was retained in immunoaffinity columns. A group-specific antibody to 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua) bound to carrier protein via the carboxyl group. In a competitive ELISA, this antibody cross-reacted well with 7-CEGua, 7-ethylguanine (7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine (7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinity columns prepared from this antibody retained a number of 7-alkGua of diverse structure. 7-EtGua in calf thymus DNA treated with diethylsulphate and ethylnitrosourea was isolated by immunoaffinity purification and quantified by HPLC-fluorescence. These results illustrate the potential of immunoaffinity purification for both individual DNA adducts and groups of adducts.     

Radioimmunoassay of oestrone in plasma. A comparison of different methods with respect to the relation between assay specificity, sample purification and antibody specificity

A radioimmunoassay (RIA) for oestrone (Oe1) in plasma was developed using an ether extraction, a partition between NaOH and light petroleum, and a TLC as sample purification and an antiserum cross-reacting with Oe2 for the final assay (Method A1). The reliability criteria are given in detail. In order to simplify this method a highly specific antiserum was developed by using Oe1-3-hemisuccinate-BSA as an antigen. Using this antiserum for the final assay but omitting the TLC (Method B) the Oe1 concentration in male plasma was 76% overestimated (Method B compared with Method A1). In pregnancy plasma Oe1 could specifically be determined after a simple ether extraction (Method C). It was concluded that the use of a highly specific antiserum (as determined by cross-reaction studies) for the final assay does not necessarily imply that a chromatographic sample purification can be omitted without loss in assay specificity. This appears to be true mainly in cases where the steroid concentration of the sample is low. Normal values of Oe1 from 80 healthy adult males were ascertained by Method A1. Age group I (22-61 years, n = 48) ranged from 1.22-5.60 ng/100 ml, median 2.81; age group II (67-90 years, n = 32) from 1.55-6.40, 100 median 3.41. The small increase of Oe1 with age was highly significant.     

The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella

The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.     

Nosocomial pneumonia

The phosphoromonothioate oligonucleotide HPV (human papilloma virus) sequence (monothioate HPV) 5'-TTG,CTT,CCA,TCT,TCC,TCG,TC-3' was photocoupled via three different sites (the 5'-end, the 3'-end and the midpoint) to PPD (purified protein derivative) and OA (ovalbumin), and the three types of conjugates (5'-HPV/carrier, 3'-HPV/carrier and midpoint-HPV/carrier) were used for the immunization of mice. Furthermore, a group of mice were immunized with the HPV sequence alone. No detectable antibody response against the monothioate HPV oligonucleotide was seen in mice receiving only the unconjugated monothioate HPV sequence. The OA-coupled monothioate HPV sequence also failed to elicit a detectable antibody response against the monothioate HPV oligonucleotide. However the PPD-conjugated monothioate HPV sequences induced a significant anti-monothioate HPV antibody response in BCG (bacille Calmette Guérin)-primed mice, a result that must be ascribed to the effect of using PPD as a carrier in BCG-primed mice. The antisera from all groups were tested on plates coated with the corresponding OA conjugates. By far the strongest response was obtained in mice receiving the HPV sequence coupled at the midpoint position. Further, all three groups of antisera obtained by immunizing with the different PPD conjugates were tested on microtiter plates coated with one of the three different OA conjugates. The antisera differed in their response depending on which OA conjugate was used for coating of the plate. Again, the midpoint-HPV/PPD antiserum showed the highest response, and this conjugate apparently represents the most efficient immunogen. Results from inhibition experiments with various relevant analogs of the monothiate HPV sequence showed that the three antiserum pools contained antibodies predominantly directed against the conformation of the monothioate backbone structure, but that at least a subpopulation of the antibodies recognized structures, which depended on the specific HPV base sequence.     

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.     

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.