Investigation of autoantibody profiles for cerebrospinal fluid biomarker discovery in patients with relapsing-remitting multiple sclerosis.

Using the UNIarray® marker technology platform, cerebrospinal fluid immunoglobulin G reactivities of 15 controls and 17 RRMS patients against human recombinant proteins were investigated. Patient cerebrospinal fluids were oligoclonal band positive and reactivities were compared to that of sex- and age-matched controls. We hereby aimed at the characterization of autoreactivity in patients with RRMS. Differences in autoreactivities between control and RRMS samples were identified comprising autoantigens identified in this study only and previously reported autoantigens as well. A combination of the 10-15 most significant proteins may be investigated further as autoantigens for diagnostic purposes. Additional investigations may include minimizing the number of proteins used in such diagnostic tests.

Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography.

The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended.

A mouse model for ricin poisoning and for evaluating protective effects of antiricin antibodies.

UNLABELLED: BACKGROUND. Ricin is a potential bioterrorism agent and no specific antidote or treatment exists for ricin poisoning. For this reason, we developed ricin-specific antibodies that were tested in a murine model of ricin poisoning for use as antidotes against symptoms of ricin poisoning. METHODS: Mice were poisoned with a lethal dose of ricin (5 microg) and their temperature and general condition were monitored for determination of a surrogate and humane end point. Mice were then treated with injections of ricin and different combinations of polyclonal anti-ricin antibodies. Antibody effect was evaluated for various doses and using various time points from ricin to antibody injection. Also, the effect of adjuvant symptomatic treatment was examined. Brain, heart, intestines, kidney, liver, lung, pancreas, spleen, and stomach tissues were sampled for histopathological analysis. RESULTS: The mouse model was reproducible and easy to use. A clear protective effect of both anti-ricin A-chain and anti-ricin B-chain antibodies-but not of irrelevant antibodies-was demonstrated with no added effect of symptomatic treatment. CONCLUSIONS: These data suggest that specific polyclonal antibodies against ricin A- and B-chain may reproducibly protect mice against ricin poisoning, even when the antibodies are administered up to 1.5 h after poisoning.

Extraction and identification of electroimmunoprecipitated proteins from agarose gels.

A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.

Effect of different hapten-carrier conjugation ratios and molecular orientations on antibody affinity against a peptide antigen.

Hapten-carrier conjugate immunization is an important tool in the generation of hapten-specific antibodies for analytical purposes and in the uncovering of basic vaccine-immunological mechanisms. The affinity of antibodies is known to play an important role for the resulting sensitivity of antibody-based assay systems and in deciding whether a vaccine-induced antibody response will be protective. With ovalbumin as a carrier protein and a peptide (7.2 NY) representing a 19 amino acid sequence from the E. coli-derived Verotoxin 2e as a model hapten we investigated whether it was possible to influence the affinity and titre of antibodies raised against the hapten using different conjugation ratios and orientations. The peptide was coupled to ovalbumin in four conjugation ratios and two molecular orientations - terminal and central - and the conjugates were verified by mass spectrometry. Mice were immunised ten times at two-weeks intervals with low doses of the eight conjugates. Blood samples collected between each immunisation were analysed by ELISA for specific antibody titres and relative affinities. With both types of conjugations, the anti-peptide antibody titres increased in response to increasing conjugation ratios, but central conjugation resulted in markedly higher titres than terminal conjugation. The overall anti-peptide antibody affinities reached approximately similar levels with both orientations, whereas a reversed proportionality was observed between conjugation ratio and antibody affinity for terminal conjugation. Thus, it appears that the molar ratio of a peptide and its carrier may affect the resulting antibody affinities, and that a conjugation ratio between a terminally conjugated peptide and its carrier approaching one will result in relatively high antibody affinities. Furthermore, the molecular orientation of the coupled peptide has a major effect on the anti-peptide antibody titres induced.

[Botulinum and ricin toxins]. / Botulinum- og ricintoksiner.

Due to their acute toxicity, accessibility and history of use, ricin and botulinum toxins are at the present time the most relevant bioterror toxins. We give a brief review of their toxicity and possible uses in bioterror attacks and describe two cases in Denmark in which immunochemical, PCR and mass spectrometric assays developed in-house were used to confirm a foodborne botulinum toxin E case and to identify bovine serum albumin as the powder enclosed in a letter sent to the U.S. Embassy in Copenhagen.