Methodology of drug trials in migraine: History and suggestions for the future.

There is a multitude of scientific papers and guideline papers on the methodology of drug trials in migraine. Here, we try to condense this into a single paper and to make proposals for future consideration. Literature known by the authors and from reference lists of relevant publications was used for the history.

Auto-immuno-deficiency syndromes.

Autoimmune diseases constitute a broad, heterogenous group with many diverse and often overlapping symptoms. Even so, they are traditionally classified as either systemic, rheumatic diseases or organ-directed diseases. Several theories exist about autoimmune diseases, including defective self-recognition, altered self, molecular mimicry, bystander activation and epitope spreading.

Cytochemical and Histochemical Staining with Peptide Antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/absorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Polyclonal Peptide Antisera.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages.The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare.

Solid Phase Peptide Carrier Conjugation.

Conjugation to carrier proteins is necessary for peptides to be able to induce antibody formation when injected into animals together with a suitable adjuvant. This is usually performed by conjugation in solution followed by mixing with the adjuvant. Alternatively, the carrier may be adsorbed onto a solid support followed by activation and conjugation with the peptide by solid-phase chemistry.

Peptide Antibodies: Current Status.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry.