An epitope, or antigenic determinant, was defined as the site on an antigen at which an antibody binds, by virtue of the antibody's antigen-combining site (called the paratope) 1. The word epitope derives from the Greek epi, meaning "upon", and topos, or "place", and thus it is the place on the antigen upon which the antibody binds.
As antigens can be recognised by two distinct groups of receptor molecules of the immune system, namely antibodies (Ab's) or T-cell receptors (TCR's), we need to distinguish whether we are talking about epitopes defined by antibodies or by TCR's. This article will deal only with antibody-defined epitopes. See T-Cell Epitope Mapping for a guide to mapping of peptide epitopes defined by TCR's.
The primary structure (amino acid sequence) of many proteins is now known. However, for most of them, the detailed 3- dimensional structure is unknown. The 3-D structure of proteins is only readily solved by crystallographic methods, such as X- ray crystallography, although the rapid development of NMR methods may soon provide an alternative source of such structural information. Mapping epitopes by solving the structure of antigen-antibody complexes using crystallography is impractical in the vast majority of cases, because antigen- antibody crystals are very difficult to make. Therefore, a number of other methods are used to deduce the nature of individual epitopes 2,3,4,5. One of these methods is the use of synthetic fragments (peptides) of the protein antigen, which can be similar enough to the homologous parts of the whole antigen to permit binding by the antibody. For this method to be practical, the affinity of the antibody for the peptide has to be such that the peptide/antibody complex does not dissociate significantly under the conditions of an immunoassay. This situation occurs with linear epitopes, thus allowing the use of peptides to define those epitopes.
Based on our unpublished epitope mapping studies with monoclonal antibodies, it appears that approximately 5-10% of all antibodies directed to native antigens bind to linear epitopes. For those antibodies, and for polyclonal antisera in general, Multipin Peptide Synthesis Technology provides a rapid, cost-effective and adaptable means of identifying linear epitopes 6.
In the following sections, strategies for epitope location and characterization on protein antigens will be outlined. Clearly, there are two prime requisites before epitopes can be mapped using synthetic peptides:
1. The amino acid sequence of at least part of the protein antigen must be known. Sequences are available for a vast number of antigens due to the sequencing of cloned genes, and for some antigens via protein sequencing.
2. A defined antibody population with specificity for that antigen must be available. The cleanest examples of such antibody populations are monoclonal antibodies (MAb's). "Polyclonal" antisera can be used, particularly if they are from animals/subjects hyperimmune to the particular antigen in question. Other antibody preparations worthy of study include: affinity-purified fractions from sera, sera depleted of particular (unwanted) antibody specificities, and defined (classified) sets of sera from unimmunized subjects. These defined sets of sera can be identified in a conventional serological test, and can be used to establish a correlation between positives in that serological test and binding to peptide epitope(s). Control antibody preparations should always be studied in parallel wherever possible.
The first and simplest approach to defining linear epitopes of a protein antigen is referred to simply as "scanning" 6. This scanning strategy has two distinct phases. The first phase allows location of the area of the sequence in which an epitope is to be found. The second phase defines the limits or boundaries of each epitope at a resolution of a single amino acid, i.e. indicates which parts of an antibody-binding peptide are "inside" and "outside" the epitope.
The General Net or "Gnet" synthesis consists of a set of peptides designed to catch all the antibody-binding regions of a protein (hence the term "net"). All overlapping peptides of a defined length, homologous with the protein, are synthesised. The use of highly overlapping peptides ensures that epitopes are not missed by being "cut" at a critical point, which could easily occur if abutting rather than overlapping sequences were made. As long as the peptides are of sufficient length, no linear epitopes should be missed. One approach is to synthesize overlapping octamers (8mers), offset by one residue. The terms offset and overlap are often confused, overlapping residues are those common to two peptides covering an area of a protein sequence while the offset is the distance in residue number between the N-terminal ends of two overlapping peptides. For example the sequences:
ACDEFGHI and CDEFGHIK
overlap by seven and are offset by one.
ACDEFGHI and EFGHIKLM
overlap by five and are offset by three.
For long protein sequences, where cost of peptide synthesis is a major consideration, a larger offset e.g. 2 to 4 residues, can be used. This reduces the number of peptides which need to be made. In this case, we would recommend longer peptides e.g. 9- to 11-mers respectively, to eliminate the chance of cutting an epitope at a critical point. With the use of longer peptides/larger offsets, there will be a loss of resolution of individual epitopes.
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This—the evolution of man into superman—was always the purpose of the ancient Mysteries, and the real purpose of modern Masonry is, not the social and charitable purposes to which so much attention is paid, but the expediting of the spiritual evolution of those who aspire to perfect their own nature and transform it into a more god-like quality. And this is a definite science, a royal art, which it is possible for each of us to put into practice; whilst to join the Craft for any other purpose than to study and pursue this science is to misunderstand its meaning. Hence it is that no one should apply to enter Masonry unless from the deepest promptings of his own heart, as it hungers for light upon the problem of its own nature. We are all imperfect beings, conscious of something lacking to us that would make us what, in our best moments, we fain would be. What is that which is lacking to us? "What is that which is lost?" And the answer is "The genuine secrets of a Master Mason," the true knowledge of ourselves, the conscious realization of our divine potentialities.
character: one the nature of which can hardly be made intelligible, or even credible, to those unfamiliar with the subject. I refrain, therefore, from more than mere mention of it, observing only that it is one not involving physical death, and in this respect only is our ceremony in accord with the experience symbolized. For if you follow closely the raising ceremony, although distinct reference to the death of the body is made, yet such death is obviously intended to be merely symbolical of another kind of death, since the candidate is eventually restored to his former worldly circumstances and material comforts, and his earthly Masonic career is not represented as coming to a close at this stage. All that has happened in the third degree is that he has symbolically passed through a great and striking change: a rebirth, or regeneration of his whole nature. He has been "sown a corruptible body"; and in virtue of the self-discipline and self-development he has undergone, there has been raised in him "an incorruptible body," and death has been swallowed up in the victory he has attained over himself. I sometimes fear that the too conspicuous display of the emblems and trappings of mortality in our Lodges is apt to create the false impression that the death to which the third degree alludes is the mere physical change that awaits all men. But a far deeper meaning is intended. The Mason who knows his science knows that the death of the body is only a natural transition of which he need have no dread whatever; he knows also that when the due time for it arrives, that transition will be a welcome respite from the bondage of this world,
other than a spark of God Himself immanent within us. Over the old temples of the Mysteries was written the injunction "Man, know thyself, and thou shalt know the universe and God." Happy then is the Mason who has so far purified and developed his own nature as to realize in its fulness the meaning of the "sacred symbol" of the second degree, and found God present not outside but within himself. But in order to find the "perfect points of entrance" to this secret (and we are told elsewhere that "straight is the way and narrow the gate, and few there be that find it") emphasis again is laid in our teaching upon the necessity of complete moral rectitude, of utter exactness of thought, word and action, as exemplified by rigid observance of the symbolic principles of the square, level and plumb-rule.
Brethren, I charge you to regard your apron as one of the most precious and speaking symbols our Order has to give you. Remember that when you first wore it it was a piece of pure white lambskin; an emblem of that purity and innocence which we always associate with the lamb and with the newborn child. Remember that you first wore it with the flap raised, it being thus a five-cornered badge, indicating the five senses, by means of which we enter into relations with the material world around us (our "five points of fellowship" with the material world), but indicating also by the triangular portion above, in conjunction with the quadrangular portion below, that man's nature is a combination of soul and body; the three-sided emblem at the top added to the four-sided emblem beneath making seven, the perfect number; for, as it is written in an ancient Hebrew doctrine with which Masonry is closely allied, "God blessed and loved the number
The next phase of the strategy, called a Window net or "Wnet", is to identify the precise boundaries of the epitope(s) located from a Gnet scan. The Window net terminology comes from the concept of this process as "looking at the linear protein sequence through a series of moving windows of different sizes". This involves the synthesis of all of the shorter overlapping sequences covering antibody-binding peptides identified in the Gnet synthesis, e.g. 4mers, 5mers, 6mers and 7mers could be made in a Wnet if the Gnet scan had been done at 8mer length.
Example: The strongly-binding area of sperm whale myoglobin as described above was used as the basis for synthesizing every overlapping 4-, 5-, 6-, and 7-mer starting on residues 118 to 125 of myoglobin:
Myoglobin sequence ...118RHPGDFGADAQGAM131...
Peptide 1: RHPG
Peptide 2: RHPGD
Peptide 3: RHPGDF
Peptide 4: RHPGDFG
Peptide 5: HPGD
Peptide 6: HPGDF
Peptide 7: HPGDFG
Peptide 8: HPGDFGA
Peptide 29: ADAQ
Peptide 30: ADAQG
Peptide 31: ADAQGA
Peptide 32: ADAQGAM
The result of testing these peptides with the same rat antiserum as scanned in the Gnet test show that the sequence of the shortest peptide to bind strongly was the pentapeptide starting on residue 122, DFGAD. All the longer peptides containing this pentapeptide also strongly bound antibodies from this polyclonal serum, showing that DFGAD comprised a dominant linear epitope as defined by this serum.