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Publisher Summary This chapter presents an overview of the workshop analyzing homocytotropic antibodies. It highlights that the word reagin has been used in the past but recently the designation homocytotropic has been proposed. The chapter discusses the passive transfer experiment by Prausnitz and Kustner which shows that human reagins fix to human skin. This is a unique property of reaginic antibody and also of IgE. Very small amounts of IgE antibody are needed; as little as 0.4 ng of AbN/ml. In contrast, 30 ng of AbN/ml of rabbit IgG antibody is the minimal dose capable of eliciting passive cutaneous anaphylaxis (PCA) in guinea pigs. An IgE myeloma protein, ND, can inhibit passive sensitization in very small doses, and this property is retained in the 5S Fc fragment obtained by papain digestion. Reaginic antibodies are retained in the skin after injection for a considerable time. A half-life of about 14 days has been found. In contrast, turn-over studies of lableed IgE indicate a half-life for circulating IgE of 2–4 days. Little is known about the affinity of IgE and especially whether or not this affinity is the same for all IgE molecules.

RECENTLY, Osserman and Takatsuki1 have described a group of γG myeloma proteins the Fc fragment of which was completely destroyed by the action of papain. A similar protein (myeloma N) was independently examined in this laboratory. This protein was found to be susceptible to papain and trypsin2.

Crystals of the plasmin Fc fragment of human immunoglobulin G have been examined in the electron microscope at various stages during dissociation and recrystallization. Two characteristic shapes are recognized in the micrographs. One is a double, concentric annulus in which the outer annulus is seen to consist of 12 masses disposed radially and attached to the inner annulus by short stalks. The centres of maximum density of the concentric annuli lie at radii of 40 A and 75 A respectively. The other shape seen frequently in micrographs is a rod-shaped object appximately 150 A in diameter and varying in length from 200 to 1000 A. The rods are composed of segments 70 A, each divided longitudinally into 35 A sections. It is believed that the segments represent side views of the annuli. A model of Fc consistent with these findings and with the present state of knowledge is presented.

A sensitive, reproducible method to quantitate human IgG, utilizing 125I Fc, derived from either the papain sensitive (ps) or papain resistance (pr) conformational forms of IgG, as the test antigen in the ammonium sulfate technique is described. Since 125I Fc fragments so obtained are innately soluble in 45 per cent saturated ammonium sulfate, free antigen may be separated from bound antigen-antibody complexes. The test system takes advantage of the capacity of IgG containing materials to block the interaction between human 125I Fc and a standard rabbit anti-human Fc.

An immunoglobulin IgG (Sackfield) which is known to have polypeptide chains shorter than those of typical proteins of its class has been subjected to fragmentation by papain in the presence of cysteine. One fragment was recovered which was indistinguishable from normal Fc fragment. The other fragment was related to normal Fab fragment but differed from it in several of its properties. The molecular weight was only one-half that of normal Fab. The optical rotatory dispersion spectrum of IgG (Sackfield) had features which differed from those of typical IgG myeloma proteins. The optical rotatory dispersion spectrum of Fc (Sackfield) was identical with those of other myeloma proteins, while the Fab (Sackfield) spectrum reflected the differences observed in the parent protein.

The eighth known case of heavy-chain disease is reported. Immunochemical studies of abnormal Fc fragment gamma-G3 protein and detailed morphologic, chromosomal, and electron microscopic ob...

1. The sedimentation coefficients of rabbit immunoglobulin G, four types of Fc fragments, univalent Fab and bivalent F(ab)2 fragments were measured as a function of pH. 2. In conjunction with molecular-weight determinations by sedimentation equilibrium, and with the behaviour on gel filtration, this enabled the state of association of the Fc fragments to be followed. 3. The type possessing an interchain disulphide bond, 1Fc fragment, changed extensively in structure, but not in molecular weight. 4. There was good correlation between the readiness to dissociate and the chain length of the shorter Fc fragments that do not contain the interchain covalent bond. 5. The increasing resistance to dissociation as the fragments became shorter ran parallel with the ability to resist enzymic attack. 6. The site of the strong association between component chains of Fc fragment is located in the C-terminal half. 7. The gel-filtration behaviour of the Fc fragments clearly confirms that the process is governed by the Stokes radius rather than molecular weight. 8. The ultracentrifugal results were used to estimate the separations of the hydrodynamic subunits in intact immunoglobulin G, and as a basis for a schematic structure.

IgM 1 immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.

Using antisera made against porcine S-sulfonated heavy chains two subclasses of porcine IgG could be distinguished on the basis of antigenic determinants located on the Fc fragment. The antisera prepared against the porcine gamma chains reacted with other mammalian IgG proteins, but not with avian, amphibian or fish immunoglobulins. Antigenic determinants responsible for cross-reactivity are further localized on the Fc′ fragment of the porcine IgG molecule. Comparable antisera, specific for porcine light chains, did not cross-react with IgG from any of the other species tested. Porcine IgG and F(ab′)2 fragment reacted with both the anti-heavy and anti-light chain sera, while the Fab fragment reacted only with the anti-light chain sera. Important antigenic determinants are thus located on the light chains and the Fc portion of the heavy chain of the porcine IgG molecule.

When antibodies are bound to Sepharose, they will specifically bind immunoglobulins of the IgG class. This interaction depends on the presence of the Fc fragment in the soluble immunoglobulin. The affinity between the soluble and the Sepharose-linked immunoglobulin is apparently similar to that between a bound antibody and its specific antigen. The effects of this interaction on the utility of affinity chromatography are discussed.

Publisher Summary This chapter analyzes some antigenic determinants that are characteristic of native and aggregated human IgG. The majority of the antigenic determinants of human IgG appear to result directly from the primary and secondary structure of the molecule rather than from its tertiary and quaternary configuration. Not only do most determinants persist following denaturation procedures that cause gross changes in the configuration of the molecule but they also survive mild proteolytic degradation. Thus, for example, the Fab and Fc fragments formed following papain digestion of human IgG not only possess the clearly defined antigenicites of the parent molecule but a mixture of these fragments can absorb about 80% of the precipitating activity of guinea-pig antisera raised against the intact human IgG molecule. This chapter analyzes a number of antigenic determinants of human IgG which appear to be dependent on the overall configuration of the molecule that is on its tertiary–quaternary structure for their expression. A determinant characteristic of native human IgG is examined. Antibodies specific for denatured human IgG are also described.

Chymotryptic hexadecapeptides have been isolated from the Fc-fragments of myeloma proteins possessing either Gm-a, f, or b allotypic activity. The structure of the peptide from a Gm-a source is: Thr-Leu-Pro-Pro-Ser-Arg-[ Asp -Glu- Leu -Thr-Lys]-Asn-Gln-Val-Ser-Leu. The homologous peptides from Gm-f and Gm-b allotypic proteins differ from those of the Gm-a source only in the two positions italicized in the bracketed tryptic pentapeptide and both have the -[Glu-Glu-Met-Thr-Lys]- sequence. These results confirm the studies reported from other laboratories. Pooled normal γG-globulins contain the two types of this tryptic pentapeptide. The sequencen of the tryptic peptides characteristics of the N -terminal eleven residues of the hexadecapeptide have been demonstrated in the pepsin Fc′-portions of various myeloma proteins. These fragments appear to contain about 111 amino acid residues. The starch gel electrophoretic properties and the amino acid contents of Fc′-fragments from different myeloma globulins show only small differences which may reflect allotypic variations.

A transplantable BALB/c mouse plasmacytoma, S63, produces an immunoglobulin with antibody activity directed specifically against the C-carbohydrate of the pneumococcus. This immunoglobylin is of the IgA class and possesses a kappa light chain. The combining site resides in the Fab not the Fc fragment. Some of the antibodies of BALB/c mice induced by C-carbohydrate have the idiotypic determinants which characterize S63-immunoglobulin.

Summary The urines of four patients have been studied. Three of these were suffering from myeloma, and the fourth from generalized amyloidosis. The presence of the complete M γG molecule has been detected in these four proteinurias. Moreover, in some of them, light chains and/or F'c and Fc fragments which are related with the heavy chains of γG, have been found present. The methods and the experimental technics are discussed and also the meaning of such proteins.

To identify certain peptides which might play a role in the biological activities of immunoglobulins and to estimate the degree of conservation of the primary structure of the Fc fragment ∗ , nine different H chains from six animal species were compared by peptide mapping. Fifty-one peptides were shared by the H chains of at least two different species, and most of these were derived from the Fc portion as determined by comparison with the maps of Fc fragments of rabbit and guinea-pig immunoglobulins. A number of peptides in combination were present only in H chains that can fix complement or sensitize the guinea pig for anaphylaxis; it is tentatively suggested, therefore, that some or all of these peptides are in some way related to biological activities.

The efficacy of biological therapies currently used in psoriatic arthritis depends not only on their blocking effect on the targeted molecule but also on the affinity of genetically defined variants of the Fc-gamma receptors for the constant portion (Fc fragment of IgG1).

MOST fibrous proteins do not crystallise. An artful way to promote protein crystallisation is to modify the structure by proteolytic attack. For example, papain cleavage of γ-globulin yields the homogeneous Fc fragment which readily crystallises1. Following a similar approach, we have crystallised fibrinogen.

An immunoglobulin molecule is a serum glycoprotein synthesized by vertebrates as antibodies against various antigens. The antigen binding domain lies in the Fab fragment containing the amino terminal while the effector function resides in the Fc fragment containing the carboxyl terminal. The Fab fragment can be separated from the Fc fragment by proteolytic treatment with papain (1). The structures of the gamma immunoglobulin and of the Fab fragment from various sources have been determined by x-ray crystallographic technique (2). The x-ray crystallographic study of a human Fc fragment at 3.5 Å shows that the Fc structure has an approximate shape of a “mickey mouse.” (2) A preliminary investigation of the rabbit Fc fragment by x-ray diffraction method was reported previously (3). We have used a similar method as Poljak and coworkers to crystallize the rabbit Fc fragment by dialysis against dilute phosphate buffer at neutral PH. A milky solution can normally be observed after 24 hours of dialysis. However, the crystal thickness was found to increase as a function of its age. The two dimensional size of the freshly prepared crystal is well over 1 μm2 which may make it suitable for low dose electron diffraction analysis.

Bovine pulmonary endothelial cells do not possess receptors for the 3b component of complement (C3b) or for the Fc portion of immunoglobulin G. The lack of these receptors may help explain the nonthrombogenic function of endothelial cells. Our findings rule out the possibility that endothelial cells participate in pulmonary immune complex disease through the binding of C3b or Fc fragments.

THE capacity of passively administered serum antibody to inhibit specific antibody formation1 has suggested that serum antibody may act as a type of “feedback” mechanism2–5. It is not known whether this effect of antibody results from its interaction with antigen or from a “direct” effect of some kind on the antibody-producing cells themselves5. A “direct” effect might be mediated by the Fc fragment of antibody, because this fragment, which does not contain the combining sites for antigen, is known to be essential for many other biological functions of antibody, such as complement fixation6, combination with skin7 and passage across the placenta8. The purpose of our experiments was to determine whether this fragment of the antibody gamma-globulin plays a part in the inhibition by antibody of specific antibody formation.

We describe a new binding test of anti-allotype antibodies anti-Igla from C 57 Bl/6 mouse serum with Fc fragments of CBA mouse IgG labelled with 131 (or 125) iodine. The precipitation of bound antibodies is achieved by rabbit anti-mouse Fab serum.