John E. Hopper

Evidence by radioimmunoassay that mitogen-activated human blood mononuclear cells secrete significant amounts of light chain Ig unassociated with heavy chain

The culture supernatant (CS) Ig of PWM-activated human blood mononuclear cells was quantitatively determined using a panel of nanogram-sensitive radioimmunoassays (RIAs) that separately measured IgG, IgM, IgA, total kappa Ig, and total lambda Ig. After initial RIA quantitation, separate CS aliquots were exposed to either a polyisotypic anti-heavy (H) chain or a nonimmune IgG solid-phase immunoabsorbent, and then reassayed for Ig content. The reassay results revealed that the anti-H chain-absorbed CS aliquots retained significant amounts of kappa and lambda Ig, but yet had a virtual absence of isotypic IgG, IgM, and IgA. Comparisons of the absorbed CS aliquots suggested that as much as one-fourth to one-third of the total secreted L chain Ig in PWM-activated cultures lacked RIA-detectable associated H chain. This unexpected finding of significant amounts of unbound L chain in mitogen-stimulated cultures raises important theoretical issues relative to the functional role of secreted free L chain and the prospects that free L chain levels may represent useful quantitative markers of B-cell stimulation.

Clinical relapse in systemic lupus erythematosus: Correlation with antecedent elevation of urinary free light-chain immunoglobulin

This paper reports preliminary evidence suggesting that measurements of free light-chain Ig (FLIg) in urine may represent quantitative markers ofin vivo polyclonal B-cell activation. Thus, longitudinal levels of urinary FLIg in patients with systemic lupus erythematosus (SLE) may be used to track or monitor thein vivo immunopathologic B-cell activity of SLE and be helpful in predicting a disease relapse. Our findings showed that dramatic rises in urinary FLIg occurred during asymptomatic intervals that preceded by 4–8 weeks the first symptomatic signs of acute SLE relapse. These results suggest that a sizable lead time may exist between the occurrence of immunopathologic B-cell stimulation and the resultant symptoms and tissue damage of immune complex-induced acute inflammation. In these studies the measurement of urinary FLIg was accomplished by an indirect method using ng-sensitive radioimmunoassays (RIAs) that measured isotypic IgG, IgA, IgM, total κ-Ig, and total λ-Ig. As a control for the assessment of renal tubular function and the excretion of low molecular weight proteins in SLE patients, longitudinal measurements of beta-2-microglobulin (B2M) and lysozyme were made using a novel solid-phase3H-biotin RIA technique.

Urine free light chains in SLE: clonal markers of B-cell activity and potential link to in vivo secreted Ig.

As a marker of in vivo B-cell activity, urine levels of free light chain (FLC) were measured twice weekly by radioimmunassay (RIA) and correlated with disease activity over periods of 5–10 months in seven patients with systemic lupus erythematousus (SLE). In addition, RIA-measured urine albumin was used to track glomerular injury, and α1-microglobulin (α1-M) levels, 28- to 32-kDa protein, provided control measurements on excretion of low-molecular-weight proteins. As controls, urine FLC levels were obtained from healthy normals and in subjects with acute pharyngitis, sickle-cell anemia, and acute sepsis or pneumonia. The control results showed that with acute sepsis/pneumonia had marked increases in urine FLC, while pharyngitis and sickle-cell controls had normal FLC levels. In SLE, active patients receiving intravenous cyclophsophamide and high-dose steroids exhibited highly increased urine FLC that fluctuated widely during therapy and fell to normal range levels with disease remission. During active SLE, urine albumin often was increased, while α1-M levels remained in normal range. In contrast to the increased FLC of active disease, inactive patients on low-dose maintenance therapy had predominantly normal FLC levels throughout the collection period. These results support our hypothesis that longitudinal levels of urine FLC can be used to track disease-related B-cell activity in SLE. Furthermore, we suggest that the urine FLC of active SLE would share LC idiotype with the clonal associated in vivo secreted Ig, and thus permit the identification of these antibodies that are targeted to the culprit immunogen(s) responsible for the pathogenesis of SLE.

Idiotypic Specificities of Immunoglobulins

This chapter reviews idiotypic specificities of immunoglobulins. Monotypic immunoglobulins, such as myeloma and Bence Jones proteins, and antibody populations from individual animals possess individually specific antigenic determinants. For example, one can prepare an antiserum in a rabbit that when appropriately absorbed will react with the human myeloma protein used as immunogen but, with rare exceptions, not with any other myeloma protein or immunoglobulin. Alternatively, one can immunize a rabbit with antibody from an individual human or rabbit and elicit specific anti-antibodies. The restriction of idiotypic determinants to antibodies of a single specificity from an individual donor clearly differentiates idiotypy from allotypy, as allotypic determinants are shared by various antibodies of an individual. A contribution of the constant region of a polypeptide chain to an idiotypic determinant is conceivable, but idiotypic differences among proteins must reflect differences in the variable regions. Anti-idiotypic antibodies can be prepared against antibodies from an individual donor, despite the heterogeneity of the immunogen.

Evolution of the Immunoglobulins

This chapter discusses the evolution of the immunoglobulins. Many invertebrates have been found to possess natural agglutinins, active against protozoa, bacteria, and the red blood cells of various invertebrate and vertebrate species. In all vertebrate species studied, with the possible exception of the lamprey and the hagfish, immunoglobulin molecules comprise one or more subunits, each consisting of two H and two L chains. The chains are linked by non-covalent interactions and in most, but not all instances, by inter-chain disulfide bonds. Assignment of an immunoglobulin to a particular class is made by comparison with mammalian or, more specifically, with human immunoglobulins. It is not certain that a knowledge of the amino acid sequence of the C H region would immediately permit identification of the class to which the immunoglobulin of a primitive species belongs. Even if, for example, the gene controlling the C μ region of human IgM evolved directly from a corresponding gene in fish, the divergence in sequence may prove to be so great as to make a definite assignment difficult.

Homogeneous Antibodies and Myeloma Proteins with Antibody Activity

Publisher Summary This chapter reviews homogeneous antibodies and myeloma proteins with antibody activity. Homogeneous proteins with antibody activity have a major practical use in the study of the relationship of amino acid sequence and 3-dimensional structure of an antibody to its specificity. It is generally accepted that homogeneous antibody populations and myeloma proteins are synthesized by individual clones of cells that have undergone extensive proliferation, frequently at the expense of the biosynthesis of other immunoglobulins. In the case of antibodies to bacterial vaccines induced in experimental animals, the antibody concentration in serum often exceeds the total concentration of immunoglobulin in normal, non-immunized animals. This concentration relationship also applies to myeloma proteins. Continuous immunization is necessary to maintain a high concentration of homogeneous antibody; and, in the face of repeated challenge by antigen, clones producing homogeneous antibody may on occasion diminish and be replaced by other clones of cells manufacturing antibody differing in structure but having specificity for the same antigen.

Nature of the Active Site of an Antibody Molecule and the Mechanism of Antibody-Hapten Interactions

This chapter will summarize information relating to the chemical nature of the active site of an antibody molecule and mechanisms of interaction with hapten. It will consider studies relating to the fine specificity of an antibody; the concept of an immunodominant antigenic grouping; forces involved in antibody–hapten interactions; the size and structure of the antibody combining site; kinetics and thermodynamics of antibody–hapten interactions; and affinity labeling of the active site as a probe for amino acid sequences in or near the site. Details of the structure of the combining regions of myeloma proteins, as ascertained by X-ray crystallography, are discussed in Chapter 5.

Allotypes of Rabbit, Human, and Mouse Immunoglobulins

This chapter discusses the genetically controlled polymorphism of immunoglobulins, a property first discovered through studies of antigenic determinants of human and rabbit immunoglobulins, respectively. Allotypy has been studied most extensively in the rabbit, man, and mouse. In each species, allotypic variation is seen in both H and L chains. In the rabbit and in man, for which data are available, genes controlling allotypic determinants on H and L chains are unlinked. In each species, the inheritance of genes controlling allotypic determinants is strictly Mendelian and is not sex-linked. An individual antibody-producing cell normally expresses only one of a pair of allelic genes. This is in accord with the fact that most, if not all normal antibody-forming cells can synthesize only a single species of L chain and a single species of H chain; that is, a cell behaves with respect to antibody biosynthesis as if it were functionally haploid.

Amino Acid Sequences in Human Immunoglobulins and in Mouse Light Chains

This chapter discusses amino acid sequences in human immunoglobulins and in mouse light chains. A large proportion of the available data on amino acid sequences of immunoglobulins relates to human and mouse proteins. This is attributable to the availability of myeloma proteins in both species and to the intrinsic interest in human proteins. Multiple myeloma, which is a relatively rare disease in humans, can be induced in BALB/c or NZB mice by prolonged treatment with mineral oil, certain other hydrocarbons, plastics, or immunological adjuvants. Tumors can be maintained by transplantation into mice of the same inbred strain; this makes it possible to accumulate large amounts of serum or ascites fluid containing a homogeneous myeloma protein. Multiple myeloma is frequently accompanied by the presence of a high concentration of homogeneous, urinary L chains—Bence Jones proteins—that correspond in structure to the L chains of the individuals serum myeloma protein. Owing to their high concentration, myeloma or Bence Jones proteins can generally be isolated in reasonably pure form.

Immunoglobulins of the Rabbit, Mouse, Guinea Pig, and Horse

Publisher Summary This chapter describes properties of immunoglobulins of four mammalian species that have been studied quite extensively—the rabbit, mouse, guinea pig, and horse. A protein is designated κ or λ on the basis of sequence homology to the corresponding human chain. In most, but not all immunoglobulins, one disulfide bond joins an H chain to an L chain. However, there are marked variations, with respect to H–H disulfide bonds. The relative serum concentrations of the immunoglobulin classes vary among species. As in the case of human immunoglobulins, large fragments, some of which have active binding sites, can readily be obtained. One reliable method for identifying a class as homologous to that of a human immunoglobulin is antigenic cross-reactivity. Amino acid sequence homologies, which are more definitive, are also available in some instances. The terms “reaginic” or “homocytotropic” antibody (HCA) are in general usage to denote antibodies capable of mediating passive cutaneous anaphylactic reactions within the autologous species. In a number of species there is more than one class of molecule which has this property.