Joseph F. Heremans

Glutaradehyde-activated aminohexyl-derivative of sepharose 4B as a new versatile immunoabsorbent

Abstract 6-Aminohexyl-Sepharose 4B can be activated with glutaraldehyde to give a stable intermediate Sepharose 4B derivative which after removal of excess glutaraldehyde, covalently binds proteins during a second step, thereby furnishing Sepharose-protein conjugates of high protein content. Such conjugates constitute effective immunosorbents, having a 13-atom long spacing arm between the Sepharose and the protein. Their preparation is safe, easy, reproducible and particularly fast.

Immunoglobulin Formation and Function in Different Tissues

There is no reason to doubt that textbooks of immunology are correct when they teach that the immune response to antigenic stimulation is a reaction in which all the lymphatic tissues of the body have their share. However, work initiated shortly after the turn of the century and gaining increasing momentum in recent years has brought to light that nearly all individual tissues and organs, when confronted with an antigen, display the capacity to set up their own local immune response which is largely independent of the systemic reaction.

Studies on human secretory IgA (II). Comparative studies on a fragment of secretory component derived from secretory IgA and fragments obtained by enzymatic digestion of free secretory component

Abstract Reduced and alkylated secretory IgA (SIgA) from human milk released a fragment resembling the secretory component (SC) but having a smaller mol. wt (34,000–45,000) while also lacking certain antigenic determinants of SC. Reductive cleavage did not affect the molecular weight or antigenic specificity of free SC (FSC) present in milk. However, enzymatic digestion of FSC by various proteases released fragments which closely resembled the above described fragment from bound SC with respect to mol. wt, antigenic specificity and electrophoretic mobility. Trypsin, chymotrypsin and subtilisin were all found to be very effective in producing these changes, whereas pepsin was less efficient. Compared to FSC, the SC bound to SIgA was resistant to the attack by chymotrypsin. All the SC fragments studied here, whether obtained by proteolysis of FSC or by reduction of whole SIgA, showed marked multi-banded electrophoretic polymorphism.

The immunoglobulins of the dog—I. Identification of canine immunoglobulins homologous to human IgA and IgM

Abstract Selected antisera against human IgA were found to cross-react with a protein from canine serum and milk, which had the physicochemical properties attributed to ‘intermediate-sedimenting γ1’ immunoglobulin. It is concluded that this protein may be called the canine homologue of IgA. Similarly, extensive cross-reaction was observed between human IgM and a protein from canine serum having properties typical of ‘γM’.

Automated nephelometric immunoassay II. Its application to the determination of hapten

Abstract The principle of a novel method for the immunological assay of haptens, based on nephelometry, has been tested, using ϵ-dinitrophenyl-lysine and progesterone as model substances. It is proposed to call this method the nephelometric inhibition immunoassay (NINIA). In an automated continous flow system where aliquots of antigen are discontinuosly injected into a constant stream of diluted antiserum, the nephelometric effect of the antigen-antibody complexes formed markedly increases with the concentration of antibody relative to antigen, as well as with the affinity of the antibodies. Haptens inhibiting this reaction can be titrated by measuring the resulting decrease of the nephelometric peaks. In practice, the precipitin reaction is carried out with a ‘developer antigen’, BGG, HGG or ferritin carrying numerous haptenic groups, and antiserum preabsorbed with graded, known, amounts of hapten, or with the unknown sample. Means of increasing the sensitivity include the use of (i) high affinity antibodies, (ii) large, heavily substituted, developer antigens, (iii) 4% polyethylene glycol as the diluent of the antiserum, and (iv) adjusting the proportions of the reactants to near-equivalence. The threshold of the method as applied to progesterone is about 10 ng.

Automated nephelometric immunoassay (ANIA) I. Importance of antibody affinity

Abstract The reason why certain antisera displaying good precipitating properties in gels may be quite inefficient in automated nephelometric immunoassays has been sought. Antibodies against dinitrophenyl groups, gamma chains, or haptoglobin were eluted by increasing concentrations of ammonium thiocyanate from their respective antigens coupled to Sepharose. It was found that those antibodies that required the highest concentrations of thiocyanate to dissociate them from their antigen, were also those that formed aggregates with the most elevated nephelometric effect. Antibodies against dinitrophenyl groups were also eluted increasingconcentrations of the specific hapten. After dissociation from the latter, these antibodies displayed a nephelometric effect clearly dependent on their affinity. By increasing the concentration of antibodies (isolated on immunoadsorbents), and lowering the reaction temperature, it was possible to compensate for the lack of affinity of certain antisera and to make them suitable for automated nephelometric immunoassay.

Biological Significance of IgA Antibodies in Serum and Secretions

Publisher Summary This chapter discusses the biological significance of IgA antibodies in serum and secretions. The major part of IgA found in secretions is a secretion product of the population of IgA-containing cells usually found in large numbers beneath the epithelial surface of mucosae, and exocrine glands. In the human small intestine, for instance, such cells number nearly 400,000/mm3 of lamina propria, and their population density far exceeds that of all other immunoglobulin-containing cells combined. The rates of biosynthesis of plasma IgA and IgG in man are both about 30 mg/kg/day, which is surprising because IgA-containing plasma cells appear to be definitely less numerous in lymphoid tissues than are cells of the IgG type. A search for an extra source of plasma IgA would naturally be directed in the first place to the digestive mucosae, which are a vast depository of IgA-producing plasma cells. Two lines of evidence showed that the bowel does indeed contribute IgA to the plasma.

The immunoglobulins of the dog—II. The immunoglobulins of canine secretions

Abstract IgA was found to be the predominant immunoglobulin in canine milk, saliva, tears, hepatic bile and intestinal fluid. The molecular size of canine serum IgA was larger than that of human serum IgA, whereas the molecular size of canine exocrine IgA was similar to or slightly larger than that of 11S human milk IgA. Both exocrine and serum IgA from the dog were readily and completely reduced to subunits of the size of human IgG by treatment with 0·1M mercaptoethanol.

Amino acid sequence of cysteic peptides of lactoferrin and demonstration of similarities between lactoferrin and transferrin

Abstract Six cysteic peptides from human lactoferrin were isolated and their amino acid sequence determined. For each of these peptides at least one sequence with a reasonable degree of similarity could be pointed out in the primary structure of chicken ovotransferrin. This substantiates the idea that lactoferrin and transferrin were originally derived from a common ancestral iron-binding protein.

Improved procedure for the isolation of J-chain from human polymeric immunoglobulins

Abstract Highly purified J-chain was isolated from S-sulfonated human polymeric myeloma IgA by gel filtration and DEAE-cellulose chromatography, without using any dissociating agents. The purified J-chain was homogeneous upon immunoelectrophoresis and electrophoresis in agarose gel, alkaline-urea polyacrylamide gel, and sodium dodecyl sulfate polyacrylamide gel. The molecular weight of the J-chain, estimated by electrophoresis in sodium dodecyl sulfate polyacrylamide gel, was found to be 24 000 ± 1200 (S.D.). The J-chain dimerized in non-dissociating solvents, even in the presence of weak dissociating agents, such as 1 M propionic acid. During the isolation of the J-chain, two minor protein components which upon electrophoresis migrated, respectively, faster and slower than the J-chain itself were identified. These two components were antigenically unrelated to each other but both were clearly precipitated with anti-J-chain antisera, giving a reaction of partial identity to the J-chain. They were considered as two different fragments of J-chain, produced by some unknown protease(s).