Pierre L. Masson

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.

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.

Studies on bovine cervical mucin I. The amino acid composition and N-terminal amino acid of bovine oestrus cervical mucin

Bovine oestrus cervical mucin, isolated by gel filtration on Sepharose 6B, was found to be homogeneous (i) in electrophoresis on cellulose acetate at pH 8.6 after release of sialic acid and in acrylamide-agarose gel at pH 8.7 in the presence of sodium dodecyl sulphate, where not a single compact zone was found after staining for proteins, (ii) by analytical ultracentrifugation and equilibrium centrifugation in a CsCl density gradient; as well as (iii) by immunoelectrophoresis, which revealed two kinds of antibodies against mucin, reacting, respectively, with the primary structure and with determinants depending on the integrity of the glycoprotein structure. Alanine was identified as the N-terminal amino acid of the peptide core, which appeared to consist of a repetition of a 32-35 residue sequence, viz. (Thr)8, (Glu, Pro, Ala, Val)3, (Asp, Leu)2 and (Arg, Cys, Ileu)1. No integral numbers could be assigned to (Ser)3.4, (Gly)2.3 and (Phe)0.6 which were also present.

The involvement of bicarbonate in the binding of iron by transferrin

Transferrin coupled to Sepharose was found to be able to bind iron (59Fe3+) only after the addition of HCO3−, indicating that HCO3− does not merely cause the development of the red color of the iron-transferrin complex but that it is essential for the formation of this complex.

Esterase activity associated with secretory IgA and free secretory component preparations from human milk

Secretory immunoglobulin A (IgA) and free secretory component were purified from human milk using methods involving (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography. It was found that the preparations, although apparently pure by conventional criteria based on immunoelectrophoresis, zone electrophoresis, or ultracentrifugation, were consistently contaminated by two different esterases. These enzymes could apparently not be removed by further ion-exchange chromatography and/or gel filtration. They could, however, be eliminated by passage on an immunoabsorbent made from anti-free secretory component antiserum.

Studies on the proteins of human seminal plasma: I. Characterisation of two esterase systems

Abstract Two esterases were demonstrated to be present in seminal plasma. The enzymes were differentiated in terms of electrophoretic mobility, immunogenicity and sensitivity to organophosphorus esters. According to their respective mobilities in the α and βγ zone they were called α-esterase and βγ-esterase. Gel filtration also separated the two esterases. The βγ-enzyme showed marked heterogeneity in starch-gel electrophoresis. It was eluted in a single peak from Sephadex G-200. Its molecular weight was estimated to be 69 000-77 000. The α-esterase, which was not detectable in all seminal plasma samples, was shown to form a complex with the iron-binding protein lactoferrin.