Natural antibodies to avidin in human serum.

Human serum was found to contain natural antibodies to the egg-white glycoprotein avidin. Of 270 samples tested, all contained antibodies to different extents, mainly of the IgG and IgM classes. Anti-avidin antibodies could be isolated by affinity chromatography.

Rapid purification of HBsAg from human plasma.

A method is described for the rapid isolation of purified HBsAg from infected donor plasma. This method uses polyethylene glycol precipitation and a single isopycnic ultracentrifugation step in which the gradient is formed in situ. It works equally well using a small swingout rotor or a zonal rotor.

The importance of quaternary structure in the expression of the C1-binding site of IgM.

The ability of C1 to bind to the Fc5mu-fragment of a monoclonal IgM was determined by means of a haemolytic C1-inhibition assay. Fc5mu fragments were produced by trypsin digestion at five different temperatures ranging from 54-62 degrees (2 degrees increments) and were purified by immunoadsorption through a column of monospecific anti-Fabmu and by molecular exclusion chromatography. Analytical ultracentrifugation showed the final preparations to be free of aggregates. A plot of mug Fc5mu required to inhibit 50% of available C1 versus temperatures of production of the fragment yielded a curve with a minimum at 58-60 degrees. Upon mild reduction and alkylation of these Fc5mu fragments their C1-fixing capacity became approximately the same irrespective of temperature of production. Fc5mu was also prepared at 25 degrees in the presence of 5 M urea, purified by immunoadsorption as before and aliquots then exposed to temperatures ranging from 40-70 degrees (5 degrees increments) for 15 min. After aggregates had been removed by chromatography a similar minimum in C1-fixation was again observed at 60 degrees. Reduction and alkylation once more abolished these differences. Fc5mu and its reduced and alkylated subunits, produced at 60 degrees and then exposed to various concentrations of urea (0-7 M) for 24 hd did not yield a minimum in C1 fixation. Reduced and alkylated Fcmu incubated at various temperatures (40-70 degrees) also did not fix C1 differentially. Examination in the near and far u.v. region of the circular dichroism spectra of different Fc5mu preparations showed a gradual loss of structure associated with restricted aromatic chromophores and secondary (beta) structure with increased temperature. Urea denaturation had a more pronounced and irreversible effect on Fc5 mu conformation. These changes could not be correlated with the CU-fixation patterns observed. It would therefore appear that elevated temperatures induce a static change in the pentameric FC-part of IgM which in turn directly influences or modulates the availability of the C1-binding site. The importance of disulphide bonds in maintaining these temperature-induced changes in Fc5mu was also indicated.

Isolation and identification of the Cmicron4-domain of IgM.

Intact Cmicron4-domain was isolated by molecular exclusion chromatography of reduced and alkylated Fc5 micron prepared by tryptic digestion at 60 degrees C of a monoclonal IgM. Two fragments were obtained of which one contained carbohydrate and the other none. These fragments were successfully separated by chromatography on insolubilised Concanavalin A. Cmicron4-domain was identified by means of amino acid sequence, amino acid composition, molecular weight and immunological analyses. It has a molecular weight of 14,700 daltons and results from tryptic cleavage at Lys-445 of the micron-chain.

Hepatitis B surface antigen in donated blood--screening and confirmation by enzyme-linked immunosorbent assay.

Principles of and test procedures enzyme-linked immunosorbent assay (ELISA) screening and confirmatory tests for hepatitis B surface antigen (HBsAg) in donated blood are described. The assay is of "third-generation' sensitivity and is practical and economical for large-scale screening of blood donors. Results from 119,000 tests show ELISA to be superior to the passive haemagglutination inhibition (PHAI) test for HBsAg. The ELISA described here is considerably cheaper than any comparable commercially available isotopic kit for HBsAg screening but is slightly less sensitive.

Use of jacalin as a solid phase in ABO reverse grouping.

A major problem of using red cells as the solid phase in assay systems is the difficulty to bind them strongly to appropriate surfaces. We report here on a number of lectins of different specificities which were examined for their ability to bind red cells to polystyrene 96-well microtitre plates. The use of the Thomsen-Friedenreich antigen-specific lectins, jacalin, mushroom and Maclura pomifera agglutinin proved the most useful for ABO reverse grouping. Jacalin-coated plates were also compared with plates coated with poly-L-lysine and bovine serum albumin/glutaraldehyde for the binding of erythrocyte membranes and were found to be superior. We also describe the colorimetric detection of the solid phase red cell antibody reaction by using an indicator erythrocyte and peroxidase chromogenic substrate.

Two methods for the introduction of amino groups into agarose-based matrices: their use in immunoaffinity chromatography.

In this study two methods of coupling antibody to Sepharose CL2B are described. They involve the introduction of amino groups either via an amino acid such as glycine or through polyethyleneimine. After introduction of amino groups into the matrices, their activation and simultaneous fixing were accomplished by treatment with glutaraldehyde. Monoclonal antibody raised against von Willebrand factor was used as a model ligand to demonstrate the stability and performance of the affinity supports. Both methods examined in this study resulted in good retention of the antibody's binding capabilities and excellent stability of the derivatized matrices. Leaching of the insolubilized protein was considerably less with the polyethyleneimine-glutaraldehyde than with cyanogen bromide.

Mouse monoclonal anti-HBs and its use in the screening of donated blood by Elisa.

A method is described for the production of mouse monoclonal antibodies to HBsAg. 4 clones produced antibodies directed at the a and one at the d determinant of HBsAg. These antibodies were conjugated to horse radish peroxidase and used in an Elisa for the detection of HBsAg in donated blood. Antibody C10 produced conjugate which rendered the Elisa as sensitive as a commercially obtainable immunoradiometric assay with which it was compared. Conditions for this assay were optimised and it may be used as a rapid (1 h) test for detecting HBsAg-positive blood. It is practicable to read this Elisa by eye instead of photometrically and it can be thus used under field conditions or in emergencies.

Safer blood--supplementation of blood products with anti-HBs.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitation of antibodies (anti-HBs) to the surface antigen of the hepatitis B virus (HBsAg). The ELISA uses HBsAg as the solid phase, and, after conjugation to horseradish peroxidase, also as the conjugate. Conditions for this assay were optimized and a rapid (1.5 hours) ELISA has evolved which works very satisfactorily for the large-scale screening of donated blood. We have used this ELISA to examine donated blood in Natal and concluded that we cannot initiate a programme of anti-HBs supplementation of parenteral blood products without hyperimmunization and plasmapheresis of selected, voluntary donors.