Takakazu Suzuki

Assay of classical and alternative pathway activities of murine complement using antibody-sensitized rabbit erythrocytes

Methods for measurement of classical complement pathway activity (CH50) and alternative complement pathway activity (ACH50) in mouse serum using rabbit erythrocytes sensitized with guinea pig anti-rabbit erythrocyte antibody have been established. The assays measured CH50 values in mouse sera that could hardly be determined by the conventional method using antibody-sensitized sheep red blood cells. Mouse serum ACH50 values determined by the method were also 5-7 times higher than those obtained in conventional assays with rabbit erythrocytes. Both the CH50 and ACH50 values varied with the strain among the 25 different strains of mice studied. BALB/c (nu/nu, male), LT/SuJ and Jcl-ICR27 strains exhibited higher CH50 values, and NIH (nu/+), ICR (nu/nu), NOD (male) and AKR strains showed lower values. The ACH50 was higher in C3H/HeN (male), C57BL/6J (male), Jcl-ICR27 and BALB/c (nu/nu, male) mice, and lower in ICR (nu/nu), NOD (female) and AKR mice. Sera from 16 out of the 25 mouse strains showed ACH50 values comparable to or higher than those in man. As for CH50, however, even the highest value seen in BALB/c (nu/nu, male) mice corresponded to about three-fifths of an average value in man. It is concluded that the complement system of mice, especially the alternative pathway of complement activation, functions as actively as that in man. It was also found that male mice have higher CH50 and ACH50 values than female mice. The differences in these parameters between males and females were only slight at the age of 4 weeks and became conspicuous after 6 weeks at which time both the CH50 and ACH50 virtually reached their respective peak levels of activity.

Studies on the hemolytic activity of the classical and alternative pathway of complement in various animal species.

The classical complement pathway (CP) activity (CH50) of sera of 13 animal species was compared by parallel assays using rabbit erythrocytes sensitized with immune antibody (RaE-Ab) and sensitized sheep erythrocytes (ShE-Ab). The alternative complement pathway (AP) activity (ACH50) in various species of animals was also measured using 13 species of erythrocytes and RaE-Ab as target cells. The results have demonstrated that virtually all species of animals studied possess CP and AP activity levels comparable with or higher than those in human sera. Concomitant measurement of the hemagglutinin titer to various species of erythrocytes carried out in the screening ACH50 assays on various species of sera with these erythrocytes disclosed a significant correlation between natural antibody level and AP activity (p less than 0.05).

GENE RESPONSIBLE FOR DEFICIENT ACTIVITY OF THE BETA SUBUNIT OF C8, THE EIGHTH COMPONENT OF COMPLEMENT IS LOCATED ON MOUSE CHROMOSOME 4

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radical hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had n o lytic activity, and Ouchternoly analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored serum deficient in C8α-γ, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the β subunit of C8. Typing of (DBA/2J ×MSM)F1 hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a singles recessive gene, designated C8b; the allele with hemolytic activity is C8b1; and the allele with no activity C8b0. Because of synteny homologies in mouse and human , we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.

An improved method for preparation of stable antigen coupled erythrocytes for passive hemolysis

Abstract The mild fixation of erythrocytes with glutaraldehyde prior to coupling to them with bis-diazotized benzidin, gave stable cells for passive hemolysis without affecting their hemolytic behavior. Furthermore, the use of proper amounts of bis-diazotized benzidin and antigen was antoher important factor in obtaining stable cells. Cells prepared in this way could be stored in phosphate buffer at low temperature, for at least 2 weeks without loss of maximal hemolytic titer and showed slight spontaneous hemolysis. The conditions described could be used with several proteins which differed in their physicochemical properties. Antigen coupled cells were also useful for immune adherence hemagglutination.

Detection of toxoplasma IgM antibody by passive latex agglutination reaction

Three major components (designated Sp-1, -2 and -3) of the microsomal pellet of Toxoplasma gondii (Tp) were isolated by Ultragel AcA 44 gel filtration chromatography from the microsomal pellet solubilized with detergents. Of these, Sp-2 proved to be most reactive with anti-Tp antibodies and its reactivity with IgM and IgG antibodies varied with the concentration at which it was used for sensitizing latex particles. Sp-2 antigen reacted with IgM antibody alone when latex particles were sensitized with less than or equal to 100 micrograms of this antigen/mg of particles, and its reactivity with IgG antibody appeared and increased progressively with increasing sensitizing concentrations of this antigen. Based on this finding, a method of direct measurement of anti-Tp IgM antibody in serum by passive latex agglutination has been developed. Polyacrylamide disc gel electrophoretic analysis of Sp-2 antigen in the presence of SDS revealed four constituents of 43, 35, 28 and 14 kDa. All these components reached with both IgM and IgG when tested by immunoblotting.

Separation of an adjuvant-active glycolipid lacking peptide moiety from wax D preparation of mycobacterium tuberculosis strain aoyama B

Abstract A macromolecular and adjuvant-active glycolipid lacking the peptide part was separated from wax D preparation of M. tuberculosis strain Aoyama B by silica gel column and preparative thin-layer chromatographies. Although a glycolipid from wax D preparation of M. bovis BCG could also be separated by the same procedure, it was not adjuvant-active. Infrared spectra of both glycolipids were similar but not identical. The main absorptions showed the presence ofhydroxy, methylene and carbonyl groups, and absorption of primary amide and P-O groups in both spectra were very weak or slight. The nitrogen contents of both glycolipids were not detectable (below 0·01%). By alkaline hydrolysis, the glycolipid from M. tuberculosis was separated finally into the hydrophilic and hydrophobic parts forming intermediate products in the process. The molecular weight of the hydrophilic part was around 9300 daltons by gel filtration and it belonged to a fraction of lower molecular weight component(s) in the hydrophilic part of wax D preparation of M. tuberculosis strain H37Rv.