Shirley E. Maddison

Quantitative capacities of glutaraldehyde and sodium m-periodate coupled peroxidase-anti-human IgG conjugates in enzyme-linked immunoassays

Covalently linked peroxidase-anti-human IgG conjugates were prepared by either glutaraldehyde or NaIO4 coupling techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows that the glutaraldehyde coupled conjugate is composed of generally lower molecular weight components than the NaIO4 coupled product. The NaIO4 conjugate, when used to quantitate human immunoglobulin (Ig) in enzyme-linked immunoassays, appears to be highly sensitive in that small amounts of Ig elicited relatively high reactivities. The quantitative range of this type of conjugates, where reactivities are linearly proportional to the amount of human Ig present, is, however, extremely narrow (0.01-0.10 micrograms/ml of human IgG). Conversely, the glutaraldehyde coupled type conjugate is capable of sustaining a much wider range of linearity (0.01-0.6 micrograms/ml), but with a more gradual rise of reactivity which corresponds well to the amount of human Ig present. Conjugates prepared with glutaraldehyde are thus more useful in quantitative assays where wide quantitative ranges are desirable. NaIO4 conjugates on the other hand, are more suited to qualitative assays where sensitivity is more important.

Systematic fractionation of Schistosoma mansoni urea-soluble egg antigens and their evaluation by the single-tube, kinetic-dependent, enzyme-linked, immunosorbent assay (k-ELISA).

The application of a single-tube, kinetic-dependent, enzyme-linked, immunosorbent assay (k-ELISA) is described. The k-ELISA is simple, highly sensitive, and quantitative. With this test, the antigenic activities and cross-reactivities of several fractions from Schistosoma mansoni eggs were quantitated and compared. Particulate egg components were solubilized readily by 8 M urea, yielding antigenic fractions of high specific activities and low corss-reactivities. SDS-PAGE and activity profiles of these antigens clearly showed that they were separate and distinct form the soluble egg antigens (SEA) group. The urea-solubilized antigens appeared to be composed of two major protein bands of high molecular weights. The yield for these antigens was significantly greater than the SEA group, thus making them worthwhile candidates for serological antigens. The systematic and quantitative nature of the present study allows for the critical comparison of every antigenic fraction from S. mansoni eggs. From data of this type, a diagnostic antigen of high efficacy can be selected.

IMMUNOLOGIC STUDIES ON HAMSTERS INFECTED WITH ENTAMOEBA HISTOLYTICA

The serologic and cell-mediated immune responses of hamsters exposed to 2 strains of Entamoeba histolytica (HM-1 and HM-19) were evaluated by a series of in vitro tests. The pathogenicity of the 2 strains was evaluated in terms of their ability to produce liver abscesses and spleen enlargement. Antibody response was evaluated by the indirect hemagglutination test. The cellular immune response was assayed by increased DNA synthesis by lymphocytes and migration inhibition of macrophages.

Schistosoma mansoni: reduction in clinical manifestations and in worm burdens conferred by serum and transfer factor from immune or normal rhesus monkeys.

Abstract Although delayed hypersensitivity to Schistosoma mansoni was conferred on rhesus monkeys (Macaca mulatta) by means of dialyzable transfer factor prepared from peripheral leukocytes or lymph node cells of infected immune donors, when such animals were challenged with 1000 cercariae of S. mansoni they developed worm burdens similar to those of nontreated controls. However, recipients of transfer factor that, in addition, received hyperimmune serum showed minimal clinical symptoms and significantly reduced worm burdens after subsequent infection with S. mansoni irrespective of whether the donors used for the transfer factor were immune or uninfected. A significant but lower degree of protection was conferred by combinations of either S. mansoni transfer factor or normal transfer factor and normal serum. Neither transfer factor nor hyperimmune serum alone conferred protection to recipients. Susceptibility to infection was assessed by observing the signs of the disease, determining the worm burdens by perfusion 10 weeks after exposure, and by observing the appearance of the intestine at autopsy. The animals which received transfer factor and immune serum were protected against clinical disease. Good correlation between worm burdens and severity of disease was observed.

Schistosoma mansoni infection in the rat. II. Effects of immunosuppression and serum and cell transfer on innate and acquired immunity.

The reproducible pattern of Schistosoma mansoni infection in the rat was confirmed. A peak worm burden was reached at the 4th week after exposure and was followed by a rapid reduction in the number of adults between the 4th and 8th weeks; thereafter a low, stable worm burden level was maintained. This pattern was not altered by X-irradiation of the rats either prior to or 3 weeks after exposure to S. mansoni. Immunosuppression by thoracic duct drainage, antilymphocytic globulin (ALG), or a combination of ALG and Imuran failed to alter the susceptibility of the rat to S. mansoni infection, as determined by worm burdens present 4 weeks after infection. ALG was not able to alter the immune state of the host acquired as the result of previous infection. The transfer of serum or cells from immune donors did not confer protection to isologous recipients. Experiments in which a combination of immune serum and sensitized cells were transferred gave equivocal results. Infection with Schistosoma mansoni in the laboratory rat follows a reproducible pattern. The host responds to the infection by a rapid reduction in adult worm burden between the 4th and 8th weeks (Stirewalt et al., 1951; Ritchie et al., 1963; Smithers and Terry, 1965a, b). After the decline in worm burden, the animal is refractory to reexposure with cercariae (Ritchie et al., 1963; Sadun and Bruce, 1964; Erickson and Caldwell, 1965; Smithers and Terry, 1965b). The mechanism responsible for initiating the worm loss or for maintaining acquired immunity has not been elucidated. Maddison et al. (1970), using five serologic tests and the passive cutaneous anaphylaxis reaction, could not show a correlation between antibody development and the rapid loss of worms. Vogel and Minning (1953), working with S. japonicum in the rhesus monkey, Stirewalt and Evans (1953), working with S. mansoni in mice, and Kagan (1958), working with Schistosomatium douthitti in mice, could not demonstrate passive transfer of immunity with hyperimmune serum. Ogilvie et al. (1966) showed that intradermal injection of specific antischistosomal reaginic antibody afforded protection to rats that were exposed to cercariae placed over the antibody depot. The same type of antibody was not protective in the skin of the rhesus monkey. We have attempted to study the immune Received for publication 9 December 1969. mechanisms responsible for the elimination of worms and for the acquired immunity in the rat. Animals were subjected to immunosuppression before primary infection, and, in one instance, before secondary exposure. Hyperimmune serum, lymphocytes from immune rats, and a combination of these two factors were investigated for their ability to confer protection to normal recipients. The results of these experiments are now reported. MATERIALS AND METHODS Inbred Lewis and ACI strains of rats (obtained from Microbiological Associates), weighing 150 to 200 g, were employed. The sexes of the cell donors and respective recipients were in accord with the sex compatibility X and Y chromosomes. Randombred Sherman rats were raised in our laboratory. In each experiment, treated and control animals were exposed at the same time to 1,000 cercariae of a Puerto Rican strain of S. mansoni by the ring method, and the worm burdens were determined by perfusion following the technique used by Smithers and Terry (1965a). The rats were exsanguinated at the time of killing 4, 6, 8, 10, or 12 weeks after exposure, and the sera were stored at -20 C.

Studies on putative adult worm-derived vaccines and adjuvants for protection against Schistosoma mansoni infection in mice.

Intraperitoneal transfer of viable adult worms of Schistosoma mansoni did not confer protection against a challenge infection to recipient mice. Antigens of schistosome origin were evaluated for their ability, with and without concomitantly administered nonspecific adjuvants, to stimulate protective immunity against S. mansoni. Freshly perfused ground worms or a putative membrane antigen extracted with 0.5 M KC1 from adult worms, when injected together with Corynebacterium parvum (or in a single experiment with poly [A : U]), resulted in a significant reduction in worm burden of a challenge infection with S. mansoni as compared with that of untreated controls. The membrane antigen was maintained carefully at low temperatures in buffers capable of retarding enzymatic degradation while it was being prepared.

Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse.

Passive transfer of immune serum alone did not confer protection to recipient mice irrespective of the routes of serum transfer or cercarial challenge of Schistosoma mansoni. Mice that received both sensitized cells and immune serum were protected against challenge by subcutaneous injection of cercariae but not by percutaneous exposure. The immune serum could be transferred as late as 8 days after subcutaneous challenge, suggesting that the protection was afforded in part by a late parasite killing mechanism which functions after the schistosomula have migrated through the lungs.

STANDARDIZATION OF FIAXTM FOR SCHISTOSOMIASIS USING CRUDE CERCARIAL AND ADULT WORM ANTIGENS

A fluoroimmunoassay (FIAXTM) has been developed for quantitating the antibody response to schistosomiasis by using cercarial and adult worm antigens of Schistosoma mansoni. The FIAXTM assay was calibrated by using an enzyme-linked immunosorbent assay (ELISA) performed with the same antigens. Studies of reproducibility and preliminary data on a battery of sera from proven cases of schistosomiasis an uninfected control sera are presented.

Immediate, Arthus, and delayed-type skin reactions in rhesus monkeys infected with Schistosoma mansoni or mycobacteria

Abstract Gross and histologic observations of immediate, Arthus, and delayed-type skin reactivities in rhesus monkeys infected with Schistosoma mansoni or mycobacteria are described. Edema without erythema occurred in the eyelid and on the chest of animals with immediate-type hypersensitivity. Eosinophils characterized the cell infiltrations. Arthus reactivity resulted in blanching of the skin test sites and in a polymorphonuclear neutrophilic infiltration. Erythema was stimulated by intrapalpebral testing of animals with delayed hypersensitivity, but in these animals the chest site or back again showed a blanched area of induration; a large mononuclear, epithelioid cell type of infiltration was characteristic of this reactivity.

Comparative isoenzyme analysis of various stages of Brugia malayi and Brugia pahangi

teinase (Asch and Dresden, 1977, Comp. Biochem. Physiol. 58B: 89-95). Cleavage products from the action of cercarial proteinase, trypsin, or chymotrypsin on the 66 and 60K polypeptides of keratin were identified by SDS polyacrylamide gel electrophoresis (Weber and Osborn, 1969, J. Biol. Chem. 244:44064412). First, 120 ,tg of substrate (purified 66 or 60K keratin) and 0.4 ,ug of purified enzyme were incubated at 22 C for 1, 4, or 20 hr in 90 ,ul of the 100 mM Tris/HCl buffer described above. The reaction mixture was then boiled for 5 min in the presence of 1% mercaptoethanol, 2% SDS and 4 M urea. Then 50 Atg of substrate were subjected to electrophoresis on a 9% polyacrylamide gel (10 x 0.6 cm), and the gel was stained with Coomassie brilliant blue, R250. The purified cercarial proteinase degraded whole purified keratin, and this activity was inhibited by a1-proteinase inhibitor (Fig. 1). The extent of keratin degradation was limited by the amount of enzyme present and stability of the enzyme at 37 C. The proteinase also degraded both the 66 and the 60K keratin polypeptides. Cleavage products were visible after 4 hr of incubation. By 20 hr, cleavage products ranging from 18,000 to 50,000 mol. wt. were visible (Fig. 2). The cleavage pattern differed from that observed with trypsin or chymotrypsin at 1, 2, 6, and 20 hr. Our results, using purified substrate and purified, secreted enzyme, confirmed previous reports suggesting that cercariae of S. mansoni can enzymatically degrade skin keratin. Our analysis of keratin cleavage products by SDS polyacrylamide gel electrophoresis indicated that the sites of cleavage by cercarial enzyme differ from the sites of cleavage by the serine proteinases trypsin and chymotrypsin. Aside from its biologic role of facilitating cercarial penetration of skin, cercarial proteinase may be useful in studying the structural domains of keratin. Supported by The Edna McConnell Clark Foundation, The Burroughs Wellcome Fund, and National Institutes of Health (AM 12433).