Studies on artificial oligosaccharide-protein antigens: induction of precipitating antibodies to defined epitopes on natural and synthetic dextrans and mannans.

Seventeen di- and trisaccharides, composed of alpha-1-6-, alpha-1-2-, alpha-1-3- and alpha-1-4-linked glucosyl and alpha-1-6- and alpha-1-2-linked mannosyl residues, were synthesized. The oligosaccharides (OS) were transformed into the corresponding 2-(4-aminophenyl) ethyl alpha-D-glucosides or mannosides, which were either diazotized or converted into isothiocyanato derivatives and then coupled to BSA, edestin or hemocyanin to give artificial antigens. In this way immunogenic analogues of branched natural and linear synthetic dextrans, linear synthetic mannans and glucomannans were obtained. Upon immunization of rabbits with these conjugates, antibodies to the OS moieties carrying alpha-1-6, alpha-1-2 and alpha-1-3 glucosyl and alpha-1-6 mannosyl residues were elicited. These antibodies cross-reacted with and precipitated natural or synthetic polymers carrying the corresponding epitope pattern. The minimal size of an immunogenic OS residue required for cross-reactivity with the corresponding polymer was found to be either two or between one and two monosaccharide units. To obtain maximum and reliable elicitation of anti-OS antibodies of IgG isotype the use for coupling of an OS density corresponding to 10-25 moles of OS/mole of BSA is recommended. Other strongly immunogenic carriers may be used. In the case of homopolymers, each OS residue should have a length of at least six-eight sugar units.

Immunogenic properties of alpha (1----6) dextran, its protein conjugates, and conjugates of its breakdown products in mice.

Mice were immunized with alpha (1-6) dextran, either as such or coupled to protein carriers, and their anti-dextran response was measured by a solid-phase radioimmunoassay and the Farr assay. Like earlier investigators we found that protein-conjugated dextran was more antigenic than plain dextran. Our novel findings were that (1) a standard dose (30 micrograms of dextran per injection) coupled to strongly antigenic protein (chicken serum albumin (CSA) was three times more antigenic than dextran coupled to weakly antigenic bovine serum albumin (BSA); (2) dextrans of low molecular weight (1000-10,000 daltons) coupled to CSA induced at least ten times stronger secondary responses than did a similarly coupled macromolecular dextran (5-40 million daltons); (3) variation of the CHO/protein ratio from 0.3 to 1 had little effect on the antigenicity of the dextran. Increase of the ratio from one appeared to decrease immunogenicity when BSA was the carrier but not when CSA was the carrier.

Polyethylene glycol reactive antibodies in man: titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors.

Antibodies to polyethylene glycol (PEG) were analyzed in patients with various allergies and in healthy blood donors employing passive hemagglutination. In untreated allergic patients and in healthy blood donors, naturally occurring anti-PEG antibody titers between 32 and 512 were seen in 3.3 and 0.2%, respectively. During hyposensitization with monomethoxy polyethylene glycol modified ragweed extract and honey bee venom, respectively, the patients showed an anti-PEG antibody response. Titers of 32-512 were found in 50% of the patients directly after the first treatment course. After 2 years of treatment the percentage of patients with such titers declined to 28.5%. Mercaptoethanol treatment of sera indicated that the anti-PEG antibodies predominantly were of the IgM isotype. The weak IgM response found in treated patients is considered to be of no clinical significance.

Dextran-reactive antibodies in children. Appearance during the first 4 years of life and relation to E. coli infections in the urinary tract.

The appearance of dextran-reactive antibodies (DRA) was investigated in 88 children with sampling of serum at birth (n = 87), at 3 months of age (n = 87), at 8 months (n = 88), at 2 years (n = 86) and at 4 1/2 years of age (n = 87). Serum DRA appeared at 3 months of age and a peak level exceeding the levels in adults was noted at 8 months of age. At 4 1/2 years the titres were close to those in adults. Investigation of DRA in 8 children with acute pyelonephritis and in 8 children with asymptomatic bacteriuria caused by E. coli did not indicate that the appearance of DRA was a consequence of Gram-negative infections. The practical conclusion drawn is that if therapy with i.v. dextran is considered for infants or children, they should undergo preventive therapy with hapten dextran similar to the procedure recommended for adults.

Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins.

Antibodies to polyethylene glycol (PEG) were raised in rabbits by immunization with monomethoxy polyethylene glycol modified ovalbumin (OA), bovine superoxide dismutase (SOD), and ragweed pollen extract (Rag), given in Freund's complete adjuvant (FCA). Immunogenicity depended on the nature of the protein and the degree of modification. With modified OA, in the presence of FCA, the majority of animals showed an anti-PEG response. With modified SOD and Rag only a small proportion of animals responded. In the absence of FCA, modified OA, given s.c., did not elicit any anti-PEG antibody response in rabbits and only a weak response in mice. PEG of MW 10,000 and 100,000 given in FCA was found nonimmunogenic in rabbits, and PEG of MW 5.9 X 10(6), given s.c. to mice, showed no or very poor immunogenic properties. Gel diffusion, heterologous passive anaphylaxis and passive hemagglutination were used to demonstrate anti-PEG antibodies raised to PEG-modified proteins. Specificity was confirmed by hapten inhibition of precipitation, inhibition of passive hemagglutination and cross-reactivity tests. PEG of MW greater than or equal to 4,000 produced specific precipitates, smaller molecules acted as monovalent haptens. From hapten inhibition of precipitation by PEG of MW 300 it appears that the antigenic determinant of PEG may be a sequence of 6-7 -CH2CH2O-units. Anti-PEG antibodies can be used analytically. By gel diffusion, Peg was detected in minimal concentrations of 0.1-1 microgram/ml. The clinical relevance of these findings with regard to therapy with PEG-modified enzymes and allergens in humans remains to be established.

Dextran hypersensitivity.

Dextran, a common plasma substitute, sometimes induces life-threatening hypersensitivity reactions. In this article Wolfgang Richter and Harriet Hedin discuss recent evidence that these Type III anaphylactic reactions, caused by natural antibodies, can be abolished by pretreatment o f patients with monovalent hapten dextran.

A new immunochemical purity test for clinical dextran. Methodology and studies on clinical dextran preparations.

A simple immunochemical procedure, based on reversed single radial immunodiffusion (RSRI) was developed to detect commonly occurring traces of antigenic contaminants in clinical dextran. High-titred antisera against non-dextran components of Leuconostoc mesenteroides NRRL B512 were produced and served as analytical reagents. Dextran samples were incorporated into a gel layer. Presence of contaminants is revealed by precipitate formation following application of antiserum to a well cut into the gel. Antigenic contaminants can be detected in concentrations exceeding 10 ppm by the Leuconostoc-RSRI test. Screen results on 119 clinical dextran samples from manufactures in different countries disclosed presence of antigen traces in 82% of preparations. Introduction of the new test and improved purification measures at Pharmacia AB resulted in purer clinical dextran with negative RSRI test scores in 99% of batches produced. Only batches passing this test are released for clinical use. Although no direct correlation was found between contaminant levels and incidence of dextran reactions, antigenic contaminants may play a contributory role in elicitation of mild reactions.

Non-immunogenicity of a purified sodium hyaluronate preparation in man.

The immunogenicity of a sodium hyaluronate preparation for clinical use (Healon, Healonid, Pharmacia AB, Sweden) in ophthalmology and certain joint diseases has been tested in man. For immunization, two subcutaneous injections of 10 mg of sodium hyaluronate were given at an interval of 1 week. The following criteria were used as indicators of immunogenicity: skin prick test, microprecipitation test and complement analyses. No evidence for immunogenic properties was obtained in any of these tests. This refers to sodium hyaluronate itself and to the trace amounts of protein components contained in the preparation.

Antibodies against hydroxyethylstarch produced in rabbits by immunization with a protein-hydroxyetylstarch conjugate.

Antibodies (ABS) against hydroxyethylstarch (HES) were raised in rabbits by immunization with HES-bovine serum albumin conjugate. ABS against HES were demonstrable by gel diffusion, passive hemagglutination (PH) and passive cutaneous anaphylaxis (PCA); specificity was confirmed by inhibition of PH and neutralization of PCA. No ABS against starch could be induced in comparative experiments. Non-immunogenicity of starch was attributed to its structural similarity with glycogen, widely distributed in animal species. Absence of cross-reactivity of anti-HES ABS with starch, amylopectin and glycogen and strong reactivity with 2-hydroxypropylstarch (DS = 0.65) and HES (DS = 0.7-1.2) indicate that hydroxyethyl substitution created antigenic determinants and conferred new immunochemical identity on the modified starch molecule. Anti-HES ABS represent specific analytical tools for the identification and quantitation of HES in biological material.

Anaphylactoid reactions in connection with infusion of invert sugar solutions are due to macromolecular contaminants.

13 untoward mold anaphylactoid reactions were observed in patients infused with invert sugar solutions in Sweden during a 7-month period in 1973: an incidence of 1/31,000 infusions. Immunological and physicochemical analysis of invert sugar solutions and of the raw material, sucrose, revealed traces of native alpha-1,6-glucan with molecular weight of 10-100 millions as contaminant. This indicates, that the sucrose had been exposed to microbial contamination during its manufacture from sugar-beet or sugar-cane. Reversed single radial immunodiffusion was used for alpha-1,6-glucan detection and screening of all sucrose batches. Rejection of contaminated sucrose as raw material reduced the incidence of anaphylactoid invert sugar reactions to about 1/575,000 infusions. Examination of all sucrose raw material for traces of crude alpha-1,6-glucan is recommended as a test for detection of microbial contamination.