Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine.

During inhalation of allergens and experimental sepsis, formation of brominated tyrosine has been reported. In this study, we first examined the immunogenicity of brominated protein prepared by treatment of N-bromosuccinimide (NBS). The immunized serum obtained reacted with brominated bovine serum albumin (BSA). The NBS dose-dependent formation of immunoreactivity, which was estimated by enzyme-linked immunosorbent assay, was observed, and the increase coincided with 3,5-dibromotyrosine (DiBrY) formation in the modified BSA, which was chemically determined by liquid chromatography/quadrupole tandem mass spectrometry (LC/MS/MS). Second, by use of immunized mice, monoclonal antibodies to the brominated one were prepared. The two established novel monoclonal antibodies obtained from the immunized mice reacted with DiBrY, 3,5-dichlorotyrosine (DiClY), and 3,5-diiodotyrosine (DiIY). Moreover, 3,5-dihalo-4-hydroxybenzoic acids (3,5-dichloro-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzoic acid) were recognized by these antibodies. These results suggest that dihalogenated tyrosines (DiBrY, DiClY, and DiIY) are the epitopes. Lastly, we used the antibody in an immunohistochemical study. Lipopolysaccharide (LPS) was intraperitoneally administered to mice, and livers were removed. Positive staining of LPS-treated mouse liver tissues by both the anti-dihalotyrosine antibody and anti-myeloperoxidase antibody was estimated, suggesting that inflammatory tissue damage induces the formation of dihalotyrosine in vivo.

Studies on experimental iodine allergy: 1. Antigen recognition of guinea pig anti-iodine antibody.

It has generally been thought that iodine allergy is cross-sensitive to various iodine-containing chemicals. However, this concept seems to deviate from the immunological principle that immune recognition is specific. To solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to iodinated autologous proteins produced in vivo by iodination reaction from various iodine-containing chemicals. Antisera to iodine were obtained from guinea pigs immunized subcutaneously with iodine-potassium iodide solution emulsified in complete Freund's adjuvant (CFA). The specificity of guinea pig anti-iodine antiserum was determined by enzyme-linked immunosorbent assay (ELISA) inhibition experiments using microplates coated with iodinated guinea pig serum albumin (I-GSA). Antibody activities were inhibited by I-GSA, diiodo-L-tyrosine, and thyroxine, but not by potassium iodide, monoiodo-L-tyrosine, 3,5,3'-triiodothyronine, monoiodo-L-histidine, or diiodo-L-histidine, or by ionic or non-ionic iodinated contrast media. The results that antigen recognition of anti-iodine antibody is specific to iodinated protein support our hypothesis. While protein iodination usually takes place both at histidine residues as well as at tyrosine residues, only iodinated tyrosine acted as an antigenic determinant and no antibody activities to iodinated histidine were detected in our experimental iodine allergy model.

Studies on experimental iodine allergy: 3. Low molecular weight elicitogenic antigens of iodine allergy.

We hypothesize that iodine allergy is an immune response to iodinated self proteins produced in vivo from various iodine-containing chemicals. Since an antigenic determinant of experimental iodine allergy is diiodotyrosine (DIT), we designed low molecular weight DIT derivatives having provocative antigenicity without sensitizing immunogenicity. Tetraiododityrosine and hexaiodotrityrosine provoked dose-dependent skin reactions in guinea pigs previously immunized with iodine. No guinea pigs immunized with hexaiodotrityrosine showed anaphylactic reaction by i.v. challenge with hexaiodotrityrosine and none of their antisera showed positive passive cutaneous anaphylaxis (PCA) reaction in guinea pigs, indicating the non-immunogenic nature of the compound. Erythrosine, one of the color additives having a structure common with DIT, was assessed for its immunological property. Enzyme-linked immunosorbent assay (ELISA) inhibition studies on erythrosine revealed that the inhibitory activity of erythrosine was stronger than that of DIT. Furthermore, erythrosine provoked a PCA reaction in animals sensitized with anti-iodine antisera. In conclusion, hexaiodotrityrosine is thought to be useful for skin testing of iodine allergy without any fear of sensitization to the allergen. Erythrosine was shown to provoke an experimental iodine allergy and, also, the relationships between the new concept of iodine allergy and features of clinical findings of adverse effects by iodocontrast media are discussed.

IgG purification to measure the level of an iodinated thyroglobulin peptide, the 3,5,3',5' tetraiodo-l-tyrosyl-l-tyrosine in human serum.

Antibodies reacting with 3,5,3',5' tetraiodo-l-tyrosyl-l-tyrosine (I2Tyr-I2Tyr) were elicited in rabbits by immunization with an oxidized yeast conjugate coupled with I2Tyr-I2Tyr. Ion-exchange chromatography was used to purify immunoglobulins, in order to improve the specificity in measurement of I2Tyr-I2Tyr level in patient serum. IgG binding capacity versus I2Tyr-I2Tyr was considerably increased after immunoglobulin purification.

Circulating diiodotyrosine: studies of its serum concentration, source, and turnover using radioimmunoassay after immunoextraction.

This report describes the application of a sensitive, specific, and reproducible RIA for diiodotyrosine (DIT) in human serum and metabolic studies on the source and kinetics of circulating DIT. Interference by cross-reactivity of T4 and other analogs was completely eliminated by isolation of DIT from serum with an efficient preparative immunoprecipitation technique. Mean (+/- SD) serum DIT levels were 161 +/- 133 pmol/liter (7.0 ng/100 ml) in 41 normal subjects, 64 +/- 30 pmol/liter in 46 pregnant women, 241 +/- 83 pmol/liter in the cord serum of 48 newborn infants, 542 +/- 494 pmol/liter in 22 hyperthyroid patients, and 101 +/- 71 pmol/liter in 15 hypothyroid patients. Mean values in pregnant, newborn and hyperthyroid subjects were significantly different from the normal mean. Very low DIT serum levels were found in four athyreotic patients during oral T4 substitution therapy, indicating that little DIT is formed by peripheral T4 degradation. In five normal subjects who received a single oral dose of 3 mg T4, serum DIT remained unchanged in one case and decreased in four cases. Radioimmunological measurements of DIT elimination from serum after the iv injection of 1 mg DIT in two normal volunteers gave MCRs of 103 and 133 liters/day and an average extrathyroidal DIT turnover rate of 19 nmol/day (8.2 microgram/day). These data indicate that circulating DIT arises predominantly from the thyroid, suggesting that peripheral formation of DIT is a minor metabolic pathway in the human.

Measurement of plasma and tissue triiodothyronine concentration in the rat by radioimmunoassay.

This paper reports a radioimmunoassay method for triiodothyronine (T3) and the application of this assay to the study of plasma and tissue T3 concentration in the rat. Several antisera formed to a T3-bovine fibrogen complex in guinea pigs and T3-thyroglobulin complex in rabbits are shown to have low or no cross reactivity with T4, monoiodotyrosine, diiodotyrosine, tetraiodothyroacetic acid (TETRAC) and reverse T3. Cross reactivity with T3 derivatives, triiodothyroacetic acid (TRIAC), and triiodothyropropionic (TRIPROP) was variable, some antisera differentiating moderately well and others not at all. An extraction method is described which removes approximately 85% of added 125I-T3 or unlabelled T3 from tissues and in the final step represents 57% of total tissue T3. Mean plasma T3 in 5 normal male rats was 58 plus or minus 6.0 ng/100 ml (SEM), in liver 769 plus or minus 56 ng/100 g, and kidney 627 plus or minus 39 ng/100 g. Tissue to plasma concentration gradients for liver and kidney were 13.3 and 10.8, respectively.