Quantification of Ponceau 4R in Foods by Indirect Competitive Enzyme-Linked Immunosorbent Assay (icELISA).

As one of the rarely allowable azo dyes, ponceau 4R can be added in some foods in some countries. However, it is necessary to develop a credible and rapid analytical method for its monitoring, because of its potentially harmful risk. The hapten of ponceau 4R was first designed and synthesized by introducing a primary amine group into the structure of ponceau 4R. Based on the well-prepared hapten, the immunogen of ponceau 4R was prepared using glutaraldehyde to link ponceau 4R to the carrier protein. The triggered polyclonal antibody was obtained and tested by ELISA to optimize the proper dilution. An icELISA was developed for ponceau 4R, and the IC50 of the method is 36.82 ng/mL. The limit of detection is 0.80 ng/mL, and the linear range is 1-10000 ng/mL. Five selected structural analogues have no cross-reactivity with the anti-ponceau 4R polyclonal antibody (<0.3). In three food samples (grape juice, carbonated beverage, and RIO cocktail), the assay exhibits good stability and reproducibility with a recovery range of 93.87-103.77%, and the intra- and interassay coefficients of variation were <11.73%. The results indicate that the proposed icELISA is sensitive, accurate, specific, and simple, which provides an alternative for the detection of ponceau 4R in foods.

Studies of the chemical selectivity of hapten, reactivity, and skin sensitization potency. 1. Synthesis and studies on the reactivity toward model nucleophiles of the (13)C-labeled skin sensitizers hex-1-ene- and hexane-1,3-sultones.

The potent skin sensitizers hex-1-ene- and hexane-1,3-sultone have been synthesized isotopically labeled with (13)C at reactive sites. The reactivity of 2-[(13)C]- and 3-[(13)C]hex-1-ene-1,3-sultones and of 3-[(13)C]hexane-1,3-sultone toward a series of model nucleophiles for protein amino acid residues, i.e., butylamine, diethylamine, imidazole, propanethiol, and phenol, was followed by (13)C NMR spectroscopy. The reactivity in water of hex-1-ene-1,3-sultone toward model nucleophiles follows the hard and soft acid and base theory with the hard nucleophiles (primary and secondary amine and phenate) mainly reacting at position 3 by S(N) substitution, and the soft nucleophiles (thiolate and imidazole) mainly reacting at position 2 by a Michael addition reaction. Hexane-1,3-sultone reacts with model nucleophiles at position 3 by S(N) substitution. Both saturated and unsaturated sultones are sensitive to hydrolysis when reacted in water.

Studies of the chemical selectivity of hapten, reactivity, and skin sensitization potency. 2. nmr studies of the covalent binding of the (13)c-labeled skin sensitizers 2-[13C]- and 3-[13C]hex-1-ene- and 3-[13C]hexane-1,3-sultones to human serum albumin.

3-[(13)C]- and 2-[(13)C]hex-1-ene-1,3-sultones (1a and 1b, respectively) and 3-[(13)C]hex-1-ene-1,3-sultone 2a were incubated with human serum albumin in phosphate buffer at pH 8.1. In both cases, the main reaction was a hydrolysis via an S(N) reaction at position 3, but several adducts were also formed. Hex-1-ene-1,3-sultone, which is a strong skin sensitizer, appears also to be a strongly oxophilic molecule reacting mainly at position 3 through an S(N) reaction to give adducts on tyrosines. This sultone was also able to react with a single lysine residue, also via an initial S(N) reaction at position 3, followed by an intramolecular Michael addition at position 2 to form a mixture of aziridinium intermediates which were subsequently hydrolyzed to give an amino alcohol derivative as the final product. The same reaction carried out on acetylated human serum albumin seems to indicate that the target lysine could be Lys199, which is known to be easily acetylated. Hexane-1,3-sultone, which is a weak sensitizer, appears to be an even more oxophilic molecule, making adducts on tyrosines through an S(N) reaction at position 3. No reaction was observed on Lys199. The difference in skin sensitization potential seems therefore to be more related to the selective ability of modifying lysine residues than to the more general ability to modify tyrosine residues.

Heterogeneity of IgE antibody response to reactive dye in sera from four different sensitized workers.

We studied RAST and RAST inhibition tests to black GR, the most frequent sensitizer among several reactive dyes in our previous study, in order to evaluate the specificity of IgE antibodies to hapten or new antigenic determinants and the crossreactivity between two reactive dyes, black GR and orange 3R, in sera from four different sensitized workers. RAST inhibition studies with black GR-human serum albumin (HSA) conjugate discs demonstrated that black GR-HSA conjugates were the most effective inhibitors. Orange 3R-HSA conjugates, unconjugated forms of black GR and orange 3R were weak inhibitors in two patients and non-inhibitory in one patient, whereas they caused strong dose-dependent inhibitions in one patient. These results suggested that the IgE response to black GR-HSA conjugates might be heterogenous and the crossreactivity between two reactive dyes differed from one patient to another.

Medium effects in antibody-catalyzed reactions.

Catalytic antibody technology has been used to explore the contribution of medium effects to the overall rate of an enzyme-catalyzed reaction. An antibody generated against a derivative of 2-acetamido-1,5-napthalenedisulfonate efficiently catalyzes the decarboxylation of 5-nitro-3-carboxybenzisoxazole. This unimolecular reaction is not susceptible to general acid-base catalysis but is highly sensitive to microenvironment; thus, it provides a simple chemical model for biologically important decarboxylations. The 10(4)-fold rate acceleration observed for the antibody reflects the kinetic advantage of the low-dielectric environment of the binding pocket acting to destabilize the substrate by desolvation and to stabilize the charge-delocalized transition state through dispersion interactions. These results are pertinent to an understanding of solvent effects in enzymic reactions in general and suggest approaches for developing antibody catalysts for numerous other reactions that involve large changes in charge distribution as the reaction coordinate is traversed.

An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers.

Methods of assaying reactive dye-specific IgE antibodies were investigated with a RAST. Sera from three patients, occupationally exposed to a reactive dye, Remazol black B (Chemical Abstract registry number 17095-24-8), were used. Directly dyed disks, that is, disks without any carrier protein, resulted in poor and unreliable measures of specific IgE. In contrast, optimized preparation of conjugates between the dye and human serum albumin resulted in efficient binding of specific IgE. The patients' RAST results were strongly positive, whereas sera from 36 exposed workers but without symptoms and sera from unexposed subjects with high levels of total IgE were negative. The hapten and carrier specificity of the IgE antibodies was studied by direct RAST and RAST inhibition. In one patient, the antibodies were principally hapten specific, whereas another patient was found to have antibodies with a high degree of specificity to the carrier. The third patient's antibodies were intermediate between the other two patients' antibodies in this respect, suggesting that antibody specificity is dependent not only on the nature of the hapten but also on individual immune response factors. The study demonstrates that it is important to use an optimized preparation of dye-protein conjugates to elicit reliable results and a high degree of specific IgE binding in the RAST.

T cell receptor variable gene usage in a specific cytotoxic T cell response. Primary structure of the antigen-MHC receptor of four hapten-specific cytotoxic T cell clones.

The primary structure of the alpha and beta chains of the T cell antigen receptor in four cytotoxic T cell clones specific for N-iodoacetyl-sulfonic-naphthyl-ethylene-diamine (AED)-haptenated target cells displaying a particular class I MHC molecule has been determined. Two of the T cell clones, 8/10-2 and 5/10-20K, recognize AED-modified targets in association with H-2Kb, while the other two clones 5/10-20D and C9 react with AED-modified cells in the context of H-2Db. Comparison of the nucleotide sequences of both the alpha and beta chain cDNAs and their deduced protein sequences indicates that a specific variable gene segment was not used to recognize the hapten and/or class I gene products. Furthermore, there does not appear to be any conserved amino acid residues used in the AED-specific response other than the framework amino acids. However, when the two clones 8/10-2 and 5/10-20D were compared, a striking similarity was seen in the J segments. These two clones that recognize AED in the context of different MHC epitopes used identical J alpha (J alpha 810) and J beta (J beta 2.6) gene segments. C9, specific for AED-Db, shared identical V beta (V beta 6) and J beta gene segments (J beta 1.1) as those of a cytotoxic T cell that recognizes allogeneic targets expressing Db. These data indicate that a simple rule governing the usage of the variable regions of either the alpha or beta T cell receptor (TcR) genes in the recognition of antigen and MHC gene products cannot be formulated. However, subtle similarities can be detected in some situations between the primary structures of the TcR and the targets they recognize.

Genetic control of hapten-reactive helper T-cell responses and its implications for the generation of augmented antitumor cytotoxic responses.

The genetic control of hapten-reactive helper T-cell activity involved in cytotoxic T-lymphocyte (CTL) responses and its implications for augmenting tumor-specific immunity were studied. C57BL/6N mice were immunized to trinitrophenyl (TNP) or N-iodoacetyl-N'-(5-sulfonic l-naphthyl)ethylenediamine (AED) hapten by inoculation of hapten-modified syngeneic spleen cells. Spleen cells from these hapten-immunized mice were tested for hapten-reactive helper T-cell activity for generation of CTL. TNP-primed spleen cells resulted in only marginal help for the generation of anti-TNP-modified syngeneic spleen cell (TNP-self) CTL response when cocultured with normal C57BL/6N spleen cells (responding cells) in the presence of TNP-self. In contrast, AED-primed spleen cells exhibited appreciable help for AED-induced CTL responses. Furthermore, AED-helper, but not TNP-helper, T-cell activity was demonstrated to augment the generation of antitumor (RBL-5 leukemia) CTL responses from normal syngeneic spleen cells when stimulated with the corresponding hapten-self plus RBL-5 tumor cells. These results indicate that the successful augmentation of syngeneic tumor immunity through T-T-cell interaction with the use of hapten-reactive helper T-cells can depend on selection of the appropriate haptenic reagent in an individual expressing a given major histocompatibility haplotype.

Restriction fine specificity of long-term, hapten-specific cytotoxic T cell clones: analysis with H-2Kbm-mutant mice and H-2Kb-specific monoclonal antibodies.

The cell-mediated cytotoxic response against autologous cells modified with the sulfhydryl reagent I-AED [N-iodo-acetyl-N-(5-sulfonic-1-naphthyl) ethylene diamine] has been described by Levy, R. B., Shearer, G. M., Richardson, J. C. and Henkart, P. A., [J. Immunol. 1981. 127: 523.]. We have established two H-2Db- and eight H-2Kb-restricted, AED-specific long-term cytotoxic T lymphocyte (CTL) clones of C57BL/10 origin. The growth of these clones has been dependent upon presence of both antigen and interleukin 2. Cytotoxicity and proliferation analysis of AED-specific Kb-restricted CTL clones with target and stimulator cells from Kbm-mutant mice demonstrated two categories of clones (A and B) based on different reactivity patterns against hapten-modified Kbm-mutant cells. AED-modified target cells of bm5, bm6 and bm9 origin were lysed by type A clones but not by type B clones, in contrast to AED-modified bm3, bm8 and bm11 target cells which were lysed by type B but not by type A clones. None of the clones lysed AED-modified bm1 target cells, but all of them lysed AED-modified bm4 and bm10 target cells. The restriction fine specificities for both cytolytic and proliferative activities of the analyzed clones were identical. A panel of monoclonal antibodies (mAb) directed against the Kb molecule was used to inhibit lysis of AED-modified target cells by CTL clones. mAb which recognize a cluster of allodeterminants located in the second external domain (C1 domain) of the Kb molecule [Hämmerling, G. J., Rüsch, E., Tada, N., Kimura, S. and Hämmerling, U., Proc. Natl. Acad. Sci. USA 1982. 79: 4737] only inhibited type B clones, whereas inhibition of both type of clones was observed with mAb specific for allodeterminants clustered in the N-terminal region of Kb. These findings suggest that different regions on the Kb molecule serve as self determinants for H-2-restricted cytotoxicity. We also demonstrate that allodeterminants recognized by mAb and self determinants involved in H-2 restriction need not be identical. Our data also support the notion that covalent AED modification of H-2 molecules is not necessary to generate the self plus X antigen for CTL recognition.

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses.

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.

Thermodynamics of hapten-antibody interactions.

Two schools of thought are currently prevalent regarding the thermodynamic mechanism(s) of hapten-antibody interaction(s). While one school is a proponent of the hapten-antibody reaction being driven predominantly by enthalpy, the second school rationalizes the mechanism as an enthalpy-entropy compensation, the magnitude of the latter being dependent on the temperature at which hapten-antibody interaction is carried out.

Cross-reactive recognition of alloantigens and certain B6 mutant antigens by antigen-specific and polyclonally activated cytotoxic effector cells.

Various B6 cytotoxic T lymphocyte (CTL) effector populations were tested for cross-reactive lysis (CRL) of unmodified third-party allogeneic as well as of various Kbm and Dbm mutant target cells. The effector cells were generated by in vitro stimulation of B6 spleen cells either from unprimed mice against alloantigen, Con A, or a pool of different allogeneic stimulators, or from in vivo hapten-self primed mice against trinitrophenyl (TNP)-self or N-(5-sulfonic-1-naphthyl)ethylenediame (AED)-self antigens. Except for AED-primed effector cells all CTL populations exhibited significant CRL on third-party allogeneic target cells. More importantly, these alloreactive and H-2-restricted effectors also lysed some of the Kb mutants, but not others. Thus, unmodified bm1 and bm11 mutants were lysed by all of the B6 wild-type effectors, whereas bm6 and bm9 were only weakly lysed by alloreactive CTL. None of the B6 CTL populations lysed bm13 and bm 14 target cells. These data indicate that (a) alloreactive and self-restricted CTL can recognize the same molecular structure of Class I antigens although the epitopes may be different; (b) CTL discriminate between a few amino acid substitutions in certain positions of the H-2 molecule, and (c) the newly generated antigenic determinants of the bm mutants allow them to be divided into three categories in terms of their allogenicity for these cross-reactive CTL populations.

The products of transferred H-2 genes express determinants that restrict hapten-specific cytotoxic T cells.

Cell lines into which cloned H-2 genes had been introduced (i.e., transformants) were used to correlate the genes and their products that are capable of functioning as H-2 restriction elements for hapten-self-(AED and TNP) specific cytotoxic T cells (CTL). These transformants provided a unique system in which major histocompatibility restricted (MHC) T cell recognition could be examined by using cells that express only H-2Ld or only H-2Dd gene products. BALB/c (H-2d) anti AED-self CTL lysed both the H-2Ld and Dd transformants, but not parental, i.e., untransformed, cells. The AED-self lysis of the Ld and Dd transformants was shown to be specifically inhibited by anti-H-2Ld and anti H-2Dd monoclonal antibody, respectively. In contrast to these results, BALB/c anti TNP-self CTL were found to lyse readily the Dd but not Ld transformed lines, supporting reports indicating that H-2Ld-restricted TNP-self CTL could not be detected. The results of this study thus demonstrate that the cell surface products encoded by these transferred MHC class I genes contain self determinants recognized by CTL.

Cell-mediated lympholytic responses against autologous cells modified with haptenic sulfhydryl reagents. V. H-2Ld self products are recognized by anti-AED-specific cytotoxic T cells.

The present studies were designed to examine the restriction elements involved in the H-2d haplotype CTL response against AED-self. BALB/c mice were immunized by in vivo administration of syngeneic BALB/c spleen cells conjugated with the sulfhydryl reactive haptenic reagent I-AEDANS. The cytotoxic T cell response generated by subsequent in vitro stimulation with AED-self was found to contain H-2K and H-2D region-specific components. In contrast to the predominant H-2D hapten-self CTL responses induced by amino-reactive haptenic reagents like TNBS and FITC, the AED-self response was predominantly directed against H-2K self products. Antibody inhibition analysis demonstrated that the H-2D region component contained Ld-AED-self CTL, as well as Dd-AED-self CTL. However, although both Ld- and Dd-restricted components could be identified shortly (less than 1 wk) after priming, the H-2D region AED-self CTL response shifted to a single restricting molecule, namely Dd, 1 wk and later after immunization. The results of this study provide the first demonstration that the H-2L locus encodes a cell surface product that can function as a restricting molecule for hapten-self-specific CTL. The shift from Ld and Dd AED-specific CTL to Dd only suggests the existence of a highly specific regulatory mechanism directed against antigen (i.e., hapten) plus self.

Induction of cell-mediated cytotoxic self responses by epidermal cells modified with a haptenic sulphydryl reagent.

Murine epidermal cells (EC) act as stimulator cells in the generation of allogeneic cytotoxic T lymphocytes (CTL) in cell-mediated lympholysis (CML) and are suitable targets for allogeneic and hapten-self CTL. To analyse the role of EC in the generation of and recognition by anti-self CTL, syngeneic hapten-modified murine EC were used as in vivo and in vitro stimulating populations and as target cells in a hapten-self CML system. Epidermal cells were modified with the sulphydryl-reactive haptenic reagent N-iodoacetyl-N'-(5-sulphonic-1-naphthyl)ethylenediamine (I-AED). C3H.SW (H-2b) AED-self CTL responses were generated by stimulation with syngeneic AED-modified EC and were readily demonstrated when tested on syngeneic hapten-modified EC. These CML responses were hapten-specific and H-2 restricted. No substantial difference was detected in the ability of AED-modified EC and spleen cells (SC) to stimulate the generation of secondary AED-self CTL. Cold target inhibition experiments with hapten-modified EC and SC blockers did not reveal tissue-specific recognition of hapten-modified EC or SC targets by AED-self CTL. These findings demonstrate that hapten-modified EC, when used for priming in vivo and subsequently for in vitro sensitization, can induce hapten-specific self CTL that are reactive against syngeneic hapten-modified EC.

Analysis of Ir gene control of cytotoxic response to hapten-modified self: helper T cells specific for a sulfhydryl hapten can substitute for an anti-TNP-H-2b self helper cell defect.

Helper T cells specific for N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine (I-AED) were generated in (C56BL/6 X C3H/He)F1 mice by immunization with I-AED-modified syngeneic cells (AED-self). The requirements for activation of hapten-induced helper cells were investigated. The results demonstrated that activation of AED and trinitrophenyl- (TNP) helper cells was strictly hapten specific. In addition, F1 AEd-helpers could be activated efficiently by either I-AED-modified H-2b or H-2k self components to enhance the anti-AED self-CTL responses. This contrasts with the previous findings demonstrating the failure of TNP-H-2b self to activate F1 TNP-helper cells. After AED-helpers were activated, they were capable of augmenting sensitization of cytotoxic T cells (CTL) against TNP-self. These results indicate that although the activation of hapten-reactive helper cells is antigen (hapten)-specific, the subsequent helper activity, as determined by augmentation of CTL responses against another hapten, is antigen nonspecific. Since helper function was antigen nonspecific, F1 AED-helper cells activated by AED-H-2b or AED-H-2k self were tested for their ability to augment the F1 and anti-TNP-H-2b CTL response. The results indicate that the Ir gene defect in the ability of F1 spleen cells to respond to TNP-H-2b self could not be corrected by these helper cells. These results are discussed in the light of Ir gene controlled differences in the activation of AED and TNP-helper cells and possible models for augmenting CTL responses against various antigens in strains that generate marginal helper activity to TNP-self.

Cell-mediated lympholytic responses against autologous cells modified with haptenic sulfhydryl reagents. I. Effector cells can recognize two distinct classes of hapten-reactive self sites on cell surface proteins.

Mouse spleen cells were modified with a sulfhydryl-reactive reagent, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine (I-AED) and used as stimulator cells in primary in vitro cultures with unmodified spleen cells of the same strain. CTL could be readily demonstrated when tested on hapten-modified syngeneic target cells. These CML responses were hapten specific and H-2 restricted. In the H-2b strain, effector cell populations were identified that recognized AED in association with K-end and D-end coded self MHC products. Functional modification of the target cells by I-AED can be effectively blocked by prereacting the cells with other SH-reagents, which is consistent with CTL recognition of the hapten on cysteine residues. Since these premodifications of the cell surface do not block the functional modification by trinitrobenzene sulfonate (TNBS), the sulfhydryl-reactive compounds and TNBS modify distinct classes of cell surface groups as determined by CTL function. The results of antibody inhibition experiments are also consistent with this interpretation. Using fluorescent labeled anti-hapten antibodies, FACS II analysis showed that there are approximately 20-fold fewer AED than TNP groups on the approximately 20-fold fewer AED than TNP groups on the cell surface when equivalent concentrations of I-AED and TNBS are compared. The possibility is discussed that these sulfhydryl-reactive compounds can be used together with the H-2Kb mutants for the localization of H-2 coded self determinants recognized in association with foreign antigens.

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. II. Analysis of the genetic control and H-2-restricted pattern of cytotoxic responses to sulfhydryl and amino reactive reagents.

N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylene-diamine modified autologous cells are shown to induce respective high and low AED-self CTL responses by spleen cells from H-2b and H-2k mice. The lysis mediated by effector populations from both haplotypes was highly H-2 restricted. Cytotoxic responses induced by a 2nd sulfhydryl- (SH) specific reagent, dithionitrobenzoic acid (DTNB), were shown not to be cross-reactive at the effector cell level with AED-self but exhibited the same high and low CTL response patterns as those induced against AED-self. These Ir-effects reported for the 2 SH reactive compounds are discussed in relation to the opposite Ir-patterns demonstrated against a number of amino (NH2) reactive compounds. The difference of self-recognition sites is suggested to account for the contrasting results observed in the response patterns induced by SH- and NH2-reactive compounds. The AED-self CTL response was found to exhibit Kk-self over Db-self Ir regulation. Thus, this latter type of genetic control appears to function independently of whether the hapten is an SH- or NH2-reactive compound.