Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy.

BACKGROUND: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. NEW METHOD: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. COMPARISON WITH EXISTING METHOD AND RESULTS: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. CONCLUSION: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.

Riboflavin photo-cross-linking method for improving elastin stability and reducing calcification in bioprosthetic heart valves.

BACKGROUND: Glutaraldehyde cross-linked bioprosthetic heart valves might fail due to progressive degradation and calcification. METHODS: In this study, we developed a new BHVs preparation strategy named as "HPA/TRA/FMN" that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA) conjugated pericardium and riboflavin 5'-monophosphate (FMN) initiated photo-cross-linking method. HPA/TRA-pericardium conjugation would provide extra phenol groups for FMN initiated photo-cross-linking. RESULTS: The feeding ratio of riboflavin 5'-monophosphate was optimized. The collagenase and elastase enzymatic degradation in vitro, biomechanics, calcification, elastin stability in vivo, and macrophage marker CD68 were characterized. We demonstrated that riboflavin photo-cross-linked pericardiums had great collagen and elastin stability, improved mechanical properties, better resistance for calcification, and less CD68 positive macrophages in rat subdermal implantation study. CONCLUSIONS: This new riboflavin photo-cross-linking strategy would be a promising method to make BHVs which have better elastin stability, less calcification, and reduced inflammatory response.

A new approach of indirect enzyme-linked immunosorbent assay for determination of D-glutamic acid through in situ conjugation.

We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 µ g/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.

Effect of mouse strain in a model of chemical-induced respiratory allergy.

The inhalation of many types of chemicals is a leading cause of allergic respiratory diseases, and effective protocols are needed for the detection of environmental chemical-related respiratory allergies. In our previous studies, we developed a method for detecting environmental chemical-related respiratory allergens by using a long-term sensitization-challenge protocol involving BALB/c mice. In the current study, we sought to improve our model by characterizing strain-associated differences in respiratory allergic reactions to the well-known chemical respiratory allergen glutaraldehyde (GA). According to our protocol, BALB/c, NC/Nga, C3H/HeN, C57BL/6N, and CBA/J mice were sensitized dermally with GA for 3 weeks and then challenged with intratracheal or inhaled GA at 2 weeks after the last sensitization. The day after the final challenge, all mice were euthanized, and total serum IgE levels were assayed. In addition, immunocyte counts, cytokine production, and chemokine levels in the hilar lymph nodes (LNs) and bronchoalveolar lavage fluids (BALF) were also assessed. In conclusion, BALB/c and NC/Nga mice demonstrated markedly increased IgE reactions. Inflammatory cell counts in BALF were increased in the treated groups of all strains, especially BALB/c, NC/Nga, and CBA/J strains. Cytokine levels in LNs were increased in all treated groups except for C3H/HeN and were particularly high in BALB/c and NC/Nga mice. According to our results, we suggest that BALB/c and NC/Nga are highly susceptible to respiratory allergic responses and therefore are good candidates for use in our model for detecting environmental chemical respiratory allergens.

Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells.

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid.

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)-ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC(50) value for PMPA being 0.027 µg mL(-1). The assay could detect PMPA even at the concentration of 0.006 µg mL(-1). The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.

The respiratory allergen glutaraldehyde in the local lymph node assay: sensitization by skin exposure, but not by inhalation.

Previously, a selection of low molecular weight contact and respiratory allergens had tested positive in both a skin and a respiratory local lymph node assay (LLNA), but formaldehyde was negative for sensitization by inhalation. To investigate whether this was due to intrinsic properties of aldehyde sensitizers, the structurally related allergen glutaraldehyde (GA) was tested. BALB/c mice were exposed by inhalation to 6 or 18ppm GA (respiratory LLNA), both generated as a vapor and as an aerosol. Other groups received 0.25% or 2.5% GA on the skin of the ears (skin LLNA). Lymphocyte proliferation and cytokine production were measured in the draining lymph nodes. GA was positive in the skin LLNA and its cytokine profile (IL-4/IFN-γ) skewed towards a Th2-type immune response with increasing dose. Inhalation exposure did not result in increased lymphocyte proliferation or increased cytokine levels, despite comparable tissue damage (irritation) in the skin and respiratory tract. We hypothesize that the highly reactive and hydrophilic GA oligomerizes in the protein-rich mucous layer of the respiratory tract, which impedes sensitization but still facilitates local irritation. Within the context of risk assessment in respiratory allergy, our results stress the importance of prevention of skin--besides inhalation-- exposure to aldehydes like GA.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines.

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Correlation between adjuvanticity and immunogenicity of cholera toxin B subunit in orally immunised young chickens.

The aim of the present study was to investigate whether the adjuvanticity of the cholera toxin B (CTB) subunit was correlated with its immunogenicity in young orally immunised chickens. Thirteen 15-day-old chickens were orally immunised with bovine serum albumin (BSA) glutaraldehyde coupled to CTB. The chicken antibody (IgG) concentrations against BSA and CTB, respectively, were quantified by ELISA. A significant positive correlation (r=0.66, n=39, p<0.001) between the concentrations of immunospecific antibodies with specificities against BSA and CTB, respectively, demonstrated that the adjuvanticity of CTB is correlated with its immunogenicity.

Antibodies directed against nitrosylated neoepitopes in sera of patients with human African trypanosomiasis.

Antibodies directed against nitrosylated epitopes have been found in sera from patients suffering from human African trypanosomiasis (HAT) but not in sera from control subjects living in the same endemic area or African control subjects living in France. We conjugated amino acids to albumin by glutaraldehyde (conjugates) and then nitrosylated the conjugates. Both conjugates and nitrosylated conjugates were analysed by enzyme-linked immunosorbent assay (ELISA). We detected antibodies directed against nitrosylated L-cysteine and L-tyrosine conjugates; antibody levels were higher in stage II patients than in stage I. Patients with severe clinical signs had higher antibody levels, and antibody levels were highest in patients with major neurological signs. Antibody response was only associated with the IgM isotype. We evaluated antibody specificity and avidity by competition experiments using conjugates and nitrosylated conjugates. Avidity was around 2 x10(-6) m for the S-nitroso-cysteine epitope and 2 x 10(-8) m for the S-nitroso-tyrosine epitope. Detection of circulating antibodies to S-nitroso-cysteine and S-nitroso-tyrosine epitopes provides indirect evidence for nitric oxide (NO) involvement in HAT and their levels are correlated with disease severity.

Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.

Survey of symptoms, respiratory function, and immunology and their relation to glutaraldehyde and other occupational exposures among endoscopy nursing staff.

OBJECTIVES: To find the nature and incidence of symptoms experienced by a large sample of hospital endoscopy nurses. To find whether nurses in endoscopy units develop asthma under current working conditions in endoscopy units. To obtain analytically reliable data on exposure concentrations of glutaraldehyde (GA) vapour in endoscopy units, and to relate them to individual hygiene and work practices. To characterise any exposure-response relations between airborne GA and the occurrence of work related symptoms (WRSs). Due to the growing concern about the perceived increase in WRSs among workers regularly exposed to biocides, all of whom work within a complex multiexposure environment, a cross sectional study was designed. METHODS: Current endoscopy nurses (n=348) from 59 endoscopy units within the United Kingdom and ex-employees (who had left their job for health reasons (n=18) were surveyed. Symptom questionnaires, end of session spirometry, peak flow diaries, skin prick tests (SPTs) to latex and common aeroallergens, and measurements of total immunoglobulin E (IgE) and IgE specific to GA and latex were performed. Exposure measurements included personal airborne biocide sampling for peak (during biocide changeover) and background (endoscopy room, excluding biocide changeover) concentrations. RESULTS: All 18 ex-employees and 91.4% of the current nurses were primarily exposed to GA, the rest were exposed to a succinaldehyde-formaldehyde (SF) composite. Work related contact dermatitis was reported by 44% of current workers exposed to GA, 56.7% of those exposed to SF composite, and 44.4% of ex-employees. The prevalence of WRSs of the eyes, nose, and lower respiratory tract in current workers exposed to GA was 13.5%, 19.8%, and 8.5% respectively and 50%, 61.1%, and 66.6% in the ex-employees. The mean percentage predicted forced expired volume in 1 second (ppFEV(1)) for ex-employees (93.82, 95% confidence interval (95% CI) 88.53 to 99.11) was significantly lower (p<0.01) than that of current workers exposed to GA (104.08, 95% CI 102.35 to 105.73). Occupational peak flow diaries completed by current workers with WRSs of the lower respiratory tract showed no evidence of bronchial asthma (<15% variation). Six per cent of the population had positive latex SPTs. Positive indications of one GA specific IgE and 4.1% latex specific IgE occurred. There was no conformity between the latex specific IgE and positive SPTs. Positive SPTs to latex were associated with WRSs of dermatitis and ocular WRSs, but no other WRSs. Exposures were above the current maximum exposure limit (MEL) of 0.2 mg/m(3) (0.05 ppm) in eight of the units investigated. A significant relation existed between peak GA concentrations and work related chronic bronchitis and nasal symptoms (after adjustment for types of local ventilation) but not to other WRSs. Peak GA concentrations were significantly higher in units that used both negative pressure room and decontaminating unit ventilation. CONCLUSION: This study documents a significant level of symptoms reported in the absence of objective evidence of the physiological changes associated with asthma. Ex-employees and current workers with WRSs warrant further study to elucidate the cause and mechanisms for their symptoms. Ventilation systems used for the extraction of aldehydes from the work area may be less effective than expected and due to poor design may even contribute to high peak exposures.

Allergic contact dermatitis from glutaraldehyde in health-care workers.

Glutaraldehyde is considered the disinfectant of choice for sterilizing medical and dental equipment. Unfortunately, glutaraldehyde has many toxic side-effects, including the ability to induce allergic contact dermatitis. In a 5-year study at the University of Kansas, 468 patients were patch tested to glutaraldehyde. A comparison of results was made between those employed in a healthcare related field and those who were not. Health-care workers (HCWs) were more than 8x more likely to be allergic to glutaraldehyde than their non-health-care working peers (NHCWs). Statistically significant differences between HCWs and NHCWs were seen in their reactivity to glutaraldehyde, thimerosal, benzalkonium chloride and methyl methacrylate. A higher than expected co-reactivity between glutaraldehyde and formaldehyde was also noted among HCWs and NHCWs, which cannot fully be explained by concomitant exposure. Allergic contact dermatitis from glutaraldehyde often causes persistent dermatitis, which frequently forces patients to leave their jobs. Although the National Institute of Occupational Safety and Health has published guidelines for safe handling of glutaraldehyde, allergy appears to continue to rise, especially among those employed in health-care professions. Until a less sensitizing disinfectant is developed, it is in the best interest of those in health-care professions, and other professions exposed to glutaraldehyde, to heighten occupational safety standards and to improve methods of barrier protection.

Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model.

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.

Glutaraldehyde: an occupational hazard in the hospital setting.

BACKGROUND: We report a series of 24 health-care workers with respiratory symptoms suggestive of occupational asthma due to glutaraldehyde exposure. METHODS: The history of asthmatic symptoms was investigated with peak expiratory flow rate (PEFR) monitoring, and in eight of the subjects, the specific bronchial provocation test (SBPT) was applied as reference standard for diagnosis of occupational asthma. Levels of glutaraldehyde were monitored in the challenge chamber during the SBPT. Work environmental levels of glutaraldehyde were measured from air samples collected at least once during the PEFR monitoring of endoscopy and theatre nurses. Specific IgE antibodies to glutaraldehyde were measured with a series of glutaraldehyde modified proteins. RESULTS: In the eight workers who underwent SBPT, the diagnosis of occupational asthma was confirmed by a positive reaction (late and dual reaction in five and in three subjects, respectively). The mean level of glutaraldehyde observed during the challenge tests was 0.075 mg/m3 (range 0.065-0.084 mg/m3). In 13 out of the 16 remaining workers, the serial PEFR monitoring showed a work-related effect. In three workers, there was no physiological confirmation of occupational asthma. Levels of glutaraldehyde from the air samples collected in the workplace were as follows: personal short-term samples (mean 0.208 mg/m3; median 0.14 mg/m3; range 0.06-0.84 mg/m3), personal long-term samples (mean 0.071 mg/m3; median 0.07 mg/m3; range 0.003-0.28 mg/m3). Measurements of specific IgE antibodies to glutaraldehyde-modified proteins were positive in seven patients (29.1%) according to a cutoff value of 0.88% RAST binding. The presence of atopy to common environmental allergens and smoking was not associated with specific IgE positivity (P>0.05; Fisher's exact test). CONCLUSIONS: Our report indicates the importance of glutaraldehyde as an occupational hazard among exposed health-care workers. Intervention in the workplace, training of personnel handling this chemical, and accurate health surveillance may reduce the risk of developing occupational asthma due to glutaraldehyde.

[Analysis of B-epitopes formed on proteins after treatment with glutaraldehyde]. / Analiz B-épitopov, formiruiushchikhsia na belkakh pri obrabotke glutarovym al'degidom.

Structural analysis of the B-epitopes formed on protein macromolecules by glutaraldehyde treatment with subsequent block of free active groups by glycine was performed. Two types of antigenic determinants recognized by different antibody populations were found by protein chemistry and immunochemistry techniques. An efficient method of blocking the antibodies to the epitopes formed on proteins by glutaraldehyde was developed.

In vivo protection against Androctonus australis hector scorpion toxin and venom by immunization with a synthetic analog of toxin II.

A synthetic peptide mimicking the North African scorpion Androctonus australis hector toxin II was designed and produced by chemical solid-phase synthesis. It contains the entire sequence of toxin II (64 amino acid residues), with each half-cystine being replaced by the isosteric residue a-aminobutyric acid, and was thus devoid of disulfide bridges. This construct was totally nontoxic in mice even if large amounts, equivalent to 1000 times the LD50 of the original toxin, were injected by the intracerebroventricular route. The synthetic peptide, either as a monomer or polymerized by means of glutaraldehyde, induced the production of antitoxin neutralizing antibodies in immunized mice and rabbits. After three injections with either the monomeric or polymerized synthetic peptide, the immunized mice were protected against several lethal doses of the corresponding native toxin or scorpion venom. Six months after immunization, the mice were completely protected against challenge with eight LD50 of the original toxin. The protection was better when the polymerized synthetic peptide was used. One month after the start of the immunization program, it showed a good correlation between antibody titer and protection. However, antibody titer decreased with time but protection remained high. This suggests that additional factors other than circulating antibodies play a role in protective activity.

Immunochemical approach to characterize post-translational modification of serum albumin using anti-glutaraldehyde-treated serum albumin antibodies.

Polyclonal antibodies against glutaraldehyde-treated rabbit serum albumin (pRSA) and human serum albumin (pHSA) were prepared from rabbit and mouse, respectively. Anti-pRSA antibody had the structural determinant depending on the polymerization process of RSA and showed only a weak cross-reactivity with the other glutaraldehyde-treated albumins. Anti-pHSA antibody (after adsorption of anti-HSA antibody) recognized only pHSA, but not HSA and the other treated albumins. The cross-reactivity of those antibodies was examined with albumins treated by other methods such as modification of glucose and fructose, carbodiimide, and transglutaminase. Among the, RSA and HSA modified with glucose and fructose had an affinity for each antibody and the reactivity depended on the extent of formation of the polymerized albumin. The results suggests that functional groups involved in cross-linking of albumin are important for formation of the cross-reactivity with the antibody and that a definite structure immunochemically similar to glutaraldehyde-treated albumin could be formed by the Maillard reaction.

Monoclonal antibodies against glutaraldehyde-conjugated histamine: application to immunocytochemistry.

We have developed mouse monoclonal antibodies (AHA-1-5, all IgG1 sub-isotype mAbs) against histamine (HA) conjugated to bovine serum albumin using glutaraldehyde-NaBH4. Among these, AHA-1 mAb was found to be the most useful for HA immunocytochemistry (ICC) in terms of specificity and sensitivity without non-specific immunobinding. AHA-1 was demonstrated to be specific to HA with an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections, and not reactive to any of the other amino acids and peptides with N-terminal histidine tested. By use of this antibody, indirect immunoperoxidase staining was observed in rat stomach fixed with glutaraldehyde (GA) in combination with NaBH4 reduction. In contrast, no immunoreactivity was seen in tissue fixed only with GA. Absorption controls indicated that the immunostaining could be completely inhibited by GA-conjugated HA, which was consistent with the results of an ELISA inhibition test. No cross-reactivity occurred with other GA-conjugated amino acids. ICC staining was dense in the cytoplasm of gastric enterochromaffin-like cells and very weak in mast cells. A new finding was that staining was noticed in some cell bands of the intermediate layer between the stratum lucidum and the stratum corneum of the stratified squamous epithelium of the gastric cardia, esophagus, tongue, and skin in rats. The results strongly suggest that the monoclonal antibody allowed highly specific detection of HA in animal tissues.

Clinical and immunologic evaluation of workers exposed to glutaraldehyde.

We describe immunologic responses in subjects exposed to glutaraldehyde (GA) who were diagnosed as having occupational asthma, or who described work-related respiratory symptoms. A series of GA-modified proteins was characterized, and used to analyse sera from 20 GA-exposed workers and 21 unexposed workers for IgE antibodies. Inhibition studies were used to determine the specificity of binding. The reaction of GA with albumin in different molar ratios produced a range of modified proteins, which were used to measure specific IgE antibodies. A significant difference between exposed and unexposed subjects with serum IgE less than 150 kU/l could be detected for GA-specific IgE antibodies (P - 0.026), and 31% of exposed workers with occupational asthma had antibody levels greater than the unexposed population (mean +2.5 SD). False-positive results were obtained with serum from unexposed workers who had total IgE levels greater than 150 kU/l, but this binding was not inhibited by GA-modified proteins. We report the first evidence of immunologic sensitization in some workers exposed to GA. However, GA may behave like many other low-molecular-weight chemicals in that specific antibodies can be detected in only a small percentage of exposed workers who report work-related respiratory symptoms.