Immunochemical Response Research Articles

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.

Optical Resonance-Enhanced Absorption-Based Near-Field Immunochip Biosensor for Allergen Detection

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.

Identifying coronary artery flow reduction and ischemia using quasistationary QT/RR-interval hysteresis measurements

Because myocardial ischemia induces QT/RR hysteresis, a correlation was hypothesized to exist between the extent of myocardial flow reduction and the magnitude of QT/RR hysteresis. Graded reductions in regional myocardial perfusion in the distribution of the left anterior descending coronary artery in open-chest pigs were used to model 1-vessel coronary artery disease. At each reduced level of left anterior descending coronary artery flow, the heart was electrically paced at progressively higher and lower rates between an initial control and maximum heart rate values. Digitized surface and intramyocardial electrograms and aortic pressure were used to measure QT/RR hysteresis, QT-interval adaptation, ST- and TQ-segment depression, and cardiac contractility. Intraexperimental blood samples were analyzed to assess inflammatory response (interleukin 6), oxidative stress (protein carbonyls), and myocyte injury (creatine kinase). Higher values of QT/RR hysteresis correlated with the severity of ischemia as assessed by TQ-segment depression in intramyocardial electrograms ( P = .002). Lower flow rates were strongly associated with higher values of QT/RR hysteresis and slower QT-interval adaptation ( P ≤ .004), and were less correlated with the magnitude of ST- and TQ-segment depression ( P ≥ .02). Significant increases in systemic measures of inflammation, oxidative stress, and cardiac myocyte injury and major decrease in cardiac contractility preceded the most severe stages of flow reduction (30% and 20% of normal flow). We determined QT/RR hysteresis index thresholds corresponding to these mechanical and immunochemical responses. QT/RR hysteresis is a strong indicator of reduced myocardial perfusion and may provide information for noninvasive assessment of mechanical and immunochemical changes associated with early stages of coronary artery disease.

Changes in peptic digestibility of bovine β-lactoglobulin as a result of food processing studied by capillary electrophoresis and immunochemical methods

Digestion studies constitute a functional tool for allergen characterisation. This strategy for investigating allergenic proteins relates to the observation of increased proteolytic resistance of some proteins recognised to exhibit allergenic potential. beta-Lactoglobulin (betaLG) is one of the major whey proteins, a potent milk allergen and shows a high stability against peptic hydrolysis in its native form. In order to study the impact of milk fermentation process on its digestibility, two complementary analytical methods were applied: capillary zone electrophoresis (CZE) to quantitatively study proteolytic degradation of betaLG isolated from different fermented bovine milk products, and enzyme linked immunosorbent assay (ELISA) to assess differences in immunoreactivity. betaLG, isolated from either raw or pasteurised cow's milk (CM), as expected, showed only minimal digestibility (less than 10% in 2 h). However, when raw milk or pasteurised milk was fermented, the rate of peptic digestion of the protein significantly increased (up to 45% in 2 h). In accordance with changes in digestibility, the immunochemical response for all fermented samples was lower than that of non-fermented references. Raw and pasteurised milk "naturally" fermented in our laboratory only resulted in a slight reduction (betaLG detected is still in the range of milligrams per gram sample), whereas the industrially manufactured sour milk as well as the "Acidophilus milk" reflected a remarkably lower level of immunoreactivity (55-56 microg/g sample).

An Introduction to Chromogenic in situ Hybridization

The determination of genetic expression has now become necessary for the diagnosis and treatment of patients. Fluorescence in situ hybridization is regarded as the gold standard for some applications, such as HER-2 expression. This method is not very practical for the routine histopathological laboratory. Chromogenic in situ hybridization (i.e., CISH) is presented as the new method and demonstrates the most promise for easy adaptability in most clinical pathology laboratories. This method enables the detection of gene expression in the nucleus using the conventional histochemical reaction. Technologists have become highly skilled in immunohistochemical techniques, and there are similarities in some aspects of the procedures between in situ hybridization and immunohistochemistry. Formalin-fixed tissue usually is pretreated and hybridized with labeled probes. in situ hybridization is the technique currently used to confirm the immunochemical response especially concerning prognostic treatment. With the advent of gene therapy, this technique is becoming in as much demand as traditional immunohistochemistry. (The J Histotechnol 28229, 2005)Submitted October 10, 2005; accepted with revisions December 6, 2005

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein

We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of ≈17 kDa monomers. It catalyses the Mg 2+-dependent hydrolysis of diadenosine triphosphate, Ap 3A, to AMP + ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, ε-(Ap 3A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap 3Aase presents a native molecular mass of ≈32 kDa and no significant differences were found in K m values (2 μM), activating effects by Mg 2+, Ca 2+, and Mn 2+, optimum pH (7.0–7.2) or inhibition by Zn 2+ and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap 3Aase and Fhit protein with K i values in the range 20–30 nM. Both human and rat Ap 3Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap 3Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a ≈17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap 3Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit–Ap 3Aase mediated by its interaction with suramin or related drugs.

Enzymatic proteolysis, under high pressure of soybean whey: Analysis of peptides and the allergen Gly m 1 in the hydrolysates

Soybean ( Glycine max) whey was hydrolyzed with Alcalase, Neutrase, Corolase 7089 and Corolase PNL during high pressure (HP) treatment at 100, 200 and 300 MPa and at atmospheric pressure for 15 min. The protein content and the degree of hydrolysis were determined. Furthermore, the allergen Gly m 1 in the treated soybean whey and the hydrolysates was detected. The results showed that HP treatments increased the hydrolysis by the four proteases used. Pressure at 200 and 300 MPa proved to be better pressures to enhance the proteolysis. The immunochemical response of soybean whey to anti-Gly m 1 monoclonal antibodies decreased after the HP treatments and this decrease was greater after the combined treatment of high pressure and enzymatic hydrolysis. Soybean whey proteins hydrolysed at high pressure could be used as sources of peptides with low antigenicity when incorporated as food ingredients.

Modulation of immunologic reactions between cultured porcine hepatocytes and human sera.

In the clinical application of a hybrid artificial liver system using porcine hepatocytes, some immunologic reactions occur between human serum and porcine hepatocytes. In this study, we investigated the immunologic mechanisms of the cytotoxic reactions, and we tried to inactivate the human serum cytotoxicity by heating the serum or the addition of nafamostat mesilate (NM). Immunologic reaction between human serum and porcine hepatocytes by evaluating the immunochemical response against human IgM, IgG, and C3 was investigated. The immunochemical analysis of inactivation by heated human serum (56 degrees C, 30 min) and adding NM were performed. The evaluation of serum cytotoxicity was as follows: when porcine hepatocytes were cultured with heating the human serum or the addition of NM, the survival ratio was observed. Immunochemical reactions against human C3 were all positive, but positive reaction against human IgM occurred in only one case (5%); those against human IgG were all negative. Both heating the serum and adding NM inhibited the immunochemical reaction of human C3. The inhibition of human C3 with NM was dependent on that concentration. Both heating of the serum and adding NM to the medium decreased damage of porcine hepatocytes. An immunologic reaction between human serum and porcine hepatocytes in a porcine bioartificial liver clearly occurred, and this reaction was controlled by heating the serum and adding NM. We believe that NM is useful in the clinical application of our hybrid artificial liver system.

Detection of the ichthyotoxic dinoflagellate Gyrodinium (cf.) aureolum and morphologically related Gymnodinium species using monoclonal antibodies: a specific immunological tool

Sixteen monoclonal antibodies which recognize different cell surface antigens of the ichthyotoxic marine dinoflagellate Gyrodinium cf. aureolum were prepared and characterized for use in identification by both immunofluorescence microscopy and flow cytometry. Based on the labeling results obtained from indirect immunofluorescence assays the monoclonals could be divided into 3 groups: (I) fluorescence of the overall cell surface, (II) fluorescence of the overall cell surface together with the flagella, and (III) fluorescence of granular-like structures randomly distributed along the cell surface and at the edges of the sulcus and cingulum. Cross-reactivity of 6 selected monoclonals with other phytoplankton species revealed that labeling was restricted to G. aureolum/G. cf. aureolum and to the morphologically closely related species Gymnodinium nagasakiense and G. mikimotoi. This suggests a taxonomic proximity of these species. Fixation of samples with paraformaldehyde produced the optimal immunochemical response, resulting in the strongest fluorescence intensities. In flow cytometric analyses, labeled cells of G. cf. aureolum could be distinguished sufficiently from unlabeled controls when monoclonals of Group II and a pooled serum (combined antibodies of Groups I, II, and III) were used. This immunochemical technique will be applied in an early warning system to detect G. aureolum cells at dilute concentrations using flow cytometry or, alternatively, image analysis.

Partial characterization of the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causative agent of chancroid, a genital ulcer disease.

The first preliminary structure of a surface lipooligosaccharide from Haemophilus ducreyi has been determined. The major oligosaccharide was released by mild acid hydrolysis and analyzed by liquid secondary ion and tandem mass spectrometry. The mass spectral data combined with composition and methylation analysis yielded the most probable structure; Gal1----4GlcNAc1----3Gal1----4Hep1----6Glc1----( Hep1----2Hep1----)3,4Hep1---- KDO, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or KDO) exists in an anhydro form. This anhydro species results from the elimination of a phosphate from C-4 of KDO during mild acid hydrolysis. The core heptose trisaccharide consists of L-glycero-D-manno-heptose, but analysis of the peracetylated sugars indicated that the 1,4-linked heptose is likely D-glycero-D-manno-heptose. The monoclonal antibody 3F11 generated against Neisseria gonorrhoeae also binds to this lipooligosaccharide and suggests that the terminal trisaccharide is Gal beta 1----4GlcNAc beta 1----3Gal beta 1----, an epitope found in the glycose moiety of the human erythrocyte glycosphingolipid lactoneotetraglycosylceramide. Mass spectrometric and composition analysis of the lipid A moiety shows that it is similar to the lipid A of Haemophilus influenzae strain I-69 Rd-/b+ proposed by Helander et al. (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). Electrospray mass spectrometric analysis of the intact O-deacylated lipooligosaccharides gave an average Mr of 2710, and supported an overall structure consisting of the above nonasaccharide linked directly to a diphosphorylated lipid A moiety through the single KDO which is phosphorylated. This structure should provide a framework to investigate the roles of lipooligosaccharides in the host immunochemical response and pathology of H. ducreyi infection, a leading cause of genital ulcer disease.

Response of cod ( Gadus morhua) larvae and juveniles to oil exposure detected with anti-cod cytochrome P-450c IgG and anti-scup cytochrome P-450E MAb 1-12-3

Cytochrome P-450c (Mr = 58 000 D) is the major cytochrome P-450 isozyme inducible by β-naphthoflavone (BNF) in cod ( Gadus morhua) liver. Polyclonal rabbit IgG against this form was used as an immunochemical probe to detect responses of cod larvae and juveniles to oil exposure. In addition, monoclonal mouse antibodies against the homologous isozyme in scup ( Stenotomus chrysops), scup cytochrome P-450E, were employed. The eggs, larvae and juveniles were reared and exposed in a system allowing controlled dosage of the water-soluble compounds from crude North Sea oil. The immunochemical response, measured with Western blotting and ELISA, in the liver of juveniles and in homogenates of whole larvae, reflected the exposure in a dose-dependent way. Larvae and juveniles that were allowed to recover in clean sea water, showed a decline towards the control levels within a few days. In this study, it is shown that cod larvae do respond to oil exposure with induced synthesis of the cytochrome P-450c isozyme, and that immunochemical detection of this induction can be a convenient tool for monitoring the pollution of aquatic environments.

Cytochromes P-450 in fish larvae: Immunochemical detection of responses to oil pollution

Abstract Rabbit antiserum against the major β-naphthoflavone-inducible cytochrome P-450 isoenzyme purified from cod (Gadus morhua L.), liver were used as an immunochemical probe to detect responses of cod larvae to oil-exposure. Cod eggs were fertilized, reared and exposed in a system allowing full control of the concentration of water soluble compounds from crude North Sea oil. The immunochemical response in homogenates of whole larvae were shown to reflect the exposure in a dose-dependent manner. The study shows that cod larvae do respond to oil-exposure with induced cytochrome P-450 synthesis, and that immunochemical probes of cytochrome P-450 induction can be a valuable tool in monitoring marine pollution.

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms

J. T. Henderson and A. L. Bieber. Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms. Toxicon 24, 473 – 479, 1986. — Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for ld 50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.

Effective control for diethylstilbestrol in cattle in the Netherlands.

In the period from August 1981 to April 1983, 10243 random samples of urine from slaughtered cattle were inspected for the presence of the stilbene derivatives diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX). Fast screening of all samples was performed by radioimmunoassay (RIA) with prior clean-up column chromatography. In this screening, 216 samples were indicated for confirmatory analyses by combined high performance liquid chromatography-gas chromatography-mass spectrometry (HPLC-GCMS) on account of an immunochemical response equivalent to 1 microgram/1 DES or more. The presence of DES was confirmed in 184 samples, DE in 5 samples and HEX in 14 samples. In the remaining 13 samples no stilbene derivative could be confirmed. From the results in 1984, after the introduction of the Meat Inspection Act, it is concluded that the use of stilbene derivatives in cattle in the Netherlands has dropped almost to zero. Of 4558 samples investigated, only 6 (0.13%) were indicated as 'stilbene' positive. The presence of DES was confirmed in 2 samples and DE in 4 samples.

The determination of lipoprotein Lp(a) by rate and endpoint nephelometry

Rate nephelometry is a fast and convenient method for Lp(a) quantification. The linearity in the range of the physiological serum concentrations is good and there is good correlation with other immunochemical methods. The antibody consumption is some 50% higher when compared to the Laurell procedure if light scatter is measured in the RU mode. Measurements in the SU mode on the other hand consume less antibody than the Laurell procedure. The endpoint method, however, is more time-consuming and considered to be less accurate. In addition, only clear samples should be used. Lipemic sera should be cleared by lipase treatment or by high speed centrifugation. The treatment of samples with detergents causes a pronounced reduction of the immunochemical response and thus should be avoided.

Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers.

Immunocytochemical characteristics of myosin have been demonstrated directly in normal and cross-reinnervated skeletal muscle fibers whose physiological properties have been defined. Fibers belonging to individual motor units were identified by the glycogen-depletion method, which permits correlation of cytochemical and physiological data on the same fibers. The normal flexor digitorum longus (FDL) of the cat is composed primarily of fast-twitch motor units having muscle fibers with high myosin ATPase activity. These fibers reacted with antibodies specific for the two light chains characteristic of fast myosin, but not with antibodies against slow myosin. Two categories of fast fibers, corresponding to two physiological motor unit types (FF and FR), differed in their immunochemical response, from which it can be concluded that their myosins are distinctive. The soleus (SOL) consists almost entirely of slow-twitch motor units having muscle fibers with low myosin ATPase activity. These fibers reacted with antibodies against slow myosin, but not with antibodies specific for fast myosin. When the FDL muscle was cross-reinnervated by the SOL nerve, twitch contraction times were slowed about twofold, and motor units resembled SOL units in a number of physiological properties. The corresponding muscle fibers had low ATPase activity, and they reacted with antibodies against slow myosin only. The myosin of individual cross-reinnervated FDL muscle units was therefore transformed, apparently completely, to a slow type. In contrast, cross-reinnervation of the SOL muscle by FDL motoneurons did not effect a complete converse transformation. Although cross-reinnervated SOL motor units had faster than normal twitch contraction times (about twofold), other physiological properties characteristic of type S motor units were unchanged. Despite the change in contraction times, cross-reinnervated SOL muscle fibers exhibited no change in ATPase activity. They also continued to react with antibodies against slow myosin, but in contrast to the normal SOL, they now showed a positive response to an antibody specific for one of the light chains of fast myosin. The myosins of both fast and slow muscles were thus converted by cross-reinnervation, but in the SOL, the newly synthesized myosin was not equivalent to that normally present in either the FDL or SOL. This suggests that, in the SOL, alteration of the nerve supply and the associated dynamic activity pattern are not sufficient to completely respecify the type of myosin expressed.

Clinical and biochemical effects of stanozolol therapy for hereditary angioedema

Stanozolol, an inexpensive anabolic steroid with a 30: 1 anabolic: androgenic ratio, was administered to 12 male and 15 female patients with biochemically proven hereditary angioedema over a 2-yr period to obtain a systematic assessment of the relationship between drug dosage and clinical response, incidence of side effects, and amelioration of complement abnormalities. All 27 patients attained the minimal effective dose, ranging from 0.5 to 2 mg daily, which controlled the frequency and intensity of symptoms with minimal side effects. At daily maintenance doses of 2, 1, and 0.5 mg the frequencies of attacks per weeks of therapy were 1 14.6 , 1 7.2 , and 1 8.2 wk, respectively. Side effects with maintenance therapy included menstrual abnormalities and virilization in four females and elevation of serum creatinine phosphokinase (CPK) in five males. In six patients on maintenance doses of stanozolol, serum levels of testosterone, free thyroxin (T4), and thyroxin binding globulin (TBG) (four males), and of estradiol, progesterone, T4, and TBG (two females) were normal. Slightly low serum levels of progesterone and TBG were found in two females who had normal menstrual cycles. Statistically significant elevations above pretherapy levels of serum inhibitor to the activated first component of complement function and C4 protein and function occurred when patients were on maintenance therapy, but these measurements remained below the lower limit of normal range. Higher doses of stanozolol (4 mg/day), which caused greater immunochemical responses, were unnecessary for control of clinical disease and were unjustified for chronic therapy because of more frequent side effects.

Allergy and Clinical Immunology

<i>Allergy and Clinical Immunology</i>provides an excellent review of the literature and research that has been accumulating, particularly in the last ten years, in this rapidly changing field. The first section, on immunology, is an unfolding of the discoveries that have led to the present understanding of cellular systems and immunochemical responses in immunology. Normal immunoprotective mechanisms are described, and the pathological variations in the responses are categorized. Some of the groundwork elucidating hereditary patterns in allergy is presented, and the accumulating evidence for genetic factors in immunity is documented. An outline and succinct description of the multiple immune deficiency disorders are helpful in conjunction with a chapter on the clinical and laboratory investigation of the immunodeficiencies. The immunologic aspects of specific organ systems are then discussed, with chapters by various authors devoted to such topics as rheumatoid arthritis, systemic lupus erythematosus, vasculitis, and immunologic aspects of the renal, hematologic,

Proteins from disassembled microtubules characterized by oligospecific antisera

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.

Treatment of Allergic Disorders with Injections of Allergen Extracts

A series of investigations did not reveal an immunochemical response to injection of allergen extracts into sensitive persons which would provide either a rationale for the treatment or a basis for a promising dosage regimen worthy of clinical trial. Properly conducted clinical trials are thus still relied on.