Comparison of low energy and high energy electron beam treatments on sensory and chemical properties of seeds.

Consumption of fresh and minimally processed foods such as seeds as a part of a healthy diet is a trend. Unfortunately, fat-rich seeds are often contaminated with pathogenic microorganisms and face frequent product recalls. Electron beams have been applied as a microbial decontamination measure for decades. Conventionally high energy electron beams (HEEB) are being used, whereas low energy electron beams (LEEB, <300 keV) have only recently been introduced to the food industry and more studies are needed. Electron beam treatment has several advantages over other decontamination technologies. The treatment is non-thermal, chemical-free, water-free, and does not use radioactive substances. The effect of electron beams on the sensory and chemical properties of seeds has not been widely studied. This study assessed LEEB and HEEB treated pumpkin and flax seeds immediately after treatments, and after three months of storage. The seeds' sensory profiles were altered after both treatments when compared with non-treated samples, with a higher dose leading to a greater level of alteration. However, the sensory profile of LEEB treated seeds was similar to the non-treated seeds whereas HEEB treated seeds differed from both. The storage period of three months further increased the observed differences between the samples. LEEB and HEEB treatments seemed to cause lipid degradation as the content of volatile aldehydes was increased. This effect was more profound in HEEB treated samples. The data presented in this study shows that LEEB as a microbial reduction solution has great potential to preserve the chemical and sensory properties of nutritious seeds.

Cooperativity in the antibody binding to surface-adsorbed antigen.

The binding to surface-adsorbed antigen of monoclonal mouse IgG-antibodies (mAbs), with two different affinities to dinitrophenyl (DNP), was measured by a calibrated ELISA. The concentration-dependence of antibody binding to surface-bound antigen of different epitope densities was analysed using Scatchard plots. The dissociation of bound tritium-labelled antibodies was measured in the presence of unlabelled antibodies in the bulk. At low surface concentration of bound anti-DNP, both high-affinity mAb and low-affinity mAb show a positive cooperativity in the binding reaction to antigen of high epitope density. Using antigen of lower epitope densities, the positive cooperativity is more pronounced for low-affinity clones. At higher surface concentrations of bound anti-DNP, the Scatchard plots indicate a negative cooperativity of binding, which is also implied by the increased dissociation found in the presence of antibodies in solution. The study confirms previous findings that the binding of antibodies to surface-bound antigen not only depends on intrinsic antibody affinity measured in solution. Other factors, such as self-interaction, also affect the heterogeneous binding reaction.

Experimental demonstration of lateral cohesion in a layer of adsorbed protein and in layers of gold-antibody complexes bound to surface immobilised antigen.

The spatial distribution of ferritin molecules adsorbed on quartz surfaces and the spatial distribution of antibody-coated colloidal gold particles over an antigen-coated surface was studied by electron microscopy. Ferritin molecules were found to initially adsorb in small clusters at 2.5 X 10(9) sites/cm2. The initial nucleation was followed by growth of the clusters. Gold antibody complexes were initially bound to the antigen-coated surface as single particles, and formation of clusters was a secondary event at higher particle densities. The results indicate that lateral cohesion between macromolecules may play a role in the stability of adsorbed layers of protein and surface immobilised antigen-antibody complexes.

A gel casting technique allowing rapid and sensitive quantitation of antibodies by diffusion-in-gel enzyme-linked immunosorbent assay.

A technique is described which allows rapid and sensitive quantitation of antibodies by diffusion towards an antigen-coated surface through a gel of wedge-shaped profile. A sensitivity of 0.16 micrograms antibody per ml solution was obtained after 45 min incubation with stirring at 22 degrees C and 0.08 micrograms/ml could be detected after 90 min incubation with stirring or with incubation at 37 degrees C.

Use of cellulose ethers as peptide antigen carriers in the ELISA.

A procedure for surface immobilisation of peptides is described. Insulin, a model for peptide antigens, was covalently coupled to alkyl-hydroxyalkyl-cellulose ethers. The cellulose-insulin conjugate was then adsorbed to the plastic surface of microtitre wells and was used as antigen in an ELISA assay. The adsorbed conjugate was shown to be stable in undiluted plasma or serum whereas adsorbed insulin was removed from the surface by incubation in undiluted serum or plasma. Adsorption of serum albumin or cellulose ether polymers to the microtitre plates followed by incubation with whole blood, showed that adsorbed albumin but not the cellulose ether was exchanged by fibrinogen at the surface. The results indicate that coupling of peptides to alkyl-hydroxyalkyl-cellulose ethers is an efficient means of immobilising peptide antigens to hydrophobic surfaces.

Kinetics of antigen-antibody reactions at solid-liquid interfaces.

The kinetics of antigen-antibody reactions is reviewed with special attention paid to the specific properties at solid-liquid interfaces. Theories of possible diffusion limitation in forward reaction rates are compared to experiments. It is found that the intrinsic forward reaction rate in the bimolecular antigen-antibody reaction is normally not limited by diffusion either in solution or at the solid-liquid interface. However, reactions at the solid-liquid interface can be diffusion limited due to depletion of reactants close to the surface. This effect depends on geometry, intrinsic reaction rate and surface concentration of receptor molecules. Normally cell surface reactions are not diffusion limited whereas reactions at artificial surfaces often are limited by diffusion. When not limited by diffusion it is also found that the intrinsic forward and reverse reaction rates are lower for surface reactions compared to reactions in solution. Antigen-antibody reactions at solid-liquid interfaces can often be considered as practically irreversible and limited by mass transport or steric interactions.

Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen.

The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG antibodies directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the antigen were used. The hapten was coupled to a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, and adsorbed to polystyrene or silicone surfaces. Four different DNP-BSA epitope densities were used. Antibodies were incubated with the antigen-coated surface overnight. The bound antibodies were detected either optically by ellipsometry or by enzyme-conjugated anti-mouse IgG antibodies in the common ELISA technique. Absorbance values from ELISA measurements were transformed to surface density through calibration by ellipsometry. The experimental data showed that the binding of a high affinity antibody (Ka = 2.0 X 10(10] was diffusion rate limited after 24 h incubation time. Identical binding isotherms were found for high and low affinity clones of anti-DNP antibodies (Ka = 4.1 X 10(7) and 3.5 X 10(5] when antigen of high epitope density was used. At low epitope density the amount of bound low affinity antibodies decreased. Electron microscopy was used for studies of the distribution of colloidal gold-antibody complexes bound to surface-immobilized antigen. The results of the experiment showed that low affinity antibodies were bound in clusters whereas high affinity antibodies bound as single particles. These findings were related to the ELISA measurements. The results indicate that the binding isotherm of antibody to surface adsorbed antigen is not merely a reflection of the intrinsic antibody affinity measured in solution. Other macromolecular properties of antibodies, e.g., lateral intermolecular interactions and phase separation, affect the heterogeneous binding reaction.

Calibration by ellipsometry of the enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) was analyzed with regard to possible diffusion limitations of the binding reaction. The absorbance values of the assay were found to follow the time and concentration relations that would occur when diffusion of antibody to the surface is the rate limiting step. This relationship was used in order to calibrate the absorbance values of the ELISA with antibody concentration by ellipsometry, which itself allows direct measurement of the amount of antibody bound to the solid phase.

Ultrastructural localisation of lipopolysaccharide-binding sites with peroxidase-conjugated lipopolysaccharides.

The localisation of lipopolysaccharide-binding sites on erythrocytes with peroxidase-coupled LPS is described. LPS was isolated from Fusobacterium nucleatum (Fus MC-8) by phenol-water extraction. The LPS was coupled to horseradish peroxidase by the two-step method of Avrameas and Ternynck (1971). The biological and serological activities of the conjugated LPS were compared with those of the native material. Peroxidase could be coupled to LPS without significant loss of endotoxic or serological activity. The LPS-peroxidase conjugate could be demonstrated on erythrocytes by light and electron microscopy.

Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA): a simple method for quantitation of class-specific antibodies.

A new method for quantifying class-specific antibodies is presented. The method has been named Diffusion-In-Gel-Enzyme-Linked-ImmunoSorbentAssay (DIG-ELISA), and is briefly as follows. Antiserum ia allowed to diffuse from wells in a gel layered over an antigen-coated plastic surface. The gel is then removed and the preparation is incubated with enzyme-conjugated anti-immunoglobulin. The enzyme is then visualised in situ by a colour reaction produced by pouring a substrate-containing gel over the plastic surface. Bovine serum albumin and rabbit-anti-BSA were used as a model system, and horseradish peroxidase or alkaline phosphatase as enzymes for visualization.

Dissociation of antibodies bound to surface-immobilized antigen.

The dissociation of antibodies bound to surface-immobilized antigen was investigated by the ELISA, using a hapten (TNP) as antigen. Antibody binding was found to be stable, and no half-time of dissociation could be defined within 69 h. The role of the bivalence of antibodies and the difference between a homogeneous and a heterogeneous reaction was investigated by comparing the dissociation rate of antigen-antibody complexes formed by monovalent Fab fragments from surface-immobilized antigen and the dissociation rate of TNP-lysine from antibodies in a homogeneous liquid phase. Fab fragments were found to dissociate with a half-time value of about 16 h, whereas the homogeneous binding of TNP-antibody dissociated with a half-time of less than 4 h, indicating that both the bivalence of antibodies and the solid phase contributed to the stability of surface-bound antigen-antibody complexes. Qualitative differences between antibodies produced by different clones in a polyclonal antibody response to TNP was investigated by a spot assay. The results indicated that a minority of the antibodies produced had the capacity of binding practically irreversibly to solid-phase-immobilized antigen. The impact of the results on the interpretation of data from solid-phase assays is discussed together with the biological importance of the findings.

Direct visual detection of protein antigen: importance of surface concentration.

A new type of commercially available substrate was used for visualization of antigen binding to antibodies immobilized on the substrate surface. Addition of antigen induced sufficient increase of the protein layer to allow direct visualization, provided that the antibodies immobilized at the surface were immunosorbent isolated. This finding shows the importance of the surface concentration for direct optical visualization of antigen. The results are discussed in relation to previous reports that it is not possible to visualize directly the binding of protein antigen to surface-immobilized antibodies.

Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction.

The binding of monoclonal antibodies to surface-immobilised antigen was studied. Antibodies against dinitrophenyl-benzene and O6-ethyl-2'-deoxyguanosine with a known affinity for the antigen were used. The amount of bound antibodies was measured by ellipsometry with an accuracy of +/- 0.15 pmol/cm2, and a sensitivity of 0.11 pmol/cm2. The binding rate of the initial antibody binding could become diffusion rate limited, and the binding rate at surface concentrations above 1 pmol/cm2 was affected by steric interaction between bound antibodies. Bound antibodies did not dissociate when rinsed with saline for up to 20 h, but dissociated in the presence of antigen (0.1 mM). The dissociation rate did not follow any identifiable rate constant. The results are discussed in relation to theoretical models of the kinetics of antigen-antibody reactions at solid-liquid interfaces.

Determination by ellipsometry of the affinity of monoclonal antibodies.

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies.

In the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) the quantitation of antibodies is based on their ability to form a diffusion gradient over an antigen-coated polystyrene surface. The antigen-antibody reaction is then visualized by an enzyme-conjugated anti-immunoglobulin. The enzyme-substrate reaction is finally performed by pouring a substrate-containing gel over the polystyrene surface. In this study with bovine serum albumin as antigen and a corresponding rabbit antiserum, the diffusion time of antiserum was shown to be the most critical variable of the method, while the antigen concentration used for coating, the conjugate binding time and the enzyme-substrate reaction time had a minor influence on the quantitation of antibodies. High antibody levels were measured with greater accuracy than low levels, but the standard deviation was below 10%. It was also shown that different sera containing antibodies to Salmonella typhi O LPS, Klebsiella pneumoniae K1 and O4 LPS, Escherichia coli O2 LPS, Yersinia enterocolitica Y3 LPS, cardiolipin and pneumococcus could be quantitated with the same accuracy.

A diffusion limited reaction theory for a microtiter plate assay.

Calculations are presented describing the diffusion limited kinetics of a solid-phase immunoassay in which reactants are immobilized at the inner surface of a cylindrical well. The calculations refer to an unstirred situation and simplified expressions are presented which can be used for calibration and optimization of the assay.

Solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of IgG rheumatoid factor-secreting cells.

Although IgG rheumatoid factor may play an essential role in the pathogenesis of rheumatoid arthritis, there is no precise method for its specific detection at the cellular level. A modification of the recently developed enzyme-linked immunospot assay has been devised for enumeration of cells secreting IgG rheumatoid factor (IgG RF) and simultaneous quantitation of the IgG RF secreted. Specific, sensitive and simple, this new assay should provide a valuable tool for study of isotype-specific RF secretion by single cells.

False positive results in class-specific rheumatoid factor (RF) assays due to interaction between RF and Fc fragments of anti-immunoglobulin indicator reagents.

A recently developed, simple immunological method, diffusion-in-gel ELISA, has been adapted for class-specific determination of rheumatoid factor (RF). Evidence was obtained of an analytical error due to interaction between RF and antigenic determinants on the Fc part of the indicator immunoglobulin (Ig) molecules used. This was eliminated by replacing the usual indicator reagent, complete Ig molecules, by conjugated Fab or F(ab')2 fragments. The findings imply that unless the RF-Fc interaction mentioned is avoided in the assaying technique, false positive results may be obtained for, e.g. IgG RF and IgA RF in sera containing IgM RF.

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells.

A solid-phase enzyme-linked immunosorbent assay (ELISPOT) is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of an immunoenzyme procedure in which enzyme-substrate reactions are performed in agarose. Dark-brown circular zones (spots), localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays.

Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances.

A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.