Reaction immunochemical

The remarkable selectivity that is inherent in the reaction of an antibody with the antigen or hapten against which it was raised is the basis for the extensive use of immunoassay for the rapid analysis of samples in clinical chemistry. Immunochemical reactions offer a means by which the applicability of potentiometric techniques can be broadened. A number of strategies for incorporating immunoassay into the methodology of potentiometry have been explored... 

An electrode in which an antibody or an antigen/hapten is incorporated in the sensing element is termed an immunoelectrode . The potential response of the immuno-electrode is based on an immunochemical reaction between the sensing element of the electrode and antibody or antigen/hapten in the sample solution. One example of such an electrode is the polymer membrane electrode shown in Fig. 7. The selective response of this electrode to specific immunoglobulins is based on the interaction between antibody in solution and an antigen-ionophore complex in the membrane ... 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-chemical reagents for the determination of a wide range of materials (immunogens) which can be made to produce immunological responses in animals such as sheep or rabbits. It is possible to combine the specificity of an immunochemical reaction with the extreme sensitivity of radiotracer detection. Analytical methods based upon these principles have achieved wide applicability in the determination of organic compounds at trace levels. 

V) The validity of RIA entirely depends upon the identical behaviour of standard and labelled substance unknown, and not on the identity of the labelled tracer and the unknown. Hence, the experimental conditions of incubation of standards and unknowns must be identical for any factors that might affect the extent of the immunochemical reaction, pH, ionic composition, protein content or any other substances of interest. However, these conditions may be tested conveniently and can be controlled effectively by preparing standards in hormone free plasma at the same dilution at which unknowns are assayed. 

Quaternary structures or ligand-ligate (receptor) interactions may be partially conserved during electrophoresis. Identification of a distinct protein is possible only by biochemical or immunochemical reactions or by comparison with an authentic sample. 

A very brief description of biological membrane models, and model membranes, is given. Studies of lateral diffusion in model membranes (phospholipid bilayers) and biological membranes are described, emphasizing magnetic resonance methods. The relationship of the rates of lateral diffusion to lipid phase equilibria is discussed. Experiments are reported in which a membrane-dependent immunochemical reaction, complement fixation, is shown to depend on the rates of diffusion of membrane-bound molecules. It is pointed out that the lateral mobilities and distributions of membrane-bound molecules may be important for cell surface recognition. 

Fig. 11. Evidence that a membrane-associated immunochemical reaction (complement fixation) depends on the mobility of the target hapten (IX) in the plane of a model membrane. The extent of the immunochemical reaction, complement fixation, is measured by A Absorbance at 413 nm. Temperature is always 32°C, which is above the chainmelting temperature (23°C) of dimyristoylphosphatidylcholine used for the data given in A and below the chain-melting transition temperature (42°C) of dipalmitoylphosphatidyl-choline used for the data in B. Thus A refers to a fluid membrane and B refers to a solid membrane. The numbers by each curve are equal to c, the mole % of spin-label hapten IX in the plane of the lipid membrane. It will be seen that complement fixation, as measured by A Absorbance at 413 nm is far more effective in the fluid membrane than in the solid membrane at low hapten concentrations (i.e., c 0.3 mo e%). In C the lipid membrane host is a 50 50 mole ratio mixture of cholesterol and dipalmitoylphosphatidylcholine. The immunochemical data suggest that this membrane is in a state of intermediate fluidity. Specific affinity-purified IgG molecules were used in these experiments. (For further details, see Ref. 5.)... 

In our opinion it is likely that a number of important membrane immunochemical reactions will be found to be dependent on lateral mobility. It will be a challenge to discover whether the lateral mobility of... 

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

In order to assess the utility of the immunochemical reaction for chemical sensing, we need to examine the effects of the experimental conditions on the primary association reaction. The effect of temperature is not particularly distinct for most reactions and cannot be generalized. This is due to the fact that the relative... 

It has been proposed to use a Si/Si02/electrolyte structure for the detection of immunochemical reaction. The antibodies are immobilized at the surface of the Si02 and their interaction with the antigen is monitored by observing the shift of the inflex point on the C-V curve, using 150 mV p-p, 1 KHz modulation. 

Immunosensors are affinity biosensors and are defined as analytical devices that detect the binding of an antigen to its specific antibody by coupling the immunochemical reaction to the surface of a device... 

For qualitative screening, a visual comparison of color with standards can be made. For semiquantitative determination, however, a spectrophotometer should be used to read absorbance to plot a calibration standard curve. The color should be read as soon as possible because it becomes unstable after 30 min. The required period for incubation varies from substance to substance but can range from 5 to 10 min to 1 or 2 hours. In certain analysis, the immunochemical reaction may require quenching after a specific amount of time. The reaction can be stopped by adding an acid, such as 1 N HC1, which turns the blue to yellow. The intensity of yellow too can be measured to determine the analyte concentration in the sample. 

Al. Aizawa, M., Kato, S., and Suzuki, S., Immunoresponsive membrane. I. Membrane potential change associated with an immunochemical reaction between membrane-bound antigen and free antibody. J. Membr. Sci. 2, 125-132 (1977). 

Soy sauce also contains some of these reaction products pyrraline (3.6), imida-zolones (53.8), lysylpyrropyridine (2.1), and pentosidine (0.7 nmol g 1 soy sauce powder). The first two were determined in the total soy sauce by immunochemical reaction, whereas the last two were determined in the nondialysable fraction (> 0.5 kDa) by acid hydrolysis and HPLC.360... 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

After the animal has been bled, the blood is allowed to dot by standing at room temperature for 1-2 h. The blood is centrifuged carefully at 5,000 g, avoiding lysis of the erythrocytes, to separate the serum and dotted fibrin factions. This antibody fraction can be stored at -80 °C for years without loss of activity. However, it is recommended that before aliquoting and freezing, the complement system should be inactivated, because this can interfere with many immunochemical reactions. Inactivation is carried out by simply heating to 56 °C for 10-20 min. 

Isotope dilution in combination with the substoichiometric principle is applied in various ways. The most important examples are radioimmunoassay for protein analysis and DNA analysis. In radioimmunoassay, radionuclides are used as tracers and immunochemical reactions for isolation. Radioimmunoassay was first described in 1959 by Yalow and Berson, and since then has found very broad application in clinical medicine, in particular for the measurement of serum proteins, hormones, enzymes, viruses, bacterial antigens, drugs and other substances in blood, other body fluids and tissues. Only one drop of blood is needed, and the analysis can be per-fonned automatically. Today more than 10 immunoassays are made annually in the United States. The most important advantages of the method are the high sensitivity and the high specificity. In favourable cases quantities down to 10 g can... 

In essence, the introduction of the avidin-biotin complex into a given system does not contribute a factor of specificity to an immunochemical reaction rather, its involvement serves to mediate between the recognition system (i.e., cintibody-cintigen complex) ind a given probe or reporter group. Such mediation usually results in an amplified or improved signal. 

The avidin-biotin technique is especially useful in double-labeling experiments. This can be done using two different enzymes in the final step, combined with two different andbody species (monoclonal and/ or polyclonal). One of the andbodies can be biodnylated and the other immunochemical reaction can be based on a different procedure (e.g., employing an antibody-enzyme conjugate). Alternatively, both antibodies can be biotinylated and the secondary reaction with the respective avidin-enzyme conjugate can be performed on different sides of an impermeant (e.g., plastic-embedded) tissue section. 

I, and I. Radioimmunoassay combines the specificity of an immunochemical reaction with the sensitivity of isotope analysis (F4, S19) and is currently developing rapidly for the analysis of steroids, peptide hormones, and specific proteins (G9). Enzymes can be determined with labeled substrates (01). The requirements of standardization and dosimetry make it probable that these methods will continue to be based on relatively large automatic instruments. However, the greatest problems are unlikely to be in the counting equipment but in sample handling and processing and in standardization of the system. 

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