Measurement with polyclonal antibodies

R. S., Synthetic peptide vaccine development measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy, J. Mol. Recog. 17, 540-557, 2004 Stills, H.F, Jr., Adjuvants and antibody production dispelling the myths associated with Freund s complete and other adjuvants, ILAR J. 46, 280-293, 2005 Miller, L.H., Saul, A., and Mahanty,... 

Beta-endorphin is a frequently measured opioid peptide. Immunoassay is the method of choice for the analysis of plasma (3-endorphin. Both RIAs and direct IRMAs have been developed for this purpose. Commercial reagent kits are widely available, and many commercial reference laboratories offer P-endorphin assays. The concentrations of P-endorphin are usually very low to undetectable in normal subjects, and it may be necessary to use extraction procedures to detect meaningful concentrations in plasma. The specificity of commercial antibodies for p-endorphin relative to p-LPH varies widely in some immunoassays, 50% cross-reactivity is seen with p-LPH. With polyclonal antibodies, results may be spuriously high owing to crossreactivity with serum immunoglobulin G (e.g., in patients with immunoglobulin G myeloma). 

A decrease of 0/C and P/C concentration ratios at the surface of Streptococcus sobrims was reported as a result of preincubation with polyclonal antibodies. A combination of the pH-dependent zeta potential and the XPS data suggested that polyclonal antibody adsorption occurred through blocking of surface phosphate. XPS measurements provided evidence for IgG attachment to Pseudomonas aeruginosa. The antibody-modified cells showed a reduced adhesion to glass under flow in a nutrient medium. ... 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Currently, there are several assays for the measurement of PSA. All of them contain monoclonal or polyclonal antibodies labeled with enzymatic, fluorometric, or radioactive markers. These assays have shown significant variations within the same patient specimens. These variations may result from differences in antibody specificity, reaction kinetics, calibration, or the system s sensitivity. Studies have shown that only free PSA and PSA-ACT show immunological reactivity in these assays. Also, reaction kinetics can influence the molar ratio. Some of these assays with shorter incubation times may specifically bind the free PSA molecule (which is a lower weight form of PSA). In the equimolar assays, changing the incubation... 

Immunochemical methods have been developed and placed on the market to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been developed and used to analyze tetracyclines in honey samples with a detection level of 20 pg/kg-1 [96]. A microplate-based indirect ELISA has been developed to analyze tetracyclines using polyclonal antibodies. The assay could measure tetracycline in the range between 0.1 and 6 ng mL L Other tetracycline antibiotics such as chlortetracycline, rolitetracycline, or minocycline are also highly recognized in this assay [98]. Several immunoassay kits are commercially available for the analysis of tetracyclines although, to our knowledge, none of them... 

Vandenbroeck et al.7 used an ELISA to determine the recovery of immu-noreactive porcine interferon-gamma (IFN-y) from E. coli inclusion bodies. The ELISA used a polyclonal coating antibody with detection by a MAb. The inclusion bodies were solubilized in diluted 6 M guanidine/HCl and IFN subsequently refolded by its removal. The antiviral activity of the interferon was measured with a bioassay using the cytopathic effect (CPE) of vesicular stomatitis virus (VSV) on bovine kidney cells. The results of this study showed that the immu-noreactivity measured by ELISA matched the biological activity measured by bioassay. 

Polyclonal antibodies of different types are known to show affinity for specific compounds. Thus, antibodies that can bind to a specific substance to be analyzed are immobilized to the walls of the test tubes, plates, or microwells. Such test tubes and plates are commercially available and supplied in the test kit. A measured amount (between 10 and 50 pL) of sample or sample extract is added to one such test tube containing an assay diluent (a phosphate buffer). An equal volume of analyte-enzyme conjugate (commercially available and supplied in the kit) is then added to the test tube. The enzyme conjugate is a solution containing the same analytes covalently bound to an enzyme. The solution mixture is incubated or allowed to stand for a specific amount of time. During this period, the enzyme conjugate competes with the analyte molecules for a limited number of antibody binding sites in the test tube. 

Immunological methods with polyclonal antisera or monoclonal antibodies provide the best quantitative measurements of intestinal or placental ALPs. Much more difficult is the production of antibodies that selectively react with different products of the tissue-nonspecific ALP gene, including the liver- and bone-derived isoforms, as these antibodies should recognize specific sugar side chains instead of... 

Currently, immunoassay is the practical method of choice for measuring cytokines and their receptors. As cytolanes are proteins, specific antibodies can be raised against recombinant cytokines and have also been measured as an indicator of cytokine presence, The general characteristics of cytokine immunoassays are comparable with the classical immunoassays, with monoclonal, oligoclonal, or polyclonal antibodies all being used (see Chapter 9). The most popular formats are immunoradiometric assay (IRMA) and ELISA, which use a first monoclonal antibody for the capture and a second antibody labeled with a radioisotope or an enzyme. 

Polyclonal antibodies are widely used in clinical laboratories for the measurement of plasma protein concentrations. However, immunoassays are often sensitive to the nature of the antibody used. The development of polyclonal antibodies is affected by several factors, such as the purity and dose of the antigen used, the species of host animal, and the immunization procedure. Monoclonal antibodies are viewed as a viable alternative to alleviate these problems. However, the expression of particular epitopes varies with the hpoprotein particles and among individuals in addition, the apohpoproteins themselves are polymorphic in nature. Therefore the use of a single monoclonal antibody might not detect a particular variant. If a monoclonal antibody is used in the determination of an apohpoprotein, it should he directed to an epitope that is expressed on all polymorphic forms of that particular apoprotein. Furthermore, the epitope should be equally reactive to the antibodies regardless of which hpoprotein class contains it. Alternatively a mixture of monoclonal antibodies directed at different epitopes of the apohpoprotein may also be used. Such mixtures are referred to as panmonoclonal antibodies. 

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