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Chapter 16 IMMUNOASSAYS for LEPTIN and LEPTIN RECEPTORS

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Chapter 16 IMMUNOASSAYS for LEPTIN and LEPTIN RECEPTORS Chapter 16 IMMUNOASSAYS FOR LEPTIN AND LEPTIN RECEPTORS Jehangir Mistry Department of Research and Development, Linco Research, Inc., St. Charles, MO Abstract; Availability of methods for accurate and reproducible quantification of leptin in various biological fluids in humans and in animals has greatly facilitated research leading to the understanding of its role in various physiological and pathophysiological conditions. Immunoassay techniques provide a simple and robust tool for measurement of biomolecules. In this chapter, I describe the principles behind various types of immunoassay methods, features of several commercially available leptin and leptin receptor immunoassays, and factors that influence leptin measurement using immunoassays. Key words: Immunoassays, RIA, ELISA, antibodies, immunofunctional 320 Chapter 16 1. INTRODUCTION To gain understanding of the biological functions of any molecule, it is critical to develop simple, accurate and reproducible tools that enable the measurement of the molecule in various biological fluids. Following its discovery, methods for measuring circulating concentrations of leptin in the human and other animal species were developed rapidly. The first assays were based on the immunoprecipitation/Western blotting techniques and were not only semi-quantitative but also tedious'. Therefore, there was an immediate need for precise and quantitative methods. The first commercial assay was introduced by Linco Research, Inc. as described by Ma et al.^. This simple and robust assay has been used in majority of the published literature on the measurement of circulating levels of leptin in human serum, plasma or tissue culture media. Leptin circulates in the blood as "free" hormone as well as bound to the extra cellular domain of its soluble receptor. The objective of this chapter is to familiarize the reader with (a) various immunoassay methods and formats available for human leptin and soluble leptin receptor measurement in biological fluids, (b) parameters such as calibration, analyte stability, interference from endogenous molecules, assay characteristics and lot-to-lot variability may influence leptin measurement in various biological matrices, and (c) the availability of various animal leptin assays. 2. COMPETITIVE IMMUNOASSAYS FOR LEPTIN The competitive immunoassays utilize either radioactive (usually '^^I- labeled) or non-radioactive leptin (usually enzyme-labeled or biotin labeled) as the tracer and only one antibody (called primary antibody) raised against the full-length or a peptide fragment of leptin. The method involves competition between the labeled and unlabeled leptin to bind to a fixed number of binding sites^ The amount of labeled leptin bound to the antibody is inversely proportional to the concentration of unlabeled leptin present. The separation of the free and bound leptin is achieved by using a double antibody system. The advantage of competitive immunoassays is that they do not require purified antibodies (e.g., antigen affinity purification or other chromatographic steps) and therefore, they are relatively easy to develop for the measurement of large peptides or proteins. The first commercial assay introduced by Linco utilizes '^^I-labeled human recombinant leptin, human recombinant calibrators and antiserum raised by immunizing rabbits with highly purified recombinant leptin. This radioimmunoassay (RIA) has been used extensively to determine leptin Immunoassays for leptin and leptin receptors 321 concentrations in serum, plasma or tissue culture media'*' ^. In addition, Linco has developed a sensitive leptin RIA suitable for measurement of very low leptin concentrations in biological matrices such as cerebrospinal fluid^. 3. "TWO-SITE" SANDWICH IMMUNOASSAYS FOR LEPTIN The sandwich immunoassays are non-competitive assays in which the analyte (e.g., leptin) to be measured is "sandwiched" between two antibodies^. The first antibody is immobilized to a solid support (e.g., inside walls of plastic tubes or microtiter wells) and the other antibody is labeled (e.g. ^^^I label or enzyme or fluorescence tag) for detection of the analyte. The analyte present in the standards or unknown samples is bound by both antibodies to form a "sandwich" complex. Unbound reagents are removed by washing the tubes or microtiter wells or the solid support. Examples of sandwich immunoassays include Immunoradiometric Assay (IRMA) or Enzyme-Linked Immunosorbent Assay (ELISA). Several leptin immunoassays kits are now available in the ELISA format. Table 1 shows compilation of a list of commercially available human leptin immunoassays from various manufacturers and suppliers, with their essential characteristics. Table 1. Comparison of Human Leptin Assays from Various Commercial Sources* Company Assay Sample Incubation Sensitivity Sample Format Dynamic Range Volume Time Treatment ALPCO Diagnostics/ BioVendor ELISA 1-50nQ/ml 33 pi 2.5 hr. 0.5 ng/ml Dilution Assay Designs ELISA 195-12,500 pq/ml 100 pi 2hr. 25.5 pg/ml None B-Bridge ELISA 1-60nc|/ml 1001 pi 2.5 hr. Not Described Dilution DSL IRMA 0.25-120 ng/ml 100 pi Overnight 0.1 ng/ml None DSL ELISA 0.5-50 nq/ml 25 pi 3.5 hr. 0.05 ng/ml None LINCO Researcii RIA 0.5-100 ng/ml SIOOpI Overnight 0.1 ng/ml None LINCO Research RIA 0,05-10 ng/ml SI 00 pi Two-day 0.01 ng/ml None LINCO Research ELISA 0.125-20 nq/ml 50 pi 3.5 hr. 0.05 ng/ml None 0.5-100 nq/ml 25 pi 3.5 hr. 0.2 ng/ml None Luminex LINCO Research Multiplex 6.2-4,500 pM 25 pi Overnight 8.7 pM None R&D Systems ELISA 30-2,000 pg/mi 100 pi 5hr. Not Described Dilution *Information was obtained from various manufacturers' kit inserts. 322 Chapter 16 4. MEASUREMENT OF FREE FORM OF LEFTIN IN HUMAN SERUM Circulating leptin in humans is bound to high-molecular-weight components, as demonstrated by traditional methods using '^^I-labeled recombinant leptin and size exclusion chromatography^"'°. Furthermore, a spun-column assay was used to determine leptin-binding activity in human serum''. Horn and Lewandowski'^' '^ used an innovative approach to measure selectively only the free leptin by developing an RIA with antibodies raised against a C-terminal leptin fragment (leptin126-i4o). Lewandowskin et al. also raised antibodies against an N-terminal fragment of leptin (leptin26-39) which was shown to recognize only the soluble receptor bound leptin. Free and bound forms of leptin have also been quantified by HPLC separation of serum samples followed by measurement of leptin with Linco'sRIA'". 5. SOLUBLE LEPTIN RECEPTOR ASSAYS At least two commercial immunoassays, one from Diagnostic Systems Laboratories, Inc. (Webster, Texas) and the other from Bio Vendor (Czech Republic), are currently available to measure the soluble form of leptin receptor. Both assays utilize the sandwich ELISA format in which the "capture" and "detection" antibodies are raised against the soluble receptor protein. Wu et al.'^ developed a Ligand-Mediated Immunofunctional Assays (LIFA) for measurement of (a) circulating endogenous leptin/soluble leptin receptor complexes and (b) total soluble leptin receptor. The soluble leptin receptor is captured by a monoclonal antibody which binds to an epitope on the soluble receptor away from the ligand-receptor binding site and equally recognizes both free leptin receptor and leptin/leptin receptor complexes. Addition of anti-leptin monoclonal antibody alone detects pre­ existing endogenous leptin/soluble leptin receptor complexes only, whereas addition of the anti-leptin monoclonal antibody together with an excess of recombinant leptin allows for the measurement of total soluble receptor'^. 6. ANIMAL LEPTIN IMMUNOASSAYS The study of leptin physiology in animals such as mice, rats, non-human primates and pigs has been greatly facilitated due to the availability of simple, robust RIAs for the measurement of this hormone. Linco Research, Immunoassays for leptin and leptin receptors 323 Inc has also developed a multi-species RIA; the antibody used in this kit was raised against human leptin but displays broad cross-reactivity to leptin molecules of many, but not all, species. Richards et al.'^ developed a rabbit polyclonal antiserum to an epitope containing a specific eight amino acid sequence (GLDFIPGL) found in the AB loop portion of most leptin proteins, such as pig, chicken, human, rat, mouse, bovine, sheep and dog. The antiserum apparently recognizes the full-length protein, regardless of the species of origin using Western blot. Slot blot or Immuno-histochemistry techniques. This is typical of many antisera which are raised against a small peptide where they can only detect the intact molecule in a denatured form but not in its native form in solution. The rodent leptin assays are now commercially available in the ELISA format as well. Antibodies raised against human, mouse and rat leptin do not appear to show any significant cross-reactivity to canine leptin. There is only one report in the literature where Kimura et al.^^ developed a sandwich ELISA for canine leptin. Table 2 shows compilation of a list of commercially available animal leptin immunoassays from various manufacturers and suppliers, with their essential characteristics. Table 2 . Comparison of Animal Leptin Assays From Various Commercial Sources* Dynamic Company Species Assay Sample Incubation Sensitivity Sample Format Range Volume Time Treatment ALPCO Diagnostics Mouse/Rat ELISA 25-1,600 pg/ml 25 Mi 4hr. 10 pg/ml Dilution Assay Design Mouse ELISA 12.6-800 pg/mi 100 pi 2hr. 1.74 pg/ml None Assay Design Rat ELISA 56-3,600 pg/ml 100 Mi 2hr. 46.7 pg/ml None Not B-Bridge Mouse/Rat ELISA 62.5-4,000 pg/ml 100 Ml 2.5 hr. described Dilution Not BioVendor Mouse/Rat ELISA 62.5-4,000 pg/ml 100 Mi 2,5 hr. described Dilution DSL Murine ELISA 0.5-50 ng/mi 26 pi 4.6 hr. 0.04 ng/mi None Not DSL Porcine ELISA 0.5-50 ng/mi 50 Ml 2.5 hr. described None LINCO Research Mouse RIA 0.2-20 nq/ml SI 00 Ml Two-day 0.05 ng/mi None LINCO Research Rat RIA 0.5-50 nq/ml SIOOMI Two-day 0.1 ng/ml None LINCO Research IVIouse ELISA 0.2-30 ng/ml 10 Ml 4hr.
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