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. 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 /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 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 (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. 0.1 ng/ml None LINCO Research Rat ELISA 0.2-30 ng/ml 10 Mi 4hr. 0.1 np/ml None

LINCO Luminex Research Mouse/Rat Multiplex 6.2-4,500 pM 10 Ml Overnight 6.2 pM None LINCO Research Primate RIA 0.5-100 ng/mi SIOOMI Overnight 0.1 ng/ml None LINCO Multi- Research species RIA 1-50 ng/mi SI 00 Ml Two-day 0.5 ng/ml None 31.25-2,000 R&D Systems Mouse/Rat ELISA pg/mi 50 Ml 5hr. 22 pg/ml Dilution

*Information was obtained from various manufacturers' kit inserts. 324 Chapter 16

7. FACTORS INFULENCING LEPTIN MEASUREMENT

For accurate and reproducible measurement of leptin, it is important to select an immunoassay that is analytically robust in terms of its performance characteristics such as precision, cross-reactivity, linearity of dilution of the biological sample, recovery of exogenously added leptin to the sample and, shows minimum batch-to-batch variability. The long-term batch-to-batch variability, specifically for different lots of standard preparation, should be monitored by using aliquots of quality control serum samples stored at <-20 °C containing a range of leptin concentrations, obtained by pooling samples from volunteers of variable body mass index. Similarly, serum pools for" animal leptin assays are also needed to monitor lot-to-lot consistency. The assay format (e.g., competitive vs. sandwich) and the antibodies used in various assays have significant influence in the measurement of various molecular forms of leptin (e.g., free vs. receptor-bound leptin, truncated form(s) of leptin). In the author's own experience, the RIA format can generally recognize multiple molecular forms of leptin but the sandwich format may measure only selected molecular forms of leptin. For example, in an RIA format, a rabbit polyclonal antibody was able to recognize leptin in monkey serum/plasma samples but the same antibody used in the sandwich format, was unable to recognize monkey leptin. Human leptin, as measured by RIAs in serum, plasma or cerebrospinal fluid has been found to be stable at -20 °C for over two years, at 4 OC for at least two months, and over at least five freeze/thaw cycles '' '' . However, this needs to be confirmed in all new assays. It has been documented that leptin secretion is pulsatile. The peak leptin concentration occurs between 00:00 and 4:00 hr and is approximately 30- 40% higher than the nadir occurring between 8:00 and 12:00 h'^' ^°. It is therefore important to standardize the timing of sample collection. Modest intra-individual differences may also exist in leptin concentration over a prolonged period. For example, Ma et al.^ have reported approximately 30% variability in two subjects in whom leptin was measured on eight consecutive mornings. Non-fasting samples are acceptable and normal feeding does not significantly affect circulating leptin concentrations. However, sudden and extreme alterations in feeding patterns should be avoided. Within 24 hr of fasting, leptin concentrations decline to about 30% of initial basal values whereas massive overfeeding over a 12-hr period increases leptin concentrations by about 50% of basal values^'. Immunoassays for leptin and leptin receptors 325

There is little evidence of interferences in leptin immunoassays. Leptin appears to be a unique molecule with no known circulating interfering immunoassay cross-reactants. Although there is evidence that a proportion of leptin in the circulation is bound to binding proteins, majority of immunoassays have been shown to measure total leptin most likely because the affinity of antibodies for leptin exceeds that of endogenous binding components. Commercial sandwich immunoassays that use monoclonal antibodies for capture and detection of human leptin may be prone to falsely elevated leptin values because of heterophilic interference occurring due to the presence of human anti-mouse antibodies or rheumatoid factors present in some human serum samples. The heterophilic interference can be eliminated or minimized by using normal mouse IgG in the assay.

8. CONCLUSIONS

Despite the widespread availability of other bioanalytical techniques such as HPLC, mass spectrometry and gel electrophoresis for the measurement of proteins, immunoassays remain of critical importance for protein quantitation in biological fluids due to their simplicity and ability to rapidly & reproducibly measure the concentration of proteins in multiple numbers of samples at one time. Due to the availability of robust immunoassays for leptin measurement, an incredible amount of research has been conducted since its discovery about a decade ago resulting in the emergence of a much wider physiological role of leptin in the last few years.

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