Substance P (SP) is a member of the tachykinin family and has an important role in immune responses. SP is detectable in plasma in a free and bound state. Simple modification of a commercially available SP enzyme-linked immunosorbent assay allows the dissociation and capture of plasma SP without solid-phase extraction.The undecapeptide substance P (SP) is a member of the tachykinin family. Its role as a neurotransmitter (21,22,23) includes regulation of immune responses (19). Elevated levels of serum or plasma SP have been associated with disorders such as inflammatory bowel disease (16), sickle cell crisis (20), depression and anxiety (2,11,14,19,26), rheumatologic diseases (1, 17), infectious diseases (28), including human immunodeficiency virus (7, 8, 13), and cancer (24, 27). SP exerts its influence on immune responses through the induction of regulatory cytokines (3,18).Considerable variability has been reported for SP levels in similar biological fluids from healthy subjects and patients with diverse disease processes (5,7,8,9,11,12). Corbally et al. (6) reported that endogenous human plasma-derived SP was reversibly and nonspecifically bound to both high-molecularmass proteins (Ͼ400,000 Da) and intermediate-molecularmass proteins (58,000 Da). SP was readily dissociated from proteins by repeat gel filtration. These observations suggested that SP binding to plasma proteins was mediated by weak hydrogen bonding and led to the development of methods for the extraction of SP from a variety of biological fluids (10, 25). Such extractions should be considered carefully since most are designed for the enrichment of low-molecular-mass peptides that can potentially exclude peptides nonspecifically bound to high-molecular-mass plasma or serum proteins. Such procedures may lead to underestimates of the total quantity of SP (4, 6).This study investigated the influence of hydrogen ion concentrations on nonspecific SP-plasma protein dissociations that allow for the optimal binding of dissociated SP by capturing antibody.Human plasma. Whole-blood-derived EDTA anticoagulated plasmas obtained from 28 healthy adult control subjects were stored at Ϫ70 o C until evaluated for SP levels. SP EIA. A commercially available SP antigen competition enzyme immunoassay (EIA; Cayman Chemical Company, Ann Arbor, MI) was modified to investigate the influence of hydrogen ion concentration on the dissociation and subsequent capture of SP in pooled normal adult human plasma. The SP EIA uses microtiter wells coated with monoclonal mouse anti-rabbit immunoglobulin G, an SP-specific polyclonal rabbit capture antibody, and an acetylcholinesterase-conjugated SP tracer. The substrate for acetylcholinesterase consists of acetylthiocholine and 5,5-dithio-bis-(2-nitrobenzoic acid). The colored end product is 5-thio-2-nitrobenzoic acid with absorbance read at 412 nm. A phosphate-citrate (PC) buffer was selected as the SP dissociation buffer with the evaluation of buffers having pH values of 7.3 to 2.2. Pooled normal adult human plasma was first dil...