Biol. Chem. Hoppe-Seyler Vol. 372, pp. 163-172, March 1991

Enzyme Immunoassay with Captured Hapten A Sensitive Gastrin Assay with Biotinyl-Gastrin Derivatives Roland VON GRÜNIGEN, Gabriele SIGLMÜLLER, Annamaria PAPINI, Klaus KÖCHER, BirgittTRAVING, Walter GÖHRING and Luis MORODER Max-Planck-Institut für Biochemie, Abteilung Peptidchemie, Martinsried, Germany

(Received 13 November 1990/11 January 1991)

Summary: Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on both the capture of the hapten by streptavidin or avidin adsorbed on polystyrene, and the antigenicity of the captured peptide.The observed full retainment of antibody binding capacity of the biotinyl-gastrins upon their immobilization, allowed to develop a sandwich-type ELISA with a sensitivity of one order of magnitude better than the standard ELISA with

polystyrene-adsorbed gastrin. This hapten capture system reduces desorption particularly pronounced for low mass peptides, and avoids possible modifications or suppression of epitopes by the adsorption process with concomitant reduction of antibody binding affinity of the antigen. This new type of assay procedure may also represent a useful tool particularly for epitope mapping with relatively low mass synthetic protein fragments.

Enzymimmunoassay mit komplexiertem Hapten. Ein empfindlicher Gastrinassay mit Biotinyl-Gastrin Derivaten Zusammenfassung: Es werden zwei N-terminale Biotinyl-Gastrine synthetisiert um den Einfluß der Länge und der chemischen Eigenschaften sowohl auf die Komplexierung des Haptens durch Streptavidin oder Avidin, an Polystyrol adsorbiert, als auch auf die Antigenizität des komplexierten Peptides zu bestimmen. Für beide Gastrin-Derivate war die Bindungsaffinität zu den Antikörpern auch nach Komplexbildung voll erhalten. Dadurch konnte ein SandwichELISA entwickelt werden, der eine zehnfach bessere Empfindlichkeit aufweist als der Standard-ELISA

mit dem Gastrin-Hapten direkt an Polystyrol adsorbiert. Dieses neue Immobilisierungsverfahren verringert die Desorption, die vor allem an niedermolekularen Peptiden beobachtet wird, und verhindert Modifizierung und Unterdrückung der Epitop-Ausbildung im Adsorptionsprozeß. Letztere Effekte können drastisch die Bindungsaffinität von Antigenen zu Antikörpern beeinflussen. Deshalb sollte sich dieses neue Immunbestimmungsverfahren vor allem auch für eine Epitop-Aufklärung von Proteinen mittels niedermolekularer, synthetischer Fragmente eignen.

Key terms: Biotinyl-gastrin, streptavidin biotinyl-hapten complex, enzyme immunoassay.

Abbreviations: Standard abbreviations as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature are used for amino acids and related derivatives; HOSu, N-hydroxysuccinimide; DCC, dicyclohexylcarbodiimide; TEA, triethylamine; DMF, dimethylformamide; DMA, dimethylacetamide; NMP, N-methylpyrrolidone, MeOH, methanol; EtOH, ethanol; BuOH, butanol; AcOH, acetic acid; AcOEt, ethyl acetate;THF, tetrahydrofuran;TFE, 2,2,2-trifluoroethanol;TLC, thin-layer chromatography; HPTLC, high-performance thin-layer chromatography; HPLC, high-performance liquid chromatography; HG17, human little-gastrin; HG-[2-17], human little-gastrin-[2-17]; ELISA, enzyme-linked immunosorbent assay; EIACH, enzyme immuno assay with captured hapten; PBS, phosphate-buffered saline; EDC, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride; pNPP, 4-nitrophenylphosphate; -IgG-aP, goat anti-IgG immunoglobulin conjugated toalcalinephosphatase; BSA, bovine serum albumin.

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R. von Gr nigen, G. Siglm ller, A. Papini, K. K cher, B.Traving,W. G hring and L. Moroder

Enzyme immunoassays with polystyrene-adsorbed low mass peptides have found widespread application for the identification of immunodominant epitopes of proteins or for the quantitative determination of endogenous peptides in physiological fluids or tissue extracts. The major principle of this heterogeneous ELISAis the immobilization of one reactant, e.g. the antigen [l] , or the antibody' 2 - 3J , by adsorption on polystyrene. Adsorption-induced alterations of proteic components capable of influencing the antigen antibody binding have been observed14'^; this phenomenon is expected to be substantially more critical in the case of low mass peptides. Besides easier desorption in the various incubation and washing stepsl6], adsorption-induced conformational changes, hiding or suppression of immunodominant epitopes as well as steric hindrance for the formation of the antigen antibody complex may severely hamper the efficiency of the assay. Covalent linkage to solid matrices'7"101, to protein carriers111'131 or to poly-lysine113'14' have been attempted to by-pass the problem. For this purpose rather unspecific crosslinking reagents are generally used; these may again lead to more or less pronounced chemical modifications of the antigenic peptide with concomitant alteration or suppression of the epitopes as well as to a reduced conformational space of the antigen on the carrier surface. It was therefore compelling to attempt a selective immobilization possibly via a prosthetic group with high affinity for a specific carrier and not interacting with serum components. For this purpose we have selected the streptavidin or avidin biotin capture system to immobilize low mass peptides. This system with its low dissociation constant has been introduced into a variety of biochemical methods and has extensively been exploited in immunological techniques (for review see ref. ^; it has recently been proposed for epitope mapping, too '161. In our previous studies on improvements of immunoassay methodology'17~201we have selected the gastrointestinal hormone human little-gastrin as hapten. From these we have learned that a selective Nterminal attachment of the little-gastrin sequence 2-17 to tracer moieties does not affect its antigenicity. Consequently, in the present study the biotin residue was linked to the N-terminus of the gastrin-[2-17] via the maleinimide/thiol addition principle already adopted successfully for N-terminal modifications of this hormone[17'21]. In order to investigate the critical spacer size for an efficient exposure of the gastrin-specific immune epitope upon complexation to avidin or streptavidin, the biotin binding head was also incorporated with a spacer of more peptide-like structure and of significantly larger size (see Fig. 1). Acompara-

Vol. 372 (1991)

tive analysis of the two gastrin derivatives was expected to indicate possible interferences of the streptavidin (or avidin) surface in the immune complex formation. Experimental Procedures PEPTIDE SYNTHESIS Melting points were determined on a capillary melting point apparatus (Biichi) and are uncorrected. Optical rotations were measured in a thermostated 1-dm cell on a Perkin Elmer polarimeter (model 141). Acid hydrolyses were conducted in OM HCI containing 2.5% thioglycolic acid for tryptophan-containing peptides (24 h at 110 °C). Amino-acid analyses were obtained on a Biotronic analyzer (LC 6001). Gas Chromatographie racemization tests were carried out on a Fractovap (Carlo Erba, 4160) with Chirasil-Val glass capillary columns (22).TLC and HPTLC were performed on precoated silica gel 60 plates (Merck AG, Darmstadt) using the following solvent systems: 1) MeOH/AcOEt/CH2Cl2 (1:2:3); 2) CHC1/TFE (3:1); 3) CHCl/TFE/80% propionic acid (5:1:1); 4) cyclohexane/CHCiyAcOH (45:45:10): 5) 1-BuOH/AcOH/ H2O/ AcOEt (3:1:1:5); 6) n-heptane/te/Y-BuOH/AcOH (3:2:1): 7) !-BuOH/AcOH/H2O (3:1:1); 8) !-BuOH/AcOH/pyridine/H2O (60:6:20:24); 9) l-BuOH/AcOH/pyridine/H2O (45:6:20:24): 10) CHCl3/MeOH/AcOH/H2O (80:40:5:10). Compounds were visualized with chlorine/tolidine, fluorescamine, Elmann's reagent for thiol compounds'23' and p-dimethylaminocinnamaldehyde for biotin compounds'2^. Reactions were performed at room temperature unless stated otherwise. D-(+)-Biotin was purchased from Fluka AG. Synthesis of biotinyl-gastrin-A N-Hydroxysuccinimido biotinate (1) Biotin (1 g; 4.1 mmol) was dissolved at 80 °C in DMF (20 m/) and upon cooling to room temperature HOSu (0.65 g; 5.3 mmol) and DCC (0.85 g; 4.1 mmol) were added. After 12 h the dicyclohexylurea was filtered off, the solution was evaporated to dryness and the residue was taken up in boiling isopropanol; the suspension was cooled to room temperature and the product was filtered off; yield: 1.33g (95%); homogeneous onTLC (solvent system: 1); mp 210-212 °C [Lit: 208-210 °C (25); 210 °C (26)]; [α]έ° = + 51.8° and [α]|406= + 62.0° (c = 1, DMF). Anal, calcd. for Ci4Hi9N3O5S (341.4): C 49.26; Η 5.61; Ν 12.31. Found: C 49.23; Η 5.66; Ν 12.17. Biotinyl-S-tert-butylthio-cysteamine (2) To a solution of S-tert-butylthio-cysteamine hydrochloride (27) (0.35 g; 1.76 mmol) andTEA(0.24 m/; 1.76 mmol) in DMF (10 ml) compound 1 (0.4 g; 1.17 mmol) was added. After 18 h the product was precipitated with water and recrystallized from EtOAc. Yield: 0.4 g (83%); mp 140.5 °C; [a]g = + 60.7° and [αβ°6= + 72.6° (c = 1,TFE); homogeneous onTLC (solvent system: 1,4). Anal, calcd. for ^Η29Ν3Ο283 · H2O (409.9): C 46.88; Η 7.62; Ν 10.25. Found: C 46.72; Η 7.76; Ν 1.0.22. Biotinyl-cysteamine (3) Compound 2 (0.1 g; 0.24 mmol) was dissolved in argon-saturated 98% aqueousTFE and reacted with tributylphosphine (0.2 m/) for 30 min.The solvent was evaporated and the residue was distributed between water and ten-butyl methyl ether. The aqueous phase was washed phosphine-free with ether and lyophilized. Yield: 70 mg (91%); homogeneous onTLC (solvent system: 1); mp 155 °C; [a]o = + 70.1° and [a]52406= + 82.8° (c = 0.5.TFE).

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Vol. 372 (1991)

Immunoassay of Peptides

Anal, calcd. for C,2H21N3O2S2 · H2O (321.5): C 44.84; H 7.21; N 13.07. Found: C 44.62; H 7.38; N 12.96. [Nlel5]-HG-[2-17] (4) A solution of H-[Glu(OfBu)] 5 -Ala-Tyr(fBu)-Gly-Trp-Nle-Asp-(OfBu)-Phe-NH 2 (28) (1.0 g; 0.52 mmol) and Boc-Gly-Pro-Trp-Leu-OH · 2H2O (21) (0.75 g; 1.3 mmol) in NMP (40 ml) and DMA (10 ml) was reacted at - 10 °C with HOSu (0.26 g; 2.3 mmol) and DCC (0.27 g; 1.3 mmol). After 4 days at room temperature the solution was concentrated and the product precipitated with 0.5% aqueous KHCO3. The solid was washed with water and then with warm MeOH: yield: 1.2 g (94%); homogeneous onTLC (solvent systems: 2,3); amino-acid analysis of the HC1 hydrolysate; Asp 1.01(1) Glu 4.78(5) Pro 0.96(1) Gly 1.92(2) Ala 1.00(1) Leu0.96(l) Nie +Tyr 2.05(2) Phe 1.01(1)Trp 1.83(2); peptide content: 100% calcd. for Mr = 2462.0: gas Chromatographie racemization test: DAla 0.6%; D-Pro

Enzyme immunoassay with captured hapten. A sensitive gastrin assay with biotinyl-gastrin derivatives.

Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on b...
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