h,lhlumwllemlstly. 1')75, Vol 12, pp 329 331

Pergamon Press

Printed m Great Britain

MODIFICATION OF BACTERIOPHAGE WITH HAPTENe-AMINOCAPROYL-N-HYDROXYSUCCINIMIDE ESTERS; INCREASED SENSITIVITY FOR IMMUNOASSAY M. BECKER and O. M~,KELA Department of Serology and Bacteriology. University of HelsinkL Haartmaninkatu 3, 00290 Helsinkl 29, Finland (First recewed 17 July 1974; m rewsed form 20 September 1974) Abstract--Batches of bacteriophage T4 were conjugated with one of the haptens: NIP, NP or DNP by inserting an ¢-aminocaproyl spacer between the hapten and the phage. The three haptencaproyl phages were approximately ten times more sensitive to anti-hapten antibodies than the corresponding directly haptenated phages with an optimal hapten coat. The hapten-caproyl phages could detect immune anti-hapten antibody (50 per cent inactivation) at concentrations ranging from 17 to 90pg/ml and the one tested batch of natural ann-NIP at a concentration of 210pg/ml. They can be used for the quantitation of IgM (and perhaps IgA) m body flmds at concentrations as low as 100 ng/ml.

INTRODUCTION The hapten-modified bacteriophage inactivation technique, first described by M~ikel~i (1966) and Haimovich and Sela (1966), has been employed as a sensitive tool for the detection of anti-hapten antibodies as well as for in vitro assays of substances present in biological fluids. In many instances classical small haptens such as NIP* (M~ikel~i, 1966) or DNP (Carter et al., 1968) have been coupled directly to the bacteriophage via existing functional groups on the haptenic moiety, leading to preparations capable of detecting as little as 10pg of anti-hapten antibody (Mgkel/i, 1966). For certain purposes even higher sensitivities would be desirable. For example, fine-specificity studies of natural anti-hapten antibodies are difficult to perform with existing phage preparations (Imanishi and M~ikel~i, unpublished results). Insertion of spacer between the immunodominant haptenic moiety and the bacteriophage might conceivably lead to a greater orientation flexibility or accessibility of the haptenic determinant. The result could be a greater sensitivity of the phage preparation to antihapten antibody. In the present communication, the N-hydroxysuccinimlde esters of (4-hydroxy-3-nitrophenyl) acetyl-eaminocaproic acid (NP cap), (4-hydroxy-5-iodo-3nitrophenyll acetyl-e-aminocaproic acid (NIP cap) and 2,4-dinitrophenyl-E-aminocaproicacid (DNP cap) were coupled to bacteriophage T4 and the resultant modified phage compared to phages coupled with the same haptens by the conventional methods (NIP-

T4 and NP-T4 coupled via the azide method and DNP-T4 coupled via the sulfonic acid derivative).

MATERIALS AND METHODS

Synthesis of hydroxysuccimmide esters of NIP-cap, NP-cap and DNP-cap NP-cap and NIP-cap were prepared according to Hatcher and M~ikel~i(1972). Purity was assayed by TLC on silica gel (chloroform-methanol-ammoma; 3:4:2). Melting points were 106-108 and 151°C respectively. DNP-cap was purchased from Sigma (Lot 81C-0160) and used without further purification. N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccmimide were products of Fluka AG (Buchs SG, Switzerland). All solvents used were analytical grade. The N-hydroxysuceinimide esters (OSu) of NP-cap, NIP-cap and DNP-cap were made essentially as described by Martin et al. (1971) with some modifications. NP-cap-OSu. 2.7g NP-eap and 1.5 g N-hydroxysuccinimlde were dissolved in 95ml 1,2-dimethoxyethane. To the above was added 1.79g DCC in 5ml of the above solvent and the reaction allowed to proceed overnight at 4°C. The resulting precipitate (N, N'-dicyclohexylurea) was filtered (1.5g recovered) and the filtrate evaporated to dryness under reduced pressure. The resulting oil was redissolved in dichloromethane (100ml)and extracted twice with 100ml vol of 0.1M sodmm bicarbonate at 4°C and once with water. The lower dlchloromethane phase was dried with anhydrous sodmm sulfate and nhexane was added until the mixture became cloudy, and finally NP-cap-OSu was allowed to crystallize at -20°C. The crystalline product was dried over phosphorous pentoxide. Yield: 2.1 g. DNP-cap-OSu was prepared in a similar fashion. For NIP-cap-OSu, the solvent was 1,2-dimethoxyethane contaming 10% dimethylfornamide. All compounds were pure Ltst of abbrevtattons: NP, (4-hydroxy-3-nitrophenyl)ace- as assayed by TLC on silica (chloroform-methanol, tic acid: cap, e-ammocaproic acid; OSu, N-hydroxysuc- 85:15). cmlmide ester; DNP, 2,4-dinitrophenyl; DCC, N, N'dicyclohexylcarbodiimide; PFU, Plaque forming units; Preparations of phage conjugates NIP, 4-hydroxy-5-iodo-3-mtrophenyl) acetic acid; TLC, All phage conjugates were prepared by treating a Thin layer chromatography number of identical phage suspensions (1011PFU/ml in 329

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Modification of bacteriophage with hapten-epsilon-aminocaproyl-N-hydroxysuccinimide esters; increased sensitivity for immunoassay.

h,lhlumwllemlstly. 1')75, Vol 12, pp 329 331 Pergamon Press Printed m Great Britain MODIFICATION OF BACTERIOPHAGE WITH HAPTENe-AMINOCAPROYL-N-HYDRO...
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