Design of the Immunogen and Label for Use in a Fluoroimmunoassay for Paracetamol

Gallacher, Gerard; Coxon, Ruth; Landon, J.; Rae, C.; Abukinesha, R

doi:10.1177/000456328802500105

Cited by 10 publications

(2 citation statements)

References 4 publications

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“…fore, suggests real, albeit small, variation in the ability of the IgG preparations to bind substrate. The magnitude of the variation in K , is similar to that of the binding characteristics of non-catalytic antibodies produced by using carefully prepared immunogens (El-Gamal et al, 1987) and an immunisation schedule (Bennett et al, 1987;Gallacher et al, 1988;Mellor et al, 1989) analogous to that used in the present work. Variation in the binding characteristics of non-catalytic antibodies during an immunisation schedule has recently been subjected to detailed analysis by Foote and Milstein (1991).…”

Section: Additional Evidence That the Catalytic Activity Of Igg Prepasupporting

confidence: 65%

Catalytic antibody activity elicited by active immunisation

Gallacher

Jackson

Searcey

et al. 1993

European Journal of Biochemistry

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) -EJB 92 1711121. The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two-year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4-nitrophenyl4-carboxymethylphenyl hydrogen phosphate and amino groups of keyhole-limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18-week immunisation programme using an analogous sulphone imunogen (comprising the amide conjugate bonded through the amino group of 4-nitrobenzyl, 4-(4-aminobutoxy)benzyl sulphone and carboxyl groups of keyholelimpet haemocyanin) were evaluated kinetically by using 4-nitrophenyl4-(3-aza-2-oxoheptyl)phenyl carbonate and 4-nitrophenyl 4-(2-hydroxyethoxy)phenyl carbonate as substrates.2. Catalytic activity was found in all 13 samples of anti-phosphate IgG but was absent in the sample of anti-sulphone IgG as well as in all samples of IgG isolated from the serum of nonimmunised animals. These findings, taken together with the lack of catalytic activity of the antiphosphate IgG towards the 2-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti-phosphate IgG preparation is entirely antibody-mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti-phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation 3. The nature of the, albeit small, variation in the catalytic characteristics of the anti-phosphate IgG (increase in both k,,,, the catalytic rate constant calculated as V/2[IgG] and k,,,/Km, the apparent second-order rate constant for the overall catalysed conversion of substrate to products, with increase in K,,, suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis.4. This interpretation is supported by the difference in the values of the dissociation constant K, for the competitive inhibition by the transition-state analogue 4-methylphenyl4-nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti-phosphate IgG samples : for the catalysis with K,,, = 4.5 yM, K, = 9 nM and for that with K, = 1.3 yM, K, = 80 nM.5. 4-Nitrobenzyl 4-(4-aminobutoxy)benzyl sulphone, the hapten that was used to prepare the sulphone immunogen mentioned in (l), failed to inhibit the hydrolysis of 4-nitrophenyl 4-(3-aza-2-oxohepty1)phenyl carbonate catalysed by anti-phosphate IgG. The sulphone moiety, therefore, does not appear to mimic adequately the carbonate in either ground-state or transition-state complexes.Since the discovery of catalytic antibodies by Tramontan0 et al. (1986) and Pollack et al. (1986), progress in this new field has developed rapidly (for a review see Lerner et al., 1991). Most of the sub...

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Section: Additional Evidence That the Catalytic Activity Of Igg Prepasupporting

confidence: 65%

Catalytic antibody activity elicited by active immunisation

Gallacher

Jackson

Searcey

et al. 1993

European Journal of Biochemistry

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“…It seemed important to investigate the possibility of producing polyclonal catalytic antibodies for several reasons: (i) the relative simplicity of producing polyclonal as against monoclonal antibodies, (ii) the possibility of extending the catalytic antibody field to include investigation of catalytic antibody responses in animals, (iii) the possibility that if polyclonal antibody preparations that obey the Michaelis-Menten equation could be prepared, despite the overall structural heterogeneity of polyclonal IgG, they would provide a ready means of investigating a wide range of kinetic characteristics before the detailed structural studies that would necessarily demand use of monoclonal antibodies, (iv) the potential value of polyclonal catalytic antibodies as catalysts for technological applications, and (v) the potential value of catalytic antibodies produced by immunization in the development of novel therapies. The production of polyclonal antibodies that bind small molecules has been extensively investigated (Landsteiner, 1947;Pauling & Pressman, 1945), and it is possible to produce polyclonal antibodies with particular recognition features by careful design of the immunogen employed for antibody production and ofthe ligands involved in subsequent binding interactions (Bauminger et al, 1974;Gallacher et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

A polyclonal antibody preparation with Michaelian catalytic properties

Gallacher

Jackson

Searcey

et al. 1991

Biochemical Journal

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1. 4-Nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.

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