Peter de Leuw:

Gewinnung und Einsatz multispezifischer Antikörper als Hilfsmittel zur Probenvorbereitung in der Rückstandsanalytik von Penicillinen
Production and Use of Multispecific Antibodies for Immunoaffinity Cleanup in Penicillin Residue Analysis


7.2 Abstract

Multispecific antibodies against penicillins were induced in three rabbits and five laying hens, using an immunogen with 6-aminopencillanic acid as immunogenic epitope. The immunogen was synthesized by coupling 6-aminopenicillanic acid (6-APA) via the 6-amino group to keyhole limpet hemocyanin or ovalbumin as carrier proteins.
The antibodies showed high cross reactivities with benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, oxacillin, cloxacillin and dicloxacillin, while cephalexin or a penicilloyl derivative did not react.
The antibody titers were between 50 and 100 in rabbit serum after the 6th booster injection. In order to increase the titer, immunizing of the rabbits was stopped and resumed 7 weeks later. The antibody titer was not increased and surprisingly the cross reactivities to all penicillins but benzylpenicillin had disappeared.
In egg yolk the titer development was followed up over the whole immunizing period. Two animals (no. 15 and 19) showed a rapid increase of the titer to about 2000 at the beginning of the immunization period which was followed by slow decrease to values about 100 - 200. The other three hens (no. 57, 79 and 81) showed a constant titer on a low level of about 250.
The development of the specificity was screened with benzylpenicillin and ampicillin and was different between the animals, too. In the yolks of one animal the cross reactivities were low at the beginning (20 %) of the immunization period, increased at a later stage (250 %) and decreased at the end of the period to 100 % compared with 6-APA.
The obtained rabbit antibodies were immobilized after ammonium sulphate precipitation via the oxidized carbohydrates of the Fc region with the intention of using them for immunoaffinity chromatography. However, binding capacities of the immobilized antibodies were too low (< 25 ng/ml gel) for using the column for residue analysis of penicillins.
Immobilizing the yolk antibodies via the amino groups after isolation with affinity chromatography resulted in colums without binding capacity. On the other hand, colums with capacities of about 2 g/ml gel could be obtained by an identical immobilization procedure with the only exception that the antigen binding sites were blocked by phenoxymethylpenicillin. As a drawback, the antibodies lost their activity within one week at + 4 C. The immobilization of the polyethylene glycol precipitated antibodies resulted in columns which were too low in capacity (80 ng/ml gel).
The application of an immunoaffinity cleanup in penicillin residue analysis was tested with a column which had the following characteristics: Antibodies from rabbit serum, induced with a cloxacillin-human serum albumin conjugate, crossreactivities: 4 % for oxacillin and 62 % for dicloxacillin.
The immunoaffinity column was integrated in a HPLC system with an automated solid phase extraction. Detection was at 230 nm when analyzing milk and bovine muscle and at 300 nm after photochemical post-column derivatization when analyzing bovine liver.
The crude extracts were obtained by ultrafiltration of milk at a molecular weight cut off of 30,000 Da and by extraction of homogenized muscle and liver with a mixture of sodum chloride solution and acetonitrile in an ultrasonic bath.
30 µg/kg isoxazolylpenicillin in milk and 300 µg/kg in muscle and liver (corresponding to the EU-maximum residue level) could be analyzed with coefficients of variation of 9 % from milk, 1.5 % from muscle and 6.5 % from liver.


The thesis was accepted on 28/June/1996 from the Fachbereich Naturwissenschaften II (Chemie/Biologie) of the Bergischen Universität-Gesamthochschule Wuppertal.
Die Dissertation wurde am 28. Juni 1996 vom Fachbereich Naturwissenschaften II (Chemie/Biologie) der Bergischen Universität-Gesamthochschule Wuppertal angenommen.


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© Mai 1997
Peter de Leuw PdL, , letzte Aktualisierung: 08.07.2018 - ausgedruckt am 16.10.2021