A SOLID-PHASE RADIOIMMUNOASSAY FOR THE DETERMINATION OF BACTERIAL-SPECIFIC ANTIBODIES WITHIN DIFFERENT IMMUNOGLOBULIN CLASSES: APPLICATION TO BOVINE BRUCELLA ABORTUS ANTIBODIES D. Levieux, G. Bezard To cite this version: D. Levieux, G. Bezard. A SOLID-PHASE RADIOIMMUNOASSAY FOR THE DETERMINA- TION OF BACTERIAL-SPECIFIC ANTIBODIES WITHIN DIFFERENT IMMUNOGLOBULIN CLASSES: APPLICATION TO BOVINE BRUCELLA ABORTUS ANTIBODIES. Annales de Recherches Vétérinaires, INRA Editions, 1978, 9 (3), pp.523-530. hal-00901034 HAL Id: hal-00901034 https://hal.archives-ouvertes.fr/hal-00901034 Submitted on 1 Jan 1978 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. A SOLID-PHASE RADIOIMMUNOASSAY FOR THE DETERMINATION OF BACTERIAL-SPECIFIC ANTIBODIES WITHIN DIFFERENT IMMUNOGLOBULIN CLASSES: APPLICATION TO BOVINE BRUCELLA ABORTUS ANTIBODIES D. LEVIEUX G. BEZARD. Station de Pathologie de la Reproduction, I,N.R.A., Centre de Recherches de Tours, B.P. 7, Nouzilly, 37380 Monnaie, France. Résumé DOSAGE RADIOIMMUNOLOGIQUE POUR LA DETERMINATION DES DIFFERENTES CLASSES D’ANTICORPS SPECIFIQUES D’UNE BACTERIE : APPLICATION AUX ANTICORPS BOVINS CONTRE BRUCELLA ABORTUS. &horbar; Un dosage radioimmunologique (DRI) en phase solide a été mis au point pour le titrage des différentes classes d’anticorps spécifiques d’une bactérie. Des tubes en verre sont sensibilisés par sédimentation, séchage et fixation au méthanol de corps bactériens de Brucella abortus. Les anticorps de sérum bovin qui se fixent sur les bactéries sont mis en évidence par l’addition d’un sérum de lapin spécifique d’une classe ou sous-classe d’immunoglobulines bovines puis d’anticorps de mouton anti- immunoglobulines de lapin marqués par !2e1. Cette technique en trois temps a été choisie car très sensible (< 0,3 picogramme d’anticorps/ml), donnant la meilleure discrimination entre la radioactivité spécifique et le bruit de fond et permettant d’éviter le marquage de plusieurs sérums de lapin anti-immunoglobulines bovines. Différents paramètres ont été étudiés : nature et préparation des tubes, quantité et fixation des Brucella, température et temps d’incubation, volume d’incubation, concentration ces réactifs. Ce DRI et la technique de fixation du complément (FC) ont été appliqués à des sérums prélevés à intervalles régu- liers sur des génisses infectées expérimentalement par t3rucella abortus pendant la gestation. Un sérum de lapin anti-lgG1 bovines a été utilisé dans le DRI car cette sous-classe est la plus active dans la F.C. La cinétique d’apparition des anticorps anti-Brucella est très signifi- cativement corrélée pour les deux techniques (r = 0,956 ; P < 0,001), démontrant ainsi la validité de ce DRI. Ce test est 10000 fois plus sensible que la F.C. Introduction rement of antibodies against bacterial an- tigens depend upon secondary effects of Assays presently available for the measu- the primary antigen-antibody interaction, such * Present address : Laboratoire des Maladies as agglutination or complement activation, Nutritionnelles, C.R.Z.V. de Theix, LN.R.A., and do not always estimate the total anti- 63110 Beaumont, France. body content of the antiserum. In addition, such tests do not lend themselves to the Antigen was washed twice before use in study of the different classes of antibodies phosphate buffered saline (PBS) pH 6.9 and, involved. for the RIA only, twice in distilled water. This may, for instance, be achieved by of each class 1) purification immunoglobulin Albumin diluent by physico-chemical means, 2) assessing antibody activity of each class. Quantitative Albumin diluent was made by adding purification of each class is difficult and human serum albumin (HSA) to PBS at a consequently this procedure has a limited concentration of 5 mg/ml. value only. determine the na- Alternatively, one may Sera ture of immunoglobulins released by disso- ciation from antigen-antibody complexes but Bovine sera for the determination of im- dissociation may be far from complete (Read munoglobulins with antibody activity against et al., 1974). Brucella abortus were obtained at repeated In a different approach, the concentration intervals from seven heifers experimentally of each immunoglobulin class is determined, infected by Brucella abortus, strain 544, in by means of single radial immunodiffusion, the 6th month of gestation (Plommet et al., before and after removal of its antibody 1976). Blood was collected from the jugular active portion (Nash and Heremans, 1969). vein, allowed to clot and the serum stored The accuracy of the measurements, essen- at -20°C. Before use in the RIA all the tially that of the immunodiffusion technique, sera were heat inactivated (56°C, 30 mn). limits this method to sera with high antibody Reference bovine serum « B1 », used for titers. purification of specific igG1 or IgG2 anti- Recently, a radioimmunoassay (RIA) has bodies, was a pool of 20 sera from brucel- been developed to measure antibodies losis-aborted cows with very high serological against Brucella abortus in bovine serum titers. by competition between immunoglobulins of the test sample and radiolabelled active Preparation of antisera immunoglobulins (Chappel et al., 1976). As described this method does not differentiate Rabbit antisera to bovine immunoglobulin the activity of the immunoglobulin classes classes and subclasses were obtained as and postulates that all the antibodies to be previously described (Levieux, 1974). antiserum to rabbit was tested have the same specificities as the Sheep igG1 pre- with radio-labelled one ; this is invalidated by the pared by immunisation 15 mg of puri- results given. fied IgGl (Diethylaminoethyl cellulose chro- After unsuccessful trials with different matography) for four weeks in Freund’s hemagglutination inhibition or indirect immu- complete adjuvant as multiple intradermal nofluorescent techniques, we propose a new injections. solid-phase RIA to measure specific anti- bodies within different immunoglobulin clas- Purification of antibodies ses, which avoids repeated centrifugations Bovine antibodies to Brucella were which are time consuming and difficult to antigen perform, without loss of material, for Bru- obtained as follows : 300 ml of the standar- cella antigen. dized antigen were incubated with 30 ml of heat inactivated serum B1. After cooling in an ice-bath the bacterial cells were cen- trifugated and repeatedly washed until the Materials and methods optical density (E !28°, ) of the supernatant was below 0.05. Antibodies were eluted with Preparation of antigen glycine-HC1 buffer (0.2 M, pH 2.6) for 15 mi- The antigen was a gift from Laboratories nutes at room temperature and the pH Roger Bellon (Neuilly, France) as a standar- adjusted to 7.0 with 1M dipotassium hydrogen dized Brucella abortus agglutination concen- phosphate. After filtration through 0.2 p,m trate (Alton and Jones, 1967). Antigen dilu- Millipore filters, the protein concentration tions quoted below refer to this concentrate. was determinated from absorbance measu- rements (E 1 280 = 13.5). The purification of on Sephadex G25 column (25 x 1 cm) equi- librated with PBS 0.1 M 7.2 the IgGl and IgG2 subclass antibodies was pH containing 5 HSA and the fractions checked for performed as previously described (Levieux, mg/ml 1974) using Sephadex G200 and DEAE cel- radioactivity in a Packard y counter. lulose chromatography. Purity of the pre- was checked immunoelectro- parations by Radioimmunoassay phoretic analysis both with rabbit anti-whole bovine serum and monospecific antisera for Fixation of Brucella antigen on glass igG1 or IgG2. The percentage of active tubes : 50 I-tl of a 1/10 dilution of the washed antibodies was tested by determination in standardized bacterial suspension were de- radial immunodiffusion (Mancini et al., 1965) posited at the bottom of glass tubes and of the concentration of each immunoglobulin allowed to dry overnight at 37°C. The dried preparation before and after absorption with Brucella were then fixed with 0.5 ml metha- an excess of Brucella. Each preparation nol (100 °/o) for 10 minutes and the excess was more than 90 p. 100 active. reagent was poured off. Immediately before Sheep antibodies to rabbit IgG were pre- use the Brucella antigen was rehydrated for pared from the sheep antiserum by affinity 10 minutes with albumin diluent and excess chromatography on glutaraldehyde-polyme- reagent was again poured off. rized rabbit and IgG (Avrameas Ternynck, A typical three-step RIA was set up in 1969). quadruplicate as follows : 0.2 ml of a 10-4 dilution of the serum to be tested in albumin lodination of antibodies diluent or ten fold dilutions of specific immunoglobulins were incubated with the Sheep anti-rabbit [gG antibodies and bo- rehydrated antigen at room temperature for vine IgGl or IgG2 anti-Brucella antibodies 24 hours. The tubes were washed twice were labelled with izsl (Amersham) by the with 2 ml of cold albumin diluent, allowed chloramine T method (Greenwood et al., to drain on filter paper and incubated with 1963) modified as follows. One mCi (5 jjU) 0.2 ml of a 10-4 dilution of monospecific was used to label 10 !Lg of immunoglobulin rabbit anti-bovine immunoglobulins at room (10 ¡Jo1). in PBS 0.5 M pH 7.2. The reaction temperature for another 24 hours. After was allowed to proceed for 10 sec at room two washes with albumin diluent the tubes temperature and then stopped by addition were incubated with 0.2 ml of radiolabelled of 50 ¡.¡.g of sodium metabisulfite (10 !LI). sheep anti-rabbit IgG for the final 24 hours, After addition of 50 !g of potassium iodide washed twice with albumin diluent and (50 !ti), the mixture was chromatographied counted for radioactivity.