Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Type IV fimbriae of Xanthomonas hyacinthi : characterization and application for the detection of wellow disease in hyacinths
    Doorn, J. van - \ 2002
    VU University Amsterdam. Promotor(en): B. Oudega, co-promotor(en): Piet Boonekamp. - [s.l.] : S.n. - 156
    hyacinthus orientalis - hyacinten - plantenziekteverwekkende bacteriën - monoclonale antilichamen - franjes - detectie - polymerase-kettingreactie - xanthomonas hyacinthi - hyacinthus orientalis - hyacinths - detection - fimbriae - plant pathogenic bacteria - monoclonal antibodies - polymerase chain reaction - xanthomonas hyacinthi
    Haemocytic defence in black tiger shrimp (Penaeus monodon)
    Braak, K. van de - \ 2002
    Wageningen University. Promotor(en): E.A. Huisman; W.B. van Muiswinkel; W.P.W. van der Knaap; J.H.W.M. Rombout. - S.l. : S.n. - ISBN 9789058086518 - 159
    penaeus monodon - crustacea - garnalen - immuunsysteem - immuniteitsreactie - immuniteit - verdedigingsmechanismen - via de cel overgebrachte immuniteit - rode bloedcellen - hemolymfe - monoclonale antilichamen - experimentele infectie - infectieziekten - garnalenteelt - penaeus monodon - crustacea - shrimps - immune system - immune response - immunity - defence mechanisms - cell mediated immunity - haemocytes - haemolymph - monoclonal antibodies - experimental infection - infectious diseases - shrimp culture

    Tropical shrimp culture is one of the fastest growing aquaculture sectors in the world. Since this production sector is highly affected by infectious pathogens, disease control is nowadays a priority. Effective prevention methods can be developed more efficiently when quantitative assays for the evaluation and monitoring of the health status of shrimp are available. The defence mechanisms of crustaceans are poorly understood, but knowledge about these is a prerequisite for the development of such health parameters. Therefore, the aim of this thesis was to obtain a better understanding of the defence system of the major cultured shrimp species in the world, Penaeus monodon . The present study emphasised the cellular components of the circulatory system, which play a central role in the haemolymph defence, i.e. the haemocytes.

    To study the usefulness of haemolymph for shrimp health assessment, several cellular and humoral characteristics of P. monodon were determined after haemolymph sampling from the ventral part of the haemocoel (chapter 2). Among other things, five different haemocyte types were distinguished by light microscopy, while electron microscopy revealed granular cells, semigranular cells and hyaline cells. It was concluded that haemolymph characterisation might be a useful tool for health estimation of P. monodon , but that standardisation of the techniques is a prerequisite.

    The use of monoclonal antibodies (mAbs) was proposed as a potential approach for the characterisation of haemocytes. Therefore, a set of mAbs specific for P. monodon haemocytes was produced by immunising mice with haemocyte membrane lysates (chapter 3). Four mAbs (WSH 6, WSH 7, WSH 8 and WSH 16) were selected and extensively characterised. For all mAbs, differences in amount and intensity of the labelling were found between immediately fixed haemocytes and non-fixed cells that were kept in Alsever's solution (AS, an anticoagulant which reduces haemocyte activation) and kept in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of the majority of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mAbs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS or in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different sizes and electron densities. Immuno-reactive extracellular fibrous material could be observed when cells were kept in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and lowest for WSH 16. Double labelling revealed that all mAbs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis was put forward that immuno-reactive molecules recognised by these mAbs, were related to haemocyte activation factors and that the mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon shrimp. Later on, WSH 8 indeed proved suitable for this in immuno-histochemical studies.

    A better characterisation of the immuno-reactive molecules would support the interpretation of the results. In order to investigate whether the mAbs reacted with well-conserved molecules and with haemocytes in animals with molecules that were better characterised than those of P. monodon , a comparative study was carried out (chapter 4). The mAbs also reacted on haemocyte monolayers of the freshwater shrimp Macrobrachium rosenbergii and the two freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus . Immuno-labelling on haemolymph monolayers of the terrestrial isopod crustacean Porcellio scaber (woodlouse) and on coelomic fluid of the annelid Lumbricus terrestris (earthworm) showed partial reactivity. Immuno-reactivity was not observed on haemolymph monolayers of the insect Spodoptera exigua (Florida moth) and the mollusc Lymnaea stagnalis (pond snail), or on blood cell monolayers of the freshwater fish Cyprinus carpio (carp) and of human. On histological sections of M. rosenbergii and P. clarkii , mAb labelling was observed on the haemolymph plasma and on a proportion of the haemocytes. This comparative study showed reactivity of the mAbs in a wide range of crustaceans and related animals and suggests that well conserved molecules were recognised, which may indicate functional importance. Later on, molecules of P. leniusculus that reacted with WSH 6 were better characterised and it was indicated that this molecule could be clotting protein or filamin, which both could be involved in coagulation processes. Unfortunately, the immuno-reactive molecules of P. monodon with WSH 8 could not be characterised further.

    The circulating haemocytes of crustaceans are generally divided into hyaline, semigranular or granular cells, however, this classification is still ambiguous. Not much is known about haemocyte production in penaeid shrimp, but for a better haemocyte classification it is useful to establish how these cells are produced and mature. In order to clarify this, the localisation and (ultra)structure of the haematopoietic tissue and its relation with the circulating haemocytes were studied in chapter 5. The haematopoietic tissue is located in many lobules dispersed in different areas in the cephalothorax, mainly at the dorsal side of the stomach and at the base of the maxillipeds. In order to study the haemocyte production and maturation, shrimp were either injected with LPS, while mitosis was inhibited by vinblastine, or were repeatedly sampled for haemolymph. The presumed precursor cells in the haematopoietic tissue were located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. It was proposed that the presumed young haemocytes were generally known as the hyaline cells. Moreover, a new model was proposed where the hyaline cells gave rise to two haemocytic developmental series, i.e., the large- and small-granular cell line. In addition, indications were found that the granular cells of at least the large-granular cell line mature and accumulate in the connective tissue and are easily released into the haemolymph. Light and electron microscopical observations supported the regulation of the haemocyte populations in the circulation by (stored) haemocytes from the connective tissue.

    In order to investigate the clearance reaction of P. monodon haemocytes live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled (chapter 6). Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that many haemocytes encapsulated the bacteria at the site of injection. Furthermore, a rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ, where they, or their degradation products, could be detected for at least seven days after injection. The lymphoid organ consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. The lymphoid organ of penaeids is also poorly studied. Electron microscopy of the lymphoid organ revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It was proposed that haemocytes settle in the tubule walls before they phagocytose. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessels in most decapod crustaceans that do not possess a lymphoid organ.

    Immuno-staining suggested that many of the haemocytes degranulate in the lymphoid organ, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the lymphoid organ is a filter for virtually all foreign material encountered in the haemolymph. Haemocyte degranulation in the lymphoid organ tubule walls could contribute to the filtering capacity of this organ.

    The experimental shrimp appeared to contain many lymphoid organ spheroids, where bacterial antigens were finally also observed. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.

    White spot syndrome virus (WSSV) is the pathogen that is a major cause of mortality in shrimp culture in the past decade. In contrast to the extensive study of the morphology and genome structure of the viral pathogen, the defence reaction of the host during WSSV infection is hardly studied. Therefore, the haemocyte response upon experimental WSSV infection was examined in P. monodon shrimp (chapter 7). A strong decline in free circulating haemocytes was detected during severe WSSV infection. The combination of in situ hybridisation with a specific DNA probe to WSSV and immuno-histochemistry with a specific antibody against haemocyte granules was carried out on tissue sections. Haemocytic reactions have never been reported in chronic or acute viral infections in shrimp, but the present results showed that many haemocytes leave the circulation and migrate to tissues where many virus-infected cells are present. However, a subsequent response to the virus-infected cells was not detected. During virus infection, the number of cells in the haematopoietic tissue was also reduced. Moreover, it was suggested that many haemocytes degranulated in the lymphoid organ, producing a similar but more obvious layer of fibrous material in the outer tubule wall than after bacterial injection.

    The obtained results are summarised and discussed in chapter 8. Furthermore, the results described in chapters 6 and 7 were used to refine the proposed model of chapter 5. The haemocytes of the small-granular cell line are suggested to mature and carry out their function in the lymphoid organ. The results of the present research emphasise the rapid activation of the haemocytes after stimulation of the animal and illustrate several relevant functions of those cells. The present knowledge provides reliable grounds for further discussions about production, maturation and activation of the haemocytes in penaeid shrimp and possibly also in related animals like other shrimp species, crayfish, lobsters and crabs. Knowledge of the functioning of the defence system is of extreme importance since stimulation of this system is considered as a potential intervention strategy in shrimp culture to overcome the infectious diseases.

    Development of recombinant antibody technology for application in plant pathogen diagnosis
    Griep, R. - \ 1999
    Agricultural University. Promotor(en): W.B. van Muiswinkel; A. Schots. - Nieuwegein : Griep - ISBN 9789058080301 - 109
    plantenziekteverwekkers - plantenziekten - antilichamen - recombinatie - monoclonale antilichamen - detectie - assays - plantenziektekunde - plant pathogens - plant diseases - antibodies - recombination - monoclonal antibodies - detection - assays - plant pathology

    This thesis describes the applicability of the novel phage display technique to select plant-pathogen-specific monoclonal antibodies (MAbs) from combinatorial antibody libraries. The retrieved MAbs are so specific that they can be used as diagnostic tools in sensitive immunoassays for the detection and identification of plant pathogens. Testing results, obtained from laboratories that have applied these recombinant MAbs, are discussed in this conclusive chapter.

    Background

    In the last decades, it has become clear that chemical crop protection has to be reduced. Many of the pesticides, applied to destroy plant-pathogenic fungi, bacteria, insects and nematodes, are hazardous to the environment, and form a serious health risk for animals and humans. Since breeding for disease resistance generally takes years, epidemics have to be prevented by sanitary measures in combination with diagnosis in an early stage of disease development or preferably beforehand. Consequently, sensitive diagnostic assays are required that allow healthy plant propagation material to be certified and soil to be monitored. It is obvious that these data can be used to take adequate crop management decisions.

    The problems associated with serological detection of plant pathogens

    Immunoassays are widely applied as detection methods in agriculture because they can be applied to large numbers of samples, and their application is fast, robust, sensitive and cheap. A major concern is the specificity of the assays. Polyclonal antisera are still the active ingredient of many useful immunoassays. However, the immunochemical complexity of several target organisms is the main reason that extensive cross-reactions with non-target organisms can occur when these antisera are used.

    In 1975, Köhler and Milstein showed that this problem could be circumvented by making monospecific antibodies in continuous cultures by fusing the antibody producing B-lymphocytes with myeloma cells [8]. This 'hybridoma' technique opened a new perspective for the production of specific monoclonal antibodies (MAbs) against various antigens. Today, many MAbs are used in research, as therapeutic agent and for diagnostic purposes.

    However, the hybridoma technique was not as advantageous as was expected in the field of plant-pathogen detection. From the trials in raising specific MAbs against many different plant-pathogens, it became apparent that there are 'difficult' antigens. Several plant viruses can not be sufficiently purified and contain immunodominant plant residues and, as a consequence, the immune response is mainly directed to those impurities. In addition, plant-pathogenic bacteria, fungi and nematodes contain many epitopes that are 'shared' with closely related non-pathogenic family members, which is often leading to extensive cross-reactions. Therefore, the selection of the cells producing the desired MAbs is laborious and often impossible.

    Many techniques have been described to deal with 'difficult' antigens and varied from the enrichment of the desired B-cell populations to guiding of the immune response towards the target antigen, such as:

    • Immunoadsorption of antigen-specific B-cells [2].
    • Selection of antigen-specific B-cells by fluorescence activated cell sorting [15].
    • Complement-mediated lysis of undesired B-cells [2].
    • Masking (immuno-complexing) of contaminating immunodominant plant epitopes with anti-healthy plant antibodies during immunization [2].
    • Suppression of the immune response to healthy plant extracts by injection with cyclophosphamide or other immuno suppressive drugs prior to immunization [11,16].
    • Induction of immunological tolerance through tolerisation of neonatal mice with healthy plant extracts prior to immunization [6].

    Although these methods have occasionally shown their value in obtaining specific MAbs in their respective cases, the common drawback of all these methods is that they are not generally applicable. In addition, the latter two are also repulsive from an ethical point of view.

    Bypassing immunization: A general solution to deal with 'difficult' antigens

    A way to circumvent immunization was offered by recent developments in recombinant DNA techniques [14,17]. These techniques allow cloning and expression of large naive antibody repertoires, derived from non-immunized human donors, in E. coli as single-chain antibodies (scFvs) or Fab fragments. The use of these combinatorial antibody libraries in combination with the display of functional antibody fragments at the tips of filamentous phage created a powerful selection system for MAbs [5,12]. Utilization of this phage display system allows direct selection of highly specific MAbs from naive combinatorial antibody libraries [3,13,18]. As immunization is omitted, phage display is not biased. Hence it will not lead to antibodies directed against the most immunodominant epitope but rather towards the most abundant antigenic determinant.

    Mission impossible: how to make the impossible possible

    Because application of the hybridoma technique was not successful in generating MAbs of sufficient specificity to detect the plant-pathogen Ralstonia solanacearum in reliable and sensitive diagnostic assays, the efficacy of the phage display system was challenged (Chapter 2). To achieve this, phages derived from the (naive) Vaughan combinatorial antibody library (1.4 X 10 10different scFvs) were panned against purified lipopolysaccharides (LPS) which were isolated from R. solanacearum race 3 bacteria.

    After four successive rounds of phage growth and selection for LPS binding, soluble scFvs were produced and tested by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy (IF). Four different scFvs could be distinguished on bases of RFLP analysis. Characterization of the monoclonal scFvs against several bacterial strains, indicated a specificity for R. solanacearum (biovar 2, race 3) LPS which is higher than can be obtained with conventional polyclonal antisera. The fact that the four scFvs were obtained within six weeks after starting this study emphasizes the potency of the phage display system.

    Selection of specific antibodies from the synthetic Nissim library, containing over 10 8different scFv, against beet necrotic yellow vein virus (BNYVV) was achieved (Chapter 3) through expression of the antibody fragments on the surface of bacteriophage M13 and subsequent binding of this phage-antibody to immobilized BNYVV. After several rounds of selection seven BNYVV-specific recombinant monoclonal antibodies were obtained. However, the yield of these monovalent scFv antibodies was low. In an attempt to improve the yields, the genes encoding the BNYVV-specific scFvs were genetically fused to alkaline phosphatase (AP/S) and expressed in E. coli . However, out of the seven different anti-BNYVV scFv-AP/S fusion proteins only three showed alkaline phosphatase activity and retained affinity for BNYVV and the quantity of produced scFv-AP fusion proteins was found to be low.

    Analysis of scFv encoding DNA during expression in E. coli showed the occurrence of high plasmid loss and a high incidence of recombination within the scFv encoding DNA, both for scFv and scFv-AP/S fusion proteins. Probably the scFvs are toxic and offer bacteria that recombine the plasmid DNA (and thereby disable expression of scFvs) a growth advantage. The more tightly repressed tetracycline promoter was used to replace the "leaky" LacZ promoter (Chapter 4). The co-expressed repressor protein tightly repressed the tetracycline promoter in the absence of inducer (anhydrotetracycline). When this new expression system was compared to the old expression system (LacZ induction by IPTG) for expression of scFv-AP fusion proteins, it was found to be superior as improved yields of functional recombinant antibodies were obtained. The tetracycline promoter is also more convenient to use. This is evident when large cultures or high numbers of cultures are grown because the medium has not to be changed since the use of this promoter is, unlike the LacZ promoter, glucose independent.

    Selection of tomato spotted wilt virus (TSWV)-specific scFvs from the naive Vaughan combinatorial antibody library (Vaughan) against purified nucleocapsids and against purified complete virus was also successful (Chapter 5). In contrast to previous selections the pooled scFv encoding DNA was isolated after the fourth round of selection, inserted in the vector pSKAP/S (Chapter 4) and tested directly as individual scFv-AP/S fusion proteins. Twelve different scFvs were obtained against the nucleocapsid (N) and five against the glycoproteins, G1 or G2. Six of the derived antibodies were produced with good yields: four scFvs against the nucleoprotein and two scFvs against G1 or G2. In ELISA they reacted with TSWV proteins in infected Nicotiana benthamiana and not with healthy plant extracts. When the N-reactive scFvs were evaluated for their specificity against six other tospoviruses, cross-reactions were observed with tomato chlorotic spot virus and to a lesser extent with groundnut ringspot virus. Several of the TSWV-reactive scFvs might be useful in routine testing, and an ELISA based on these recombinant antibodies is presently evaluated by routine testing laboratories.

    The ultimate goal

    The development of a sensitive double antibody sandwich ELISA format, based on recombinant antibodies, was a major objective. Whole antibodies coat very well to ELISA plates because they contain flexible hinge-regions that allow rotation of the antigen-binding site. However, scFvs are not suited for coating onto ELISA plates, as they are small molecules that are inactivated upon coating [7]. Fusion of scFv to immunoglobulin domains may enhance the coating efficiency while retaining the full binding activity. Therefore, N56, one of the TSWV binding scFvs, was transformed into a mini-antibody through addition of a constant part (Mouse K constant region) and a flexible dimerization domain (Chapter 5). These bivalent mini-antibodies proved to be very active as a coating reagent in ELISA (Chapter 5) and could compete with a conventional polyclonal antiserum for coating efficiency. As was shown in Chapter 5, sensitive detection of TSWV nucleocapsids could be achieved by application of scFv-AP/S fusion proteins as antibody-enzyme conjugate. Therefore, serological testing can thus be carried out entirely by using bacteria derived coating and detection reagents.

    Endowing antibodies with novel properties

    Routine testing for R. solanacearum is currently mainly performed by immunofluorescent cell staining (IF). However, conjugation of antibodies with the fluorochrome fluorescein isothiocyanate (FITC) is not reproducible and FITC fades rapidly upon illumination. This is not the case with the product of he genetic fusion of R.solanacearum -specific scFvs with green fluorescent protein (GFP). Bright, specific fluorescent labeling of target bacteria was observed when the obtained scFv-GFP fusion proteins (fluobodies) were tested by flow cytometry and in IF. The fluobodies proved to be more resistant to illumination, as was shown by IF after prolonged illumination (Chapter 6).

    Table 7.1.Summary of testing the recombinant scFv alkaline phosphatase fusion protein (monoclonal) in comparison to polyclonal anti- Ralstonia solanacearum antiserum FITC or alkaline phosphatase conjugates. For ELISA the plates were coated with a R. solanacearum specific antiserum (pca 9523bcd), blocked, incubated with samples (obtained from potato lots by PD regulations), incubated with either LPS7-AP/S or pca9523bcd-AP conjugate.

    potato samples

    N=206
    IF-polyclonalELISA-polyclonalELISA-monoclonal
    IF+

    Typical
    IF+

    Atypical
    IF-60 min

    OD>350
    60 min

    OD<350
    60 min

    OD>150
    60 min

    OD<150
    1NAK97 IF+, PD97+,

    Undiluted, N=19
    100%0%0%100%0%100%0%
    NAK97 IF+ ,PD97+,

    Diluted 1:10, N=17
    100%0%0%94%6%100%0%
    2Suspected, NAK97 IF±,

    PD97-,Undiluted, N=31
    16%13%71%32%68%26%74%
    Suspected NAK97 IF±,

    PD97-, Diluted 1:10, N=27
    15%11%74%19%81%15%85%
    3NAK97 Cross-reactions

    N=52
    0%12%88%4%96%0%100%
    Negative

    N=47
    0%2%98%0%0%0%100%
    PD negative control

    N=13
    0%0%100%0%0%0%100%

    1 Samples were tested positive in 1997 and it was confirmed that they contained R. solanacearum bacteria.
    2 Samples were tested positive in 1997 but did not contain R. solanacearum bacteria.
    3Samples were tested negative in 1997.

    Preliminary data of routine testing by testing laboratories

    The R. solanacearum- specific scFv, anti-LPS 7, was evaluated for its use in IF and in ELISA and was found to react with the same specificity as scFv LPS 12 (Chapter 2). It reacted with several R. solanacearum race 3 strains and showed cross-reactions with fewer strains than the polyclonal antiserum that is routinely applied for brown rot diagnosis of potato in the Netherlands. The general Netherlands inspection service for potatoes (NAK) evaluated the use of the anti-LPS7-AP/S based ELISA in their own laboratory and compared the results with those obtained with IF. The samples, obtained and already tested by the Dutch Plant Protection Service (PD) and NAK in 1997, were derived from various potato varieties such as Agria, Bildstar, Bintje Cardinal, Desiree, Diamant, Dore, Elkana, Felsina, van Gogh, Kanjer, Karnico, L. Rossetta, Monalisa, Spunta, Stefano and Symphony.

    The extraction of bacteria was performed according to the regulation of the PD and the results, summarized in Table 7.1, show that IF and ELISA gave comparable results for detection. Moreover, the ELISA that utilizes scFv-AP/S fusion proteins for detection is comparable with the polyclonal ELISA, but more importantly, the background is lower when the recombinant antibody is used for detection instead of the polyclonal alkaline phosphatase conjugate. No problems of increased background were observed regarding the sampling from the different potato varieties. The reduced number of cross-reactions that was observed for the recombinant antibodies in Chapter 2 also seems to hold for this field trial, as the number of positive samples found within the pool of suspected and cross-reacting samples was reduced. It can therefore be concluded that the recombinant scFv LPS7 is very useful under real testing conditions.

    General conclusion and future perspectives

    This thesis shows that antibody phage display is a useful technique for the selection of specific recombinant antibodies against a variety of plant-pathogens. Thus far, recombinant antibodies have been selected only against viruses and bacteria. The results described are promising, and it is expected that the phage display technique can also be used successfully for raising specific MAbs against fungi and nematodes. Therefore, the perspective that was predicted after introduction of the hybridoma technique in 1975 [8], now becomes reality with application of the novel antibody phage display technique.

    Although the synthetic naive Nissim library (10 8) was not compared directly to the naive Vaughan library (10 10), the obtained results favor the application of the latter. The variability within the retrieved scFv-antibodies was higher and the yield of the scFvs was better. Moreover, the amount of scFv-AP/S fusion protein required for a strong signal in the ELISA was up to 50-fold lower, indicating that the recombinant antibodies retrieved from the Vaughan library were of higher affinity. In general, it can be stated that large (>10 10) naive antibody libraries are a prerequisite for generating useful recombinant antibodies.

    More than 50 different antigen-specific recombinant antibodies were selected without the use of experimental animals. However, screening the selected scFv-antibodies was a major problem since E. coli produced several of those, only with great difficulty. It remains disappointing that the choice of a scFv for further characterization was based more on its yield, than on its affinity and specificity for the antigen. Development of eukaryotic expression systems might be an option. Yeast, insect-cells, fungi and plants resemble more closely the mammalian cells than the prokaryotic bacterium E. coli with regard to protein expression, and improved yields of functional scFv-antibodies can be expected when eukaryotes are used.

    There is an ongoing improvement of selection techniques from combinatorial antibody phage display libraries such as selectively infective phage [1,9], expression of antibodies on ribosomes [4] and selectively infected bacteria [10]. Meanwhile, combinatorial phage display libraries are designed to give better folding, and consequently, better producing antibodies. When these novel techniques find their way into plant pathology, it will undoubtedly result in highly specific antibodies that can be used in diagnostic assays that are fast, reliable, robust and cheap.

    Ontogeny of the immune system of fish using specific markers
    Romano, N. - \ 1998
    Agricultural University. Promotor(en): W.B. van Muiswinkel; J.H.W.M. Rombout. - S.l. : Romano - 168
    vissen - karper - zeebaars - immunologie - immuniteitsreactie - immunocytochemie - immunohistochemie - ontogenie - merkers - monoclonale antilichamen - fishes - carp - sea bass - immunology - immune response - immunocytochemistry - immunohistochemistry - ontogeny - markers - monoclonal antibodies

    A panel of monoclonal antibodies (mAbs) was used for the characterisation of leucocyte subpopulations during the ontogeny of common carp ( Cyprinus carpio , L.) and sea bass ( Dicentrarchus labrax , L.). In carp the leucocytes were monitored in different lymphoid organs by immunofluorescence and flow cytometry using specific mAbs for early T cells (WCL9), B cells (WCI12), monocytes/macrophages (WCL15) and thrombocytes (WCL6).

    Early T cells were very numerous (77 %) in thymus during the first weeks post fertilisation (p.f.), but also present in other organs, especially head kidney. Subsequently, these cells disappeared from all organs, except the thymus (40%). B cells appeared in the head kidney from the second week p.f., and later on in the spleen and blood, but their number remained low in the thymus and gut. Thrombocytes were detected in cell suspensions of spleen from the first week p.f. and their percentage increased until the 4 thweek (30%) and then decreased in spleen (10%), but increased in blood (30%). Monocyte/macrophage-like cells were present in all organs from the first week p.f. and their percentage gradually increased until the 8 thweek p.f..

    By using mAb WCL15 on fixed tissue the in situ distribution of monocytes/macrophages was studied in thymus, head kidney, spleen and gut from 2 days until 60 weeks p.f.. Macrophages were found from day 2 p.f. in the head kidney and in the dorsal part of the yolk sac epithelium. From 1 week onwards, macrophages were also found scattered in the thymus and gut and during the second week also in spleen. The number of macrophages increased in all lymphoid tissues until the 6 th-8 thweek p.f., then decreased except in the thymus, where they became localised mainly at the cortical-medullary boundary.

    By using mAb WCL38 increasing numbers of mucosal T cells were observed in cell suspensions of gills and intestine from the first week p.f. onwards. With immuno-histochemistry the early appearance of mucosal T cells could be confirmed in the gills and intestine but was also detected in skin. With mAb WCL9 the ontogeny of the carp thymus was studied and special attention was paid to the development of cortex and medulla. The differentiation between cortex and medulla started in the 4 thweek p.f.. Ultrastructural study of the developing thymus confirmed these data and permitted the analysis of the distribution and morphological differences of the epithelial cells. In addition, numerous apoptotic cells appeared in the cortex from the 4 thweek p.f. onwards, while lower numbers were observed in medulla.

    In sea bass, mAbs DLT15 and DLIg3 specific for T and B cells, respectively, were employed to describe the morphology and distribution of these cells. Enriched leucocyte fractions from different tissues (thymus, spleen, head kidney, intestine and blood) were used. High numbers of T cells were detected in thymus and intestine, whereas B cells were more numerous in blood, head kidney and spleen. Furthermore, an ontogenetic study with DLT15 revealed that the thymus was the first organ with T cells, followed by head kidney and spleen. Although some differences were observed between the two commercially important fish species studied, the data presented in this thesis are of phylogenetic interest and can be used for the development of vaccination strategies in young fish.

    Design, characterization and application of the multiple air-lift loop bioreactor
    Bakker, W.A.M. - \ 1995
    Agricultural University. Promotor(en): J. Tramper; C.D. de Gooijer; H.H. Beeftink. - S.l. : Bakker - ISBN 9789054854791 - 172
    chemische reacties - uitrusting - chemische eigenschappen - automatische regeling - instrumentatie - systemen - zuurstof - monoclonale antilichamen - hybridoma's - chemical reactions - equipment - chemical properties - automatic control - instrumentation - systems - oxygen - monoclonal antibodies - hybridomas

    A new bioreactor is introduced: the Multiple Air-lift Loop reactor (MAL). The MAL consists of a series of air-lift loop reactors within one vessel. With the MAL, a new type of geometry for air-lift reactors with an internal loop is introduced. This new geometry was characterized with respect to hydrodynamics, mixing and oxygen transfer. The hydrodynamics were described by an existing model. Hydrodynamics, mixing and oxygen transfer in the new reactor configuration were comparable to that in conventional air-lifts with an internal loop.

    The design and use of the MAL as a reactor cascade, to approximate plug-flow behaviour, were studied. Biological model systems were used to compare the reactor series to a single vessel. These model systems included immobilized invertase and nitrifying bacteria. With the immobilized invertase it was shown that a threecompartment MAL gives an improved substrate conversion when compared to a single vessel of the same overall volume. This could be described with a previously developed model. Also for the immobilized nitrifying bacteria improved substrate conversion was shown in the comparison between a series and a single vessel. Free suspended hybridomas were used for monoclonal antibody (MAb) production. It was shown that reactor series can be useful research tools for kinetic studies. In the second vessel in the series conditions were obtained that can hardly be reached in a single vessel. Not only growth, but also death could be studied under stable conditions. A model was derived that describes hybridoma growth and their MAb production.

    Vessels in a series can be of equal volume, but very often unequal volumes can be more advantageous. Therefore, choosing the appropriate reactor volumes is an important design step, which is discussed for different applications. Finally, a general procedure for choosing the optimal bioreactor cascade configuration for any application is given.

    The effect of repeated injections of murine monoclonal antibodies against progesterone on immuneresponse and plasma progresterone neutralizing efficacy in cyclic pigs
    Arriens, M.A. ; Booman, P. - \ 1989
    Zeist : I.V.O. (IVO-report B-312) - 32
    antilichamen - antigenen - hybridoma's - immuunsysteem - immuniteit - immunologie - monoclonale antilichamen - varkens - progesteron - geslachtshormonen - diergeneeskunde - antibodies - antigens - hybridomas - immune system - immunity - immunology - monoclonal antibodies - pigs - progesterone - sex hormones - veterinary science
    Monoklonale antilichamen voor de bepaling van abscissinezuur
    Davelaar, E. ; Vonk, C.R. ; Boonekamp, P.M. - \ 1989
    Wageningen : CABO (CABO - verslag nr. 104) - 21
    abscisinezuur - monoclonale antilichamen - hybridoma's - abscisic acid - monoclonal antibodies - hybridomas
    In dit verslag wordt beschreven op welke wijze met abscissinezuuur bij muizen een goede immunogene reactie werd verkregen en hoe de selectie van verkregen antilichamen moet worden uitgevoerd
    E. Coli K99 specific B-cell formation in lymphoid tissues of cattle and its significance for bovine monoclonal antibody production : some preliminary results
    Wissink, H.C.G. ; Veerhuis, R. ; Booman, P. - \ 1989
    Zeist : IVO (IVO - report B-321) - 22
    antilichamen - antigenen - rundvee - hybridoma's - immunisatie - immunotherapie - monoclonale antilichamen - vaccinatie - vaccins - diergeneeskunde - antibodies - antigens - cattle - hybridomas - immunization - immunotherapy - monoclonal antibodies - vaccination - vaccines - veterinary science
    Monoclonal antibodies in animal production : their use in diagnostics and passive immunization
    Booman, P. - \ 1989
    Agricultural University. Promotor(en): C.C. Oosterlee; E.J. Ruitenberg. - S.l. : Booman - 149
    zoötechniek - monoclonale antilichamen - hybridoma's - diergeneeskunde - vaccinatie - immunisatie - immunotherapie - vaccins - obstetrie - zwangerschap - immunologische technieken - elisa - zootechny - monoclonal antibodies - hybridomas - veterinary science - vaccination - immunization - immunotherapy - vaccines - obstetrics - pregnancy - immunological techniques - elisa
    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal antibodies have been applied to biological and medical sciences.

    In animal production monoclonal antibodies are increasingly finding application in the areas of diagnostics, passive immunization and fundamental research. In Chapter 1 of this thesis some applications of monoclonal antibodies within these areas, with emphasis on reproduction, are discussed. It has been concluded that the particular advantages of monoclonal antibodies can firstly and most easily be shown in immunodiagnosis. Once a hybridoma producing a monoclonal antibody appropriate for a particular application has been obtained, large amounts of a homogeneous and reliable reagent are available for as long as they are needed. As ingredients in test kits, monoclonal antibodies have rapidly replaced conventional polyclonal antibodies.

    An example of a typical diagnostic test in animal production in which monoclonal antibodies might be used is the milk-progesterone test for confirmation of oestrus and pregnancy diagnosis in cattle. In Chapter 2 the production and characterization of monoclonal antibodies against progesterone have been described in order to standardize an enzyme immunoassay for milk-progesterone. The antibodies differed considerably in their binding affinity for progesterone and showed distinct specificities for a variety of steroids. Results show that, although the technique of monoclonal antibody production selects antibodies specific for a single antigenic determinant, this does not always preclude the possibility of cross-reactivity. Moreover, most antibodies produced have affinities far below the corresponding conventional antisera, as has been discussed in Chapter 1. The monoclonal antibody with the highest association constant and relatively good specificity did not detect progesterone with any greater sensitivity than the conventional polyclonal sera. However, since monoclonal antibodies avoid the dependency upon animals producing high quality antisera and improve test standardization, a commercially available rapid progesterone cow-side test has been designed based on the monoclonal antibody with the best characteristics.

    Results of Chapter 2 underline the necessity to evaluate carefully the need of producing monoclonal instead of polyclonal antibodies for a given antigen, as considerable time and effort are required to obtain a monoclonal antibody with suitable properties. Preselection of antibodies on affinity and specificity in an early stage makes the monoclonal antibody technology more efficacious. In our laboratory a cocktail of related steroids was used to enable such an early selection of antibodies against either oestrone or oestrone sulphate. Testing the replacement of oestrone, coupled to horse- radish peroxidase, from the antibodies made it possible to produce high affinity monoclonal antibodies with nearly unique specificities in a relatively short time. Experiments are in progress to develop a test for pregnancy diagnosis in pigs in faecal samples based on those antibodies.

    Another application in diagnostics is the use of monoclonal antibodies against a male-specific protein, the H-Y antigen, for sexing bovine embryos before implantation. H-Y is a weak antigen and immunization with H-Y antigen in an inbred strain of mice usually results in production of low titered, low affinity antisera. Because only a low percentage of mice has a good antibody response and their sera run out quickly, monoclonal antibodies were produced (Chapter 3). Male specificity of the antibodies was tested in a variety of assays including enzyme immunoassays based on various sources of soluble H-Y and indirect immunofluorescence assays based on binding of the antibodies to H-Y antigen on the cell surface of male and female cells obtained from a number of tissues and species. Several monoclonal antibodies appeared to be positive in all assays tested, suggesting that the molecule conferring the H-Y antigenicity lacks speciesspecificity and appears to be identical for soluble and membrane-bound H-Y antigen.

    The most promising monoclonal antibodies reactive with the H-Y antigen have been evaluated for their efficiency in sexing Day 7 bovine preimplantation embryos (Chapter 4). Although in an indirect immunofluorescence assay a discrimination between male and female embryos could be made, evaluation of the staining patterns was fairly subjective because of non-specific binding of the monoclonal antibodies to the embryonic cells. After modifying the technique in order to reduce non-specific binding, the number of false positives after sexing under these conditions was greatly reduced, which suggests that the monoclonal antibodies detect a male-specific antigen. It was concluded that the occurrence of false-negative embryos might be caused by a weak expression of the H-Y antigen and/or a low affinity of the monoclonal antibody for bovine cell-surface H-Y antigen. The antibodies used in our experiments had been selected on basis of their binding to both soluble and cell-surface H-Y antigen originating from different sources. Currently, monoclonal antibodies which are selected on high affinity for protein structural determinants on bovine cell-surface H-Y antigen are being produced in order to be used in a highly discriminating sensitive fluorescence assay.

    In Chapter 1 has been discussed that it will take some time to fully realize the potential of monoclonal antibodies in the area of passive immunization or immunomodulation. The production costs are still relatively high and the reaction of the animal to the injected antibodies may limit the effects of passive immunization. Some such limitations are illustrated in the experiments in which anti- progesterone murine monoclonal antibodies were administered to cyclic pigs (Chapter 5). Intravenous injection of increasing amounts of anti- progesterone antibodies resulted in a concomitant rise in levels of antibody-bound progesterone. At the same time a significant rise in plasma concentrations of total progesterone was observed immediately after administration of higher doses of antibodies. Therefore, the net effect of progesterone binding by the antibody was relatively small and more or less independent of the quantities of antibody administered. It has been suggested that animals maintain adequate levels of free progesterone in their circulation by resorption of progesterone from a pool present in body tissues. The effects of administration of anti-progesterone antibodies on plasma levels of free progesterone are, however, not only influenced by the proposed compensating effect of resorption, but also by the possible initiation of a humoral response of the pigs to the injected antibodies. In the experiments described in Chapter 5 it is shown that a minimum dose of 32 mg anti-progesterone antibody elicited an antimouse response after the first injection, having an neutralizing effect on the anti-progesterone monoclonal antibodies administered with the second injection. When smaller quantities of antibody were used, an anti-mouse reaction was detected after the second or third injection.

    From reports concerning the therapeutic use of monoclonal antibodies in man it is known that such an immune response may be directed partially against the isotypic determinants, partially against the idiotypic determinants of the murine antibodies administered. Although anti- idiotypic responses cannot be excluded as a complicating factor, homologous antibodies might offer some advantages over their murine counterparts in terms of effectiveness for passive immunization.

    In Chapter 6 the construction of a bovine-murine heteromyeloma cell line to be used for the production of bovine monoclonal antibodies has been described. It was anticipated that a heteromyeloma would retain the superior fusion characteristics of the mouse myeloma cells and, because of the presence of bovine chromosomes, would be better able to support stable bovine antibody production than interspecies hybridomas produced by fusing mouse myeloma cells with bovine lymphocytes. First (bovine-murine) and second generation (bovine-[bovine-murine]) fusion partners were compared for fusion efficiency and the generated number of antigen- specific antibody-producing clones. In addition, the optimal time- interval between boosting and harvesting of the lymphocytes for fusion and the source of lymphocytes was studied. It could be concluded that fusion of bovine lymph node cells with the second generation heteromyelomas on Day 7 after the final booster injection resulted in the largest number of specific antibody-producing clones. Experiments with a third generation bovine-murine heteromyeloma cell line indicated that fusion efficiency could be further improved (unpublished data). Studies with anti-rotavirus and anti-pregnant mare serum gonadotrophin (PMSG) bovine monoclonal antibodies, produced with the second generation fusion partners, indicate that the heteromyeloma cell lines are very useful for the production of bovine monoclonal antibodies.

    The availability of such bovine monoclonal antibodies offers the possibility to compare the efficacy of homologous and heterologous antibodies in cattle after repeated passive immunization. The in vivo immunoneutralization of PMSG by murine and bovine monoclonal antibodies was chosen as a model for such a study (Chapter 7). Results indicate that repeated injection of murine monoclonal antibodies against PMSG (mMCA) alone did not, or only to a small degree, elicit an anti-mouse immune response. The simultaneous administration of mMCA and PMSG resulted in relatively high levels of anti-mouse antibodies after the second injection, leading to a decrease in neutralizing activity of mMCA. The results suggest that the neutralizing activity of mMCA is inhibited more by anti- idiotypic than by anti-isotypic antibodies against mMCA. After repeated administration of the bovine monoclonal antibody against PMSG (bMCA), either alone or in combination with PMSG, no anti-bMCA antibodies could be detected. In addition, no change in plasma levels of bMCA and PMSG, compared with levels after the first injection, was observed. Although it has to be confirmed by further experiments whether our findings can be generalized, the present results suggest that for repeated passive immunization in cattle homologous antibodies are to be preferred above heterologous antibodies. As far as we can see now it is necessary to evaluate carefully the need to produce homologous or heterologous antibodies, dependent on the amounts of antibody to be administered and the number of treatments.

    In conclusion, the potential of monoclonal antibodies for diagnostic use, therapy or fundamental research, discussed in Chapter 1, together with the results presented in this thesis indicate that the monoclonal antibody technology will have an important impact on the improvement of animal quality and productivity.

    Characterization of monoclonal antibodies against progesterone
    Schakenraad, J.M. ; Booman, P. ; Tieman, M. - \ 1983
    Zeist : I.V.O. (Report / Research Institute for Animal Production "Schoonoord" no. B-224) - 42
    hybridoma's - monoclonale antilichamen - progesteron - hybridomas - monoclonal antibodies - progesterone
    Monoclonal antibodies to Herpes Simplex Virus type 2
    MacLean - Pieper, C.S. - \ 1982
    Landbouwhogeschool Wageningen. Promotor(en): A. van Kammen; A.C. Minson. - S.l. : S.n. - 78
    antilichamen - antigenen - herpesviridae - hybridoma's - immunoglobulinen - monoclonale antilichamen - reticulo-endotheliaal systeem - antibodies - antigens - herpesviridae - hybridomas - immunoglobulins - monoclonal antibodies - reticuloendothelial system

    In this thesis the production and characterisation of monoclonal antibodies to Herpes Simplex virus Type 2 is described. The developement of a suitable radioimmunoassay for the detection of anti-HSV-2 antibodies, and the selection of an optimal immunisation schedule, is given in chapter 2. Three fusion experiments were performed, resulting in nine stable hybridoma lines. The monoclonal antibodies secreted by these lines were subsequently characterised. A list of these antibodies and their properties, including target antigens, is given at the back of this thesis. Four of the antibodies were directed against glycoproteins of the virus, two reacted with non-glycosylated proteins and for three antibodies no target antigen could be identified. All anitbodies were of the IgG class.

    Two antibodies, LP2 and LP3, were directed against the same protein, gD. Competition binding experiments involving two additional monoclonal antibodies against this protein showed that there are at least three different type-common antigenic sites on the gD molecule of HSV-2. LP2 and LP3 are directed against different antigenic sites.

    Using tunicamycin, an unglycosylated precursor of gD was found with a molecular weight of 49,000D.

    The reactivity of antibodies LP1 and LP4 with a number of different HSV-1 and HSV-2 strains strains was determined. LP4 proved to be specific for HSV-2, while LP1 showed comparible reactivity with HSV-1 and HSV-2 strains.

    The ability of antibodies LP2, LP3 and LP4 to protect Balb/c mice from infection with HSV-1 or HSV-2 was assessed. Antibody LP2, which is strongly neutralising, markedly reduced both the inflammatory response and virus titres in the site of infection compared to non-treated mice. Antibody LP3, which is directed against the same protein and is of the same sub- class, had no effect except for a slightly enhanced inflammatory response. Mice treated with antibody LP4 were identical to the control group.

    Treatment with antibody LP2 reduced the frequency with which latent infection was established in infected animals, and reduced the virus titres recovered from reactivated ganglia. Again antibody LP3 had no effect.

    The implications of the findings described above are discussed at the end of each of the chapters. A review of some of the current and potential applications is given in the Discussion.

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