Immunoglobulin adsorption on modified surfaces

Preservation of biological functioning of proteins during immobilisation is of special interest in various biomedical and biotechnical applications. In industry physical adsorption of immunoglobulins (IgGs) onto solid surfaces is still the predominant immobilisation procedure because it is relatively easy to perform. Physical adsorption, however, often results in an undesired loss of biological activity. This loss of activity may be caused by changes in the specific folding (the conformation) of the IgG or by a reduced accessibility of the antigen binding sites by blocking, for instance when IgG molecules are adsorbed with their antigen binding sites oriented towards the sorbent surface.To control the orientation and conformation of adsorbed IgG molecules we studied the interactions involved in physical adsorption of IgG molecules on solid surfaces. Our goal was to optimize the biological activity of adsorbing IgG molecules. For this, we introduced a new method to achieve oriented physical adsorption of IgG. This concept is based on the anisodimensionality of IgG molecules and resembles 'molecular sieving' on the sorbent surface. The 'sieve' is created by preadsorbed molecules that form a steric barrier preventing adsorption at some sites, but leaving patches of uncovered surface area. The open areas in the preadsorbed layer can be tuned in such a way that only the smaller part of anisodimensional molecules can enter and adsorb. In the case of 19G this means that only the Fc part can adsorb to the surface and thereby forcing the larger antigen binding parts (F(ab) 2 ), directed towards the solution and, hence, accessible to bind antigens. A 'sieve' formed either with preadsorbed IgG molecules or with triblock copolymers of poly(ethylene oxide), PEO, and poly(propylene oxide), PPO, of the type PEOPPO-PEO was proven to yield a higher specific biological activity of subsequently adsorbing IgG.In brief, the effect of a 'molecular sieve' on IgG adsorption is essentially threefold. Firstly, it induces oriented IgG adsorption. Secondly, it prevents extended undesirable structural changes in adsorbed IgG and thirdly, it prevents undesirable reorientation of the adsorbed IgG.In chapter 1 it is explained that in many applications there is a need to control the biological activity of adsorbed IgG. The physical properties and characteristics ofIgG molecules and the interactions involved in physical adsorption of proteins are described and, more importantly, our variant of 'molecular sieving' is introduced. Finally, an outline of this thesis is given.The influence of electrostatic interactions on the adsorption of IgG is examined both theoretically and experimentally in chapter 2. The long range interaction between IgG and the sorbent surface is treated in terms of the DLVO theory. We attempted to make use of the dipolar character of the IgG molecules to control their orientation upon adsorption. It is concluded that electrostatic interactions have a strong influence on the adsorption behaviour of IgG molecules on hydrophilic charged surfaces. Due to extensive desorption of IgG from both positively and negatively charged surfaces, electric field-induced orientation of IgG could not be established unambiguously.Chapter 3 is mainly dedicated to the orientational aspects of IgG adsorption. In this chapter the phenomenon of 'molecular sieving' is demonstrated first theoretically using a Random Sequential Adsorption (RSA) model and second experimentally by a set of reflectometry experiments on surfaces partially covered with preadsorbed layers of either IgG or triblock copolymers of PEO-PPO-PEO. The rate of IgG adsorption and the maximum adsorbed amount decreases with increasing adsorbed amount of triblock copolymer. On the precoated layers, IgG is indeed adsorbed in a preferential orientation which yielded a higher specific biological activity of the IgG molecules. Furthermore, we observed that the preadsorbed layers prevent undesirable reorientation of adsorbed IgG.The mass flux towards the surface also has a profound effect on the adsorbed amount and, consequently, on the orientation of IgG.In chapters 4, 5 and 6 conformational changes in IgG are studied. In chapter 4 a structural analysis of a monoclonal lgG adsorbed on different silica surfaces (hydrophilic, hydrophobic, hydrophobic with preadsorbed triblock copolymers) using ATR-FTIR spectroscopy is given. The secondary structure of adsorbed IgG on a hydrophilic silica surface resembles that of native 19G in solution. The presence of preadsorbed triblock copolymers on the hydrophobic silica surface cause a decrease in the adsorbed amount of IgG and, more importantly prevent substantial structural rearrangements in the adsorbed IgG molecules.In chapters 5 and 6, Circular Dichroism (CD) is used as a spectroscopic technique for studying protein structure in the adsorbed state. Chapter 5 gives information on the structural changes of IgG molecules induced by adsorption on a hydrophobicsurface and compares these changes with those induced by heat treatment. Neither heatinduced nor adsorption-induced structural changes lead to complete unfolding into an extended polypeptide chain, but leave a significant part of the IgG molecule in a globular or corpuscular form. The structural changes induced by heating and by adsorption are different.The effect of preadsorbed IgG and triblock copolymer molecules on the secondary structure of subsequently adsorbing IgG molecules is studied in chapter 6. Structural rearrangements were less extensive with increasing surface coverage of the polymer. It was found that preadsorbed IgG molecules have comparable effects on the secondary structure of subsequent adsorbing IgG; a more native-like structure is retained for the higher adsorbed amounts. Hence, partial pre-coating of a surface is an effective way to control the secondary structure of later adsorbed IgG molecules.To examine whether the model results apply to industrially manufactured diagnostic methods we implemented in chapter 7 the triblock copolymer preadsorption procedure in a microplate assay. Radio-actively labelled IgG and hCG molecules allowed us to monitor the adsorption of IgG and the subsequent specific binding of hCG. The data obtained are in agreement with our earlier model studies and demonstrate that sieving of the IgG by the polymer does take place, resulting in the creation of a more favourably oriented IgG layer. Our studies indicate that the amount of precoated material is critical in the formation of an operational 'sieve'.</p

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further stud

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin – anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries

Proficiency test for allergens in food 2014

In the autumn of 2014 a proficiency test for allergens in baby cereal was organized by RIKILT, Wageningen UR. This PT-test enabled laboratories to evaluate their competence for the analysis of allergens in baby cereal. Both quantitative and qualitative methods were accepted. The proficiency test was carried out according to ISO/IEC 17043, however this specific test is not part of the accreditation

Methoden om allergenen in voedsel te detecteren

Vanuit Europese regelgeving geldt per1 november 2005 de verplichting om de aanwezigheid van allergie opwekkende stoffen (allergenen) op de etiketten van voedingsproducten te vermelden. Om dit beleid goed te handhaven moet bekend zijn hoeveel allergenen een mens binnen mag krijgen voordat er een allergische reactie optreedt. Een extra factor om rekening mee te houden in allergenen onderzoek, zijn nieuw ontwikkelde soorten voeding ('novel foods'). Er kan bij deze voeding sprake zijn van nieuwe of onbekende eiwitten. Nu is nog moeilijk te voorspellen of deze eiwitten allergie opwekke