A simple transformation independent method for outlier definition

Abstract Background: Definition and elimination of outliers is a key element for medical laboratories establishing or verifying reference intervals (RIs). Especially as inclusion of just a few outlying observations may seriously affect the determination of the reference limits. Many methods have been developed for definition of outliers. Several of these methods are developed for the normal distribution and often data require transformation before outlier elimination. Methods: We have developed a non-parametric transformation independent outlier definition. The new method relies on drawing reproducible histograms. This is done by using defined bin sizes above and below the median. The method is compared to the method recommended by CLSI/IFCC, which uses Box-Cox transformation (BCT) and Tukey’s fences for outlier definition. The comparison is done on eight simulated distributions and an indirect clinical datasets. Results: The comparison on simulated distributions shows that without outliers added the recommended method in general defines fewer outliers. However, when outliers are added on one side the proposed method often produces better results. With outliers on both sides the methods are equally good. Furthermore, it is found that the presence of outliers affects the BCT, and subsequently affects the determined limits of current recommended methods. This is especially seen in skewed distributions. The proposed outlier definition reproduced current RI limits on clinical data containing outliers. Conclusions: We find our simple transformation independent outlier detection method as good as or better than the currently recommended methods. </jats:sec

Indirect method for validating transference of reference intervals

Abstract Background: Transference of reference intervals (RIs) from multicentre studies are often verified by use of a small number of samples from reference individuals or by the use of one serum sample (Serum X for NORIP RI). Despite recommended and appropriate methods, both have inconveniencies and drawbacks. Several attempts have been made to develop an indirect method, which uses historical data from the laboratory. These methods are retrospective relying on older test results. A near prospective method would be preferable for the laboratories introducing new methods or changing analytical platforms. Methods: We performed a data mining experiment using results from our laboratory information system covering patients from a large geographic area. Request patterns for patients with assumed healthy characteristics were identified and used to extract laboratory results for calculation of new RI by an indirect method. Calculated RI and confidence intervals (CIs) were compared to transferred NORIP RI verified by NFKK Reference Serum X. Results: We found that our indirect method and NFKK Reference Serum X in general produced similar results when verifying transference of RI. The method produces results for all stratifications. Only single stratifications and one analyte showed unexplained incongruences to the NORIP RI. Conclusions: Our results suggest using request patterns as a surrogate measure for good health status. This allows for a data mining method for validation of RI or validating their transference, which is likely to be applicable in countries with similar healthcare and laboratory information system. </jats:sec

Erkennung und Mimikry

Title 1 Summary 3 Content 4 Abbreviations 6 1 Introduction 7 1.1 Preface 7 1.2 Lewis Y 9 1.2.1 Structure and pathway of the Lewis carbohydrates 9 1.2.2 Expression of Lewis Y on normal cells 11 1.2.3 Function of Lewis Y 12 1.2.4 Lewis Y changes in cancer 12 1.2.5 Lewis Y expression and prognosis 13 1.2.6 Anti-Lewis Y therapy 14 1.2.7 Crystal structures of antibodies with Lewis Y 15 1.3 Technologies 16 1.3.1 Antibodies and antibody fragments 16 1.3.2 Phage display 18 1.3.3 Anti-idiotypic antibodies and molecular mimicry 20 1.3.4 Saturation transfer difference nuclear magnetic resonance 22 2 Results 24 2.1 Specificity ELISA 24 2.2 Cloning and sequences of the antibodies 27 2.3 Expression of antibodies and antibody fragments 29 2.4 Chain shuffling �looking for new specificities 31 2.5 Antibody purification 34 2.6 Epitope mapping of the H type 2 carbohydrate 37 2.6.1 STD-NMR on H disaccharide 38 2.6.2 STD-NMR on H type 2 39 2.7 Flow cytometry 41 2.8 Immunocytochemistry 44 2.9 Affinity measurements 46 2.10 Mimicry between histone H1 and a mixed carbohydrate epitope 48 2.11 Anti-idiotypic antibodies 50 2.11.1 Selection method for mimicry antibodies 51 2.11.2 Anti-idiotypic antibodies mimicking Lewis Y or H type 2 55 2.11.3 Generation of Ab3 58 2.12 Adjuvant effect of domain I of phage protein 3 60 3 Discussion 63 3.1 Lewis Y binding antibodies and specificity 63 3.2 Expression and purification of the antibodies 67 3.3 H type 2 epitope 69 3.4 Detection of Lewis Y on cancer cells 71 3.5 Affinities of the antibodies 73 3.6 Mimicry between histone H1 and a mixed carbohydrate epitope 75 3.7 Selection method of anti-idiotypic antibodies 77 3.8 Anti-idiotypic mimicry 79 3.9 Adjuvant effect of DI of phage p3 81 3.10 Achievements and outlook 83 4 Materials and Methods 85 4.1 General materials 85 4.1.1 Nemod Hybridoma clones 85 4.1.2 Antigens 85 4.1.3 Phage 85 4.1.4 Bacteria 86 4.1.5 Cell lines 86 4.1.6 Vectors 86 4.1.7 Primers 86 4.1.8 Antibodies and lectin 87 4.2 Phage display protocols 87 4.2.1 Phage amplification and precipitation 88 4.2.2 Selections 88 4.2.3 Phage screening 89 4.3 Cloning 89 4.3.1 Sequencing 89 4.3.2 Cloning of DNA from hybridoma cells 89 4.3.3 Construction of DI fusions 90 4.4 Expression of scFvs and antibody purification 90 4.4.1 Production of antibodies 90 4.4.2 Purification of antibodies 91 4.4.3 Production and isolation of antibodies from Prolifix supplemented media 91 4.4.4 Expression and harvesting of scFv and scFv-DI 92 4.4.5 Purification of scFv and scFv-DI 92 4.5 Antibody characterisation assays 92 4.5.1 ELISA 92 4.5.2 Periodate oxidation 93 4.5.3 Western blots 93 4.5.4 Surface plasmon resonance 93 4.5.5 Cell staining 94 4.5.6 Flow cytometry 94 4.5.7 Inhibition analysis 94 4.5.8 Saturation transfer difference nuclear magnetic resonance 95 4.6 Immunology assay 96 4.6.1 Mouse immunisation 96 4.6.2 IgG1 and IgG2a antibody immune responses and sera specificity 96 Acknowledgement 97 Curriculum Vitae 98 References 101 Summary in German 112The goal of my work was to enhance the knowledge on Lewis Y binding antibodies and their interaction with the antigen for the development of Lewis Y-specific therapeutics using advanced antibody technologies including anti-idiotypic antibodies. The Lewis Y carbohydrate antigen is overexpressed in 60-90% of cancers of epithelial origin, which makes it a target of interest for therapy. Immunotherapeutics are promising candidates for future clinical applications. Several antibodies have been developed against Lewis Y, but most of them cross-react with related carbohydrate structures. The specificity determination of the four Nemod Lewis Y binding antibodies showed that one (A70-C/C8) bound exclusively to the Lewis Y antigen, whereas the three others revealed cross-reactivities to related carbohydrate structures. A chimeric mouse-human IgG was successfully generated from the IgM antibody A70-C/C8 and this chimeric antibody (cIgG CC8) proved to be as specific towards Lewis Y, as the parent antibody. Starting from two mouse monoclonal antibodies two new chimeric antibodies were created by rearrangement of their chains. As a result new chimeric antibodies (AA9/CC8) were generated as IgM as well as IgG. These new antibodies were found to have two important features: (1) Exclusive recognition of Lewis Y and (2) enhanced reactivity to Lewis Y. Approximately a factor of 10 was estimated for the cIgG AA9/CC8 when compared to the cIgG CC8. The antibodies were evaluated in ELISA, flow cytometry, immunocytology, and surface plasmon resonance. The data generated show that the antibody cIgG AA9/CC8 is the most promising candidate for further development and optimising. A thorough investigation of the antigens recognised by the Nemod antibody A70-A/A9 revealed two related carbohydrate structures, Lewis Y and Lewis b, and surprisingly the histone H1. This is the first real hint to a naturally occurring immunological mimicry between carbohydrate structures and a naturally occurring protein. Anti-histone antibodies are often found in patients suffering from the auto-immune disease systemic lupus erythematosus (SLE). The mimicry identified here suggests a possible explanation for the cross-reaction of some SLE auto-antibodies to the surface of cancer cells. Generally carbohydrates are weak immunogens whereas it is possible to generate a strong anti-carbohydrate response by vaccination with protein mimics, e.g. anti-idiotypic antibodies. Anti-idiotypic antibodies have so far been generated by a tedious process involving immunising mice and generation of hybridomas. We developed an improved phage display selection strategy that after only a few rounds of selection generated clones with the wanted specificity from naïve scFv-phage libraries. Only two rounds of selection using specific elution with the antigen and a protolytic helper phage ensured that maximum diversity was retained in the selected clones. It could be also shown that an anti-idiotypic scFv generated with this new technique was able to induce an anti-carbohydrate response in mice. This technique is promising as a new tool for the selection of anti-idiotypic antibodies and surrogate molecules. Furthermore, it could be demonstrated that the fusion of DI of the phage protein p3 to the scFvs, previously shown to be a mediator of activity, also provides good adjuvant properties and is a new method for inducing immunogenic responses. Results of basic research presented here enhance the knowledge for the development of tailored immunotherapeutics.Immuntherapeutika gewinnen in der Krebstherapie an Bedeutung. Das Kohlenhydrat-Antigen Lewis Y (LeY) ist ein lohnendes Zielantigen, da es in 60-90 % der Tumoren epithelialer Herkunft überexprimiert ist. Eine Anzahl von Antikörpern gegen dieses Antigen wurde bereits beschrieben, aber die meisten von diesen zeigen Kreuzreaktionen mit verwandten Kohlenhydrat-Strukturen, die auch auf gesunden Geweben vorkommen. Das Ziel meiner Arbeit waren die detaillierte Spezifitätsanalyse von vier Lewis Y-bindenden Antikörpern (mAk) der Firma Nemod (darunter drei noch nicht beschriebenen), die Charakterisierung ihrer Interaktion mit dem Epitop sowie die Generierung von rekombinanten Antikörpern und antiidiotypischen Antikörpern als potentiellen Immuntherapeutika. Die Spezifitätsanalyse der vier mAk ergab, dass ein Antikörper (A70-C/C8, IgM) absolut spezifisch für Lewis Y ist, während die drei anderen unterschiedliche Kreuzreaktivitäten zu verwandten Kohlenhydrat- Strukturen aufweisen. Aus A70-C/C8 wurde ein chimärer Human/Maus-Antikörper als IgG mit gleicher Spezifität generiert (cIgG CC8). Weiterhin wurden chimäre Human-Maus-Antikörper (IgM und IgG) mit unterschiedlichen leichten Ketten (AA9/CC8) hergestellt und umfassend charakterisiert. Diese Antikörper haben sehr interessante Eigenschaften: 1. Sie reagieren ausschließlich mit LeY (wie CC8). 2. Sie zeigen eine etwa 10fach stärkere Bindung an das Antigen im Vergleich zu cIgG CC8. Die IgG-Variante ist ein vielversprechender Ausgangspunkt für die Entwicklung eines therapeutischen Antikörpers. Die Spezifitätsanalyse des Antikörpers A70-A/A9 ergab Bindung an zwei verwandte Kohlenhydrat-Strukturen, Le Y und Le b, sowie überraschend an ein Protein, Histon H1. Die Daten werden interpretiert als Erstbefund einer immunologischen Mimikry zwischen natürlich vorkommenden Oligosaccharid- und Peptid-Antigenen. Anti-Histon-Antikörper werden häufig bei Patienten mit systemischer Lupus erythematosus-Erkrankung (SLE) gefunden. Gleichzeitig reagieren SLE- Autoantikörper häufig mit Tumorzellen. Da Kohlenhydrate oft schwache Immunogene sind, eignen sie sich nicht immer für Vakzinen. Eine Möglichkeit, bessere Immunogenität zu erreichen, sind anti-idiotypische Antikörper, die das ursprüngliche Antigen immunologisch imitieren. Diese Strategie wurde mit der Generierung anti-idiotypischer single-chain-Antikörper (scFv) im Fall des Le Y-Tumorantigens angewendet. Zu diesem Zweck wurde eine verbesserte Methode zur Phagen-Display-Selektion entwickelt, die es erlaubt, nach wenigen Selektionsrunden eine hohe Ausbeute und große Vielfalt von Klonen der gewünschten Spezifität zu erhalten. Die neue Methode kombiniert die Verwendung eines Trypsin-empfindlichen Helferphagen mit antigen-spezifischer Selektion. Mit ihr konnten scFvs selektiert werden, die das Lewis Y-Antigen immunologisch imitieren und in vivo in Mäusen Antikörper gegen Le Y generieren. Weiterhin wurde durch Fusion von DI des Phagen p3 an die scFv ein sehr guter Adjuvans- Effekt erzeugt werden. Diese methodischen Neuerungen sind wichtige Voraussetzungen für weitere Arbeiten zur Herstellung massgeschneiderter Immuntherapeutika