Standardisation and commericalisation of a filarial antibody test for use in the global lymphatic

273 Pages·2016·5.89 MB·English

by  Diane Dogcio Hall

ResearchOnline@JCU

This file is part of the following reference:

Hall, Diane Dogcio (2016) Standardisation and
commericalisation of a filarial antibody test for use in the
global lymphatic filariasis elimination programme. PhD
thesis, James Cook University.

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http://researchonline.jcu.edu.au/44645/

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STANDARDISATION AND COMMERCIALISATION OF A FILARIAL ANTIBODY
TEST FOR USE IN THE GLOBAL LYMPHATIC FILARIASIS ELIMINATION
PROGRAMME

Thesis submitted by Diane Dogcio HALL
B.Sc. Biotechnology / B. Information Technology
(Charles Sturt University, New South Wales, Australia)
in March 2016

In partial fulfilment of the requirements for the
Degree of Doctor of Philosophy
in the College of Public Health, Medical
and Veterinary Sciences,
James Cook University, Queensland, Australia
STATEMENT OF SOURCES DECLARATION

I declare that this thesis is my own work and has not been submitted in any form for
any degree in another university or institution of tertiary education. All the information
from the works of others, published or unpublished has been acknowledged in the text
and in the reference list.

______________________________ _____________________
Signature Date

STATEMENT OF ACCESS

I, the undersigned, understand that as the author of this thesis, I agree to make it
available for use within the University Library and, via the Australian Digital Thesis
network for use in other approved libraries.

All users consulting this thesis will have to sign the following statement:

In consulting this thesis, I agree not to copy or closely paraphrase
it in whole or in part without the written consent of the author;
and to make proper public acknowledgement for any assistance
which I have obtained from it.

Beyond this, I do not wish to place any restriction on access to this thesis.

________________________________ ________________________
Signature Date
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PREFACE

This translational research is a collaborative work of the College of Public Health,
Medical & Veterenary Sciences, in the Division of Tropical Health and Medicine,
James Cook University (JCU) and Cellabs Pty Ltd.

This research was made possible by the support and generosity of Dr Anthony
Smithyman, managing director of Cellabs Pty. Ltd., for providing me with a research
laboratory to carry out all experiments, including equipment, accessories, computers
and financial assistance for all other associated costs for my research work. Cellabs
has approved the use of proprietary methods for development of the Filariasis Bm14
Antibody CELISA.

This research was also made possible by the full scholarship granted by the College
of Public Health, Medical & Veterinary Sciences, in the Division of Tropical Health,
under the following candidature committee:

Head of Academic Group: Dr Sue Devine
Research Student Monitor/Chair: A/Prof Jeffrey Warner
Advisory Panel: A/Prof. Wayne Melrose, A/Prof. Dr Patricia Graves, Prof Rick Speare
Associate Dean of Research Education: A/Prof Jeffrey Warner

The research-grade Bm14 ELISA was the original work of Professor Gary Weil and his
group at the School of Medicine, Washington University in St. Louis, MO, USA. This
research-grade ELISA was brought to Cellabs Pty Ltd for standardisation and
commercialisation. The design and development work commenced in 2002, headed
by Dr G-Halli Rajasekariah, Research & Development, Cellabs Pty Ltd. I worked as
the assistant technical officer. From the development of the kit described in Chapter 2
through to the release of the prototype kit in 2007, I performed and analysed a
majority of the experiments under the guidance of Dr Rajasekariah. The optimisation
and validation of the kit from 2008 through to the release of Mark II in 2010 was my
original work. All the data presented in Chapter 2 were selected experiments that I had
performed and analysed. All the other experiments including data analysis for the rest
of the chapters were my original work except for Chapter 7, which were the work of
external evaluators of the Bm14 Antibody CELISA, acknowledged accordingly.
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The recombinant Bm14 antigen was licensed to Cellabs Pty Ltd by the Barnes-Jewish
Hospital, St. Louis, Missouri, USA in an agreement made in 2008 for the development
of an ELISA for the detection of antibodies for lymphatic filariasis. The Bm14
recombinant antigen was used for researching the assay performance in a rapid test,
dipstick format that is complimentary to the ELISA, covered in Chapter 4. Additional
work on optimisation and evaluation is required for commercialisation. The current
assay is research use only and is not included for commercialisation use under the
Cellabs Bm14 recombinant protein license agreement at the time this thesis was
completed.

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DEDICATION

I would like to dedicate this thesis to my daughter Maya and my late father, James.

To Maya, you have always been my light during those nights I stayed up to finish my
writing. Thank you for being a good baby. To my father who taught me perseverance,
each time I felt like giving up, I have always thought about you - this is for you.

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ACKNOWLEDGEMENT

This thesis has been made possible through the contributions of many other people. I
would like to thank my supervisor Associate Professor Wayne Melrose for his
guidance and unwavering support. I consider myself fortunate to have a dedicated
supervisor who believed that this story needed to be told. After retirement, Wayne
chose to take an adjunct position at the University and continued to support me with
his words of wisdom and encouragement that fostered my will to get to the finish line.
For this, I am grateful. I would like to thank my advisors. Dr G-H Rajasekeriah has
been my mentor from the beginning. He believed in me and without his
encouragement, I would never have embarked on this challenging post-graduate
study. I am grateful for his guidance, teachings, leadership and patience throughout
the years of assay development and thesis writing. The guidance and feedback of Dr
Patricia Graves during the thesis writing has been extremely helpful. Being an off-
campus student, discussion with advisors can be difficult but Tricia has been quick to
reply to my emails and arrange interstate phone calls. Her advice and feedback have
given me the confidence to get through to the end. I would also thank Professor
Richard Speare for his kind support at the beginning and halfway through this
research. Dr Anthony Smithyman has been instrumental in this research work. He has
always found the time out of his busy schedule to sit down with me each time I needed
consultation. His wisdom and expertise in the field of tropical and neglected diseases
and immunodiagnostics has been monumental in the progress of this research. For
this, I am grateful.

And last but not the least, I would like to thank my colleague, Neil Marshall for all his
help in running countless ELISAs, repeatedly recovering corrupted thesis files,
proofreading and listening to my complaints and worries. My dearest family, I thank
you for the love and support throughout these challenging times of further education.
To my friends, Rebecca and Shaun, you don’t know how much your support,
understanding and encouragement meant to me, I thank you both for always believing
in me.

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ABSTRACT

This thesis addresses an important research question of how to take a diagnostic test
from the research and development laboratory to a commercially-available product
that can be used reliably in a global disease control programme. Laboratory
procedures, regulatory requirements and administrative steps required to standardise
a filarial antibody test are covered. The question is answered by following the progress
of a test developed for use in the Global Program to Eliminate Lymphatic Filariasis.
The “lessons learnt” in this translational research apply to the development and
commercialisation of any similar product.

The need for reliable diagnostic tools for defining end-points in transmission
assessment surveys and post-intervention surveillance becomes imperative as the
lymphatic filariasis elimination efforts are moving swiftly to achieve the set objectives
by 2020. A variety of diagnostic methods are used, including the detection of
microfilariae and antigen detection assays. These tests have served very well in the
mapping and preventive chemotherapy phases of the elimination programme, but for
the final stages of the programme, there is a strong belief that they may not be
adequate to confirm that the transmission of infection has ceased. After a few rounds
of preventive chemotherapy, these tests no longer have the same sensitivity due to
low mf and antigen prevalence.

Exposure to infective larvae can take months or years before an adult worm becomes
established, a patent infection develops and filarial antigen and mf can be detected.
What is required is a robust test that can detect immunological changes in any
individual after the successful implementation of preventive chemotherapy. The
absence of filarial antibody is a key point for concluding that transmission has ceased,
marking the elimination of lymphatic filariasis.

Assays for filarial antibodies have been available in the 1970s, but they are based
upon the use of crude, whole-worm antigens. These consequently cross-reacted with
other parasites, resulting with great concern for the lack of specificity. The “research-
grade” methods are varied from one laboratory to the other, making comparisons
between institutions virtually impossible. Also, research laboratories do not have the
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expertise and capability of producing industry-standard commercial kits required for a
global programme.
This thesis describes certain specific methods and techniques used in optimisation
and standardisation of an ELISA based on a recombinant antigen Bm14. The Bm14
ELISA is specific to brugian and bancroftian filariasis and, therefore, ideal for use in
lymphatic filariasis elimination programmes. The Bm14 ELISA aims to address the
general agreement that any antibody test for the global LF elimination programme has
to meet the following requirements:

• must be based upon recombinant antigens;
• needs to be reactive with all three lymphatic filariasis species;
• must have excellent sensitivity and specificity;
• must be produced as a standardised commercially-produced kit;
• produced under high-quality control standards with on-going quality assurance;
• can be produced in large-scale to satisfy demand, but at an affordable price;
• has excellent transport and storage stability under tropical conditions and;
• preferably produced in an ELISA and rapid test format.

The standardisation process for Bm14 ELISA includes evaluation of raw materials,
assay optimisation and validation. The raw materials and reagents for the assay were
evaluated for best results by experimentation. Optimisation was achieved by
experimenting on the important concepts of ELISA kinetics for each critical assay
component. Validation of the final protocols and assay performance of the optimised
ELISA was completed to demonstrate that the test will perform as intended. The
standardised test has a sensitivity and specificity of 100% and 98% respectively. No
cross-reactivity with strongyloidiasis, schistosomiasis, dengue, malaria, Chagas
disease and toxoplasmosis was found but it was cross-reactive with onchocerciasis,
therefore, cannot be used in other African countries where the disease overlaps. A
shelf-life of 12 months was validated. Repeatability and reproducibility calculated by
percentage coefficient of variation were 4.2% and 3.3% respectively and both were
within the 10% acceptable performance criteria.

To address the need for a convenient field-based test, the ELISA construct was
converted to a rapid diagnostic test (RDT) in a dipstick format. Evaluation of the Bm14
dipstick found 100% concordance with ELISA results.

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To commercialise the assays, conformity to International Standards of in vitro medical
devices manufacturing (ISO13485) (ISO, 2012) is required by the Australian
Therapeutic Goods Administration (TGA). A series of documents structured to follow a
quality management system (QMS) was prepared. Documents include scientific
methods and procedures, kit inserts and performance results to support the design
and development of the commercial product. The output aims to provide a set of
guidelines on the commercialisation process that can be used as a reference for future
developers of innovative diagnostic tests.

Antibody subclass IgG4 is the widely accepted marker for lymphatic filariasis active
infection however, studies also show that IgG1 may represent a response to incoming
infective larvaeexposure. The standardised Bm14 Antibody CELISA was used in an

experiment to investigate the role of IgG1 in antibody detection. Serum samples from
a high-transmission area of Papua New Guinea were used in an age-profile study. It
was found that both subclasses were equally present across the age groups,
regardless of antigen status although, levels of IgG4 were higher than IgG1. Follow-up
work is needed to ascertain whether a subclass switching occurs much earlier by
investigating the antibody response of infants in a low-transmission setting.

The standardisation of the Bm14 Antibody CELISA and information from this study will
fill the critical gap in one of the challenges of the lymphatic filariasis elimination
programme. There is currently the need for a reliable recombinant-based antibody test
for stopping preventive chemotherapy namely:

(1) end-point determination of preventive chemotherapy and;

(2) post-preventive chemotherapy surveillance: for the detection of hot-spots and
verification of transmission interruption.

This study will provide industry commercialisation information for diagnostic test
research, a gap that needs to be addressed to show how to convert a research-
grade test to a standardised, commercially available test that can be utilised for
both clinical and research applications. This study provides information for:

(3) industry-based standardisation and optimisation methods for diagnostic tests to
ensure product performance meet the intended purpose and;
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