Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 38109

AbstractMaterial and MethodsResultsDiscussionReferencesDOI and Others
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2012 | Month : April | Volume : 6 | Issue : 2 | Page : 200 - 206

Microarray ELISA for Autoantibody Screening in Connective Tissue Diseases

Sarita Kumble, Lok Choi, Carlos Lopez-Muedano, Krishnanand D. Kumble

1. Chief Technology Officer 2. Scientist 3. Software engineer 4. Chief Executive Officer Pictor Limited, Auckland, New Zealand

Correspondence Address :
Dr. K.D. Kumble, Pictor Limited, 24 Balfour Road,
Parnell, Auckland 1052, New Zealand.
Phone: +64 9 309 0950
E-mail: a.kumble@pictordx.com

Abstract

Objective:
This study was performed to demonstrate the use of an ELISA-based microarray technology which is termed as ‘PictArrays’, to identify autoantibody expression patterns in patients with symptoms of autoimmune connective tissue disease.
Methods:
Eight commonly tested antigens were simultaneously tested on specially designed 16-well slides for their autoantibody expression patterns. The assay specificity, sensitivity and reproducibility for each of the antigens were measured. The results were analyzed by using specially developed algorithms to identify seropositive samples.
Results:
The multiplex assay could identify specific antigen binding by autoimmune sera on the arrays. The PictArray sensitivity was similar to that which was obtained in established immunoassays, and the assay reproducibility was within limits which were acceptable for diagnostic uses. The software could correctly identify the positive antigen reactivity at concentrations as low as 2 units/ ml of the antibody.
Conclusion:
The data demonstrated the use of a multiplex platform to simultaneously measure multiple autoimmune antibodies. PictArrays offer significant advantages over other multiplex technologies, which include (i) the use of document scanners to read the test results (ii) ease of operation which requires no specialized technical training beyond that which is required for using the conventional ELISA kits (iii) reduction in errors through software-based data analysis, and (iv) inclusion of internal controls to monitor the assay performance of each sample. These features permit the use of PictArrays in resourceconstrained laboratories using existing infrastructure without significant capital expenditure.

Keywords

Microarrays, Autoantigens, Autoantibodies, Connective tissue disease, Systemic rheumatic disease, Immunoassay

Introduction
Autoimmune connective tissue diseases (ACTD) are a group of conditions which are characterized by multi-organ inflammation and autoimmunity, which affect between three to five percent of the global population (1). These chronic diseases can be life threatening and they require immediate access to medical services during their acute phases to prevent multi-organ damage. Although the symptoms vary depending upon the disease, many diseases share the common symptoms of joint aches and pains, fatigue, muscle pain and weakness, skin rashes and the inflammation of organs (2). Due to this, the correct diagnosis of ACTD depends not only on the clinical presentation of the patient, but on the determination of the autoantibody profile in the patients’ serum. The first and most essential step in the management of ACTD is the recognition of the disease itself by the attending physician. Since ACTD can affect any part of the body and have a myriad of clinical manifestations, an early diagnosis can result in effective treatment strategies that can arrest the autoimmune process before it can irreversibly damage the body. In the lesser economically developed regions, most physicians rely on the physical symptoms instead of using the available diagnostic tools to identify and distinguish between the various connective tissue diseases. As a result, people with ACTD often suffer for several years before being given a correct diagnosis and treatment (3). Currently, patients who present with the symptoms of ACTD are tested for the presence of antinuclear antibodies (ANA) by doing Original Article Biotechnology Section indirect immunofluorescent (IIF) staining of HEp-2 cells (4). This test is inconsistent and highly dependent on subjective interpretation by the technician. The ANA positive samples are then tested for their binding to specific autoantigens, usually by ELISA. The patterns of reactivity with the individual antigens are more disease specific than ANA staining patterns, thereby providing clinically useful prognostic information (5). As compared to the traditional ELISA tests that detect only a single analyte at a time, multiplexing provides diagnostic results for a number of different analytes on a single specimen, thus saving valuable time and money and leading to a faster diagnosis and start of treatment. The last decade has seen an increase in the commercial availability of multiplex diagnostic technologies for autoantibody screening (6),(7),(8). However, these commercially available multiplex tests for identifying antibodies to individual autoantigens have largely been developed to meet the requirements of wellfunded laboratories in highly automated environments, thus raising the cost of the testing (9). These tests do not address the needs of a majority of the world’s population in the less economically developed parts of the world, where the prevalence of autoimmune disease is similar to that in developed countries (10). A major challenge is to develop in vitro diagnostic tests for use as a basis for affordable, user-friendly tests that can be deployed in the regions of the world where they are most needed. PictArrays meet the requirements for accessibility, sensitivity, specificity, reliability of results and user-friendliness which have been defined by the World Health Organization by coining the term “ASSURED”, for an ideal diagnostic test that can be used in resource-restricted settings (11). PictArrays directly measure the antibodies which are associated with ACTD, while they also eliminate subjectivity associated with IIF techniques. Providing semi-quantitative and objective results by multiplexing changes how physicians interpret the patient’s results. The need to run multiple samples from the same patient is eliminated since all the tests can be performed together for the same sample, thus providing an immediate Extractable Nuclear Antigen (ENA) profile. The objective of this study was to use PictArrays to identify the antibody expression patterns to eight different autoimmune antigens in patients with the symptoms of ACTD. The data has been presented to show that PictArray performance was similar to that of established ELISA assays.

Material and Methods

Multiplex ELISA
by using PictArrays Diluted samples were added to the wells of PictArray slides (Table/Fig 1) shows the layout of the slide and the test panel), and following incubation, the wells were washed thrice with PBS which contained 0.1% Tween 20 (PBST). The sample wells were sequentially incubated with an anti-human IgG-biotin conjugate and Streptavidin-horseradish peroxidase (HRP) which was interspersed with three PBST washes. The HRP activity was measured by using 3, 3’ diaminobenzidene (Thermo, USA) as the substrate and the reaction was stopped after 5 minutes by washing the wells with PBST. The dried slides were scanned on a Canoscan 5600F flatbed document scanner at 600dpi resolution. The scanned images of the coloured array spots were saved as tagged image files (tif) and they were analyzed by using the Pictor software.
Data Analysis
A software was developed to rapidly analyze the PictArray test results by using simple graphic user interfaces (GUI) which required minimum manual input. In brief, the protocol for the image analysis begins with the opening of the the tif image file and identifying the first spot in the uppermost array of both the columns by using the cursor. The software then identifies the center of the array spots in all the wells of the slide and places a grid to measure the total intensity values of each spot. These values are corrected for background noise, based on the negative control spots within each array. The intensity values of the test spots are then corrected for non-specific signals by using the values which were obtained from well 16, which contained a sample negative control. The corrected test intensity values which were obtained within two minutes of the image capture were then used for all further analyses.
The conventional ELISA assay
Eight-well strips (Maxisorp, NUNC) were coated with 1μg/ ml antigen in PBS and they were incubated overnight at 4ÂșC. Stepwise incubations were performed at 37ÂșC for 60 minutes, starting with the blocker, followed by the addition of diluted samples to the wells and washing thrice with PBST. The wells were then sequentially incubated with an anti-human IgG-biotin conjugate and Streptavidin-horseradish peroxidase, which was interspersed with three PBST washes. The horseradish peroxidase activity was measured by using 3,3’,5,5’-tetramethylbenzidine (Moss, USA) as the substrate and the reaction was stopped after 5 minutes by adding 1N sulfuric acid. The colour intensity was measured by reading the absorbance at 450nm on a spectrophotometer (SpectraMax, Molecular Dynamics, USA).

Results

The array layout
The panel consisted of the most commonly detected antigens, following the ANA positive testing by immunofluorescence assays (Table/Fig 2). The 52kDa and 60kDa SSA antigens were spotted individually to improve assay sensitivity, since each antigen preparation was highly enriched for the individual component (Arotec Diagnostics, product data sheets). Each array also consisted of nine control spots which constituted the controls that monitored the performance of the sample and the reagents which were used in every step of the ELISA assay. The negative control spots are used for calculating the intra-array background.
Assay specificity and sensitivity
Serum samples which were positive for specific auto-antigens were the kind gift of Dr. Neil Cook (Arotec Diagnostics Limited, New Zealand). These samples were retested by using commercially available ELISA kits (Diagnostic Automation, CA) to confirm the autoantibody specificity (Table/Fig 3). Since the kit did not contain wells which were coated with the CENP-B antigen, the CENP-B positive sample was not independently tested. No detectable cross-reactivity was obtained for antibody binding to any of the printed antigens (Table/Fig 4). The sensitivity of the autoantibody detection to the PictArrays was determined by measuring antigen binding at a range of serum dilutions which started from an initial 100-fold dilution. Detectable antibodies to specific antigens were seen for all the samples which were tested (data not shown). However, in order to compare the array results with those from conventional ELISA, the sera had to be diluted 2-fold from an initial 1000-fold dilution. The results showed an array sensitivity comparable to that which was obtained by the conventional ELISA (Table/Fig 5). In an effort to measure the amount of antibody which was bound to the arrayed antigens a standard curve was generated by titrating known concentrations of human IgG on the arrayed anti-human IgG spot in four wells of the slide. The results showed that the antigen-specific antibody at levels of 5ng per ml of serum could be detected on the autoantibody arrays (data not shown).
Software-based Identification of Sera Containing Autoantibodies
One hundred and twenty five serum samples from healthy subjects (Sera Labs, UK) were tested on arrays to establish the threshold levels of serum antibodies to autoantigens in the general population. The average signal intensity values which were obtained from the healthy cohort for each antigen was added to 1.6 times the standard deviation to calculate the threshold.The samples were reported as negative if the signal intensity fell below the threshold value. Samples with intensity values between the threshold and two times the threshold were reported as ambiguous (+/-), while those which were above two times the threshold were positive for the presence of an autoantibody to the specific antigen. By using this algorithm, a positive reactivity could be detected at less than 2 units/ ml when standards with known autoantibody units were tested on the arrays (Table/Fig 6).
Assay Reproducibility
Fifteen replicates of each autoantigen-specific positive sample were tested to determine the inter-array coefficient of the variation. The results showed less than 20% variability in the normalized signal intensity (Table/Fig 7) at a 400-fold serum dilution. This level of variability did not affect reporting of test results as being either positive or negative for antibody binding to the arrayed antigen.

Discussion

Autoantibodies are central to the diagnosis and assessment of autoimmune connective tissue diseases. Several reports have shown complex autoantibody patterns in patients with connective tissue diseases and they have suggested the need to screen several tens of autoantigens to determine specific antibody patterns (12). However, interpretation of complex data on the reactivity to tens of antigens by rheumatologists and its practical importance in making treatment decisions remains to be demonstrated. The commonly followed algorithm for the diagnosis of autoimmune connective tissue diseases is based on the scores which are derived from the physical symptoms and ANA testing by IIF, which is considered to be the gold standard for ANA screening (13). A positive ANA test is followed by testing for specific autoantigen binding (4). The disadvantages of IIF include a high demand on laboratory personnel time, difficulty in standardization of the assay method due to variations in substrate and sample processing, and most importantly, the subjective interpretation of the results by pathologist (14). The ENA screen as an ELISA test was introduced into clinical practice to permit an initial evaluation of the presence of antibodies to any extractable nuclear antigen before performing individual tests (15). The inability of IIF in detecting rare antibodies against centrioles and other cellular targets was compensated by the higher sensitivity for the detection of antibodies to some antigens, which could have been missed by IIF (16),(17),(18). Maguire et al (19) reported a study in which patients who tested positive for ANA by IIF and negative by ELISA were indistinguishable in symptoms from patients who tested negative by both assays after a oneyear follow-up. Their study suggested that ELISA would reduce the number of patients who were referred to a specialist and were subjected to needless follow-up. It has also been suggested that when there is a high clinical suspicion of connective tissue disease, a focused testing for specific autoantibodies should be performed, irrespective of the ANA result (17). As an alternate approach, multiplex assays overcome many of the shortcomings of IIF while enhancing the value of traditional ELISA tests (20). They can be used to rapidly screen and concurrently characterize a wide range of autoantibodies. Additionally, the analysis of the test data by computer-generated algorithms reduces subjectivity in interpretation of the results. The ability to include internal controls in every sample in order to monitor the test performance reduces the chances of human and mechanical errors as compared to a conventional ELISA assay which is done by using one well-one test microtiter plates. Several studies which were performed over the last several years have shown a correlation between the results of the multiplex and ELISA (21). Autoantigen arrays have been suggested for use in following autoantibody profiles over time as the markers of disease remission and relapse. Most commercially available multiplex systems have significant setup and operational costs, which restrict their use to large, highly resourced and automated laboratories in regions with high labour costs. A report which compared the performance of various commercially available ELISA assays for autoantibody screening indicated that no single vendor provided tests that are vastly superior to the other, so that the ultimate selection of the assay system may be determined by factors such as differences in cost, customer service and turn-around time (22). We have designed 16-well disposable polycarbonate slides which can be reproducibly manufactured in large quantities, which require picogram amounts of antigens to be immobilized in the array spots. These slides permit the use of standard laboratory instruments for the sample processing and are also conducive to rapid scale-up by using commercially available ELISA analyzers. Pads of high-protein binding membranes which are laminated with a double-sided adhesive are inserted into the wells of these slides. Arrays of the autoantigens are spotted onto these pads, following a standardized layout in which a 5×5 array with control spots in the first column and in the last row of each array are included to monitor every step of the ELISA; controls confirm the integrity of the serum sample as well as the performance of the detection antibodies and the enzyme substrate. Duplicate spots of the eight tests complete the array. This layout can be maintained for all the ELISA-based tests with minor variations, to account for the types of tests which are included in the panel. The use of a precipitable peroxidase substrate that results in the deposition of the coloured product on the array spot enables the use of a flatbed scanner for reading the test results. This reduces the setup costs significantly, allowing access to a multiplex technology in those regions where its need is highest. The development of proprietary software to convert the image file into a clinical result within two minutes of test completion enables technicians in a clinical diagnostic laboratory to rapidly obtain test results with minimum data handling. Moreover, the test results can be obtained as a text file, which can be easily integrated into the existing laboratory databases for storage and reporting, in formats which are developed by the individual laboratories. We suggest that the PictArray ENA panel is a viable replacement for the individual ELISA assays. PictArrays can satisfy the need for both an initial screen, as well as for the subsequent determination of clinically significant autoantibodies. We have demonstrated that the performance of PictArrays was similar to that of established ELISA assays for each of the eight antigens which were tested. The array results demonstrated excellent analytical specificity and sensitivity, while software-based algorithms for the identification of samples which contained specific autoantibodies provided rapid test results. In conclusion, we present data for an affordable multiplex platform in which multiple biomarkers can be measured simultaneously. This technology can be easily integrated into diagnostic laboratories with a moderate level of resources and infrastructure. Further developments are underway to enable the use of this technology at the point-of-care. PictArrays offer significant advantages over other multiplex technologies, which include (i) low setup and operational costs due to lack of the requirement for sophisticated instruments (ii), easy operation which requires no specialized technical training since the assay is based on ELISA (iii), reduction in errors due to a software based data analysis and the inclusion of internal controls to monitor the individual assay performance. Autoimmune diseases are on the rise due to an aging population and coupled with the need for an extensive infectious disease testing, multiplex technology is a timely and much needed addition to the clinical diagnostic laboratory. There is a real need to include many different types of tests on one platform (such as infectious diseases and autoimmune diseases) to save space, time and money, and to limit medical waste. Performing ELISA tests in a multiplex panel as has been reported in this paper by using PictArrays, combines an up and coming testing format with a tried and true testing method. As the PictArray test menu increases, the potential cost and the throughput benefits which are realized by the laboratory will increase exponentially.

References

1.
McBride JD, Gabriel FG, Fordham J, Kolind T, Barcenas-Morales G, Isenberg DA, et al. Screening autoantibody profiles in systemic rheumatic disease with a diagnostic protein microarray that uses a filtration-assisted nanodot array luminometric immunoassay (NALIA). Clin Chem. 2008;54:883-90.
2.
Davidson A, Diamond B. Autoimmune diseases. N Engl J Med, 2001;345:340-50.
3.
Marrack P, Kappler J, Kotzin BL. Autoimmune disease: why and where it occurs. Nature Med. 2001;7:899-905.
4.
Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for the clinical use of the antinuclear antibody test and the tests for the specific autoantibodies to the nuclear antigens. Arch Pathol Lab Med. 2000;124:71-81.
5.
Moder KG. Use and interpretation of rheumatologic tests: a guide for clinicians. Mayo Clin Proc 1996;71:391-96.
6.
Rouquette A-M, Desgruelles C, Laroche P. Evaluation of the multiplexed immunoassay, FIDIS, for the simultaneous quantitative determination of antinuclear antibodies and for comparison with the conventional methods. Am J Clin Path. 2003;120:676-81.
7.
Buliard A, Fortenfant F, Ghillani-Dalbin P, Musset L, Oksman F, Olsson NO. Analysis of nine autoantibodies which were associated with systemic autoimmune diseases by using the Luminex technology. Results of a multicenter study. Ann Biol Clin (Paris). 2005;63:51-58.
8.
Prestigiacomo T, Humbel RL, Larida B, Binder SR. Multiplexed analysis of thirteen antibodies by using the BioPlex 2200 fully automated immunoassay analyzer. Autoantigens, autoantibodies. In: Conrad K. Sack U editors. Autoantigens, autoantibodies, autoimmunity. Lengerich; 2004;463-68.
9.
Yager P, Domingo GJ, Gerdes J. Point-of-care diagnostics for global health. Ann Rev Biomed Eng. 2008;10:107-44.
10.
Shapira Y, Agmon-Levin N, Shoenfeld Y. Geoepidemiology of autoimmune rheumatic diseases. Nat Rev Rhematol. 2010;6:468-76.
11.
Mabey D, Peeling RW, Ustianowski A, Perkins MD. Diagnostics for the developing world. Nat Rev Microbiol. 2004;2:231-40.
12.
Balboni I, Chan SM, Kattah M, Tenenbaum JD, Butte A, Utz PJ. Multiplexed protein array platforms for the analysis of autoimmune diseases. Ann Rev Immunol. 2006;24:391-418.
13.
Jaskowski TD, Schroder C, Martins TB, Mouritsen CL, Litwin CM, Hill HR. Screening for antinuclear antibodies by doing enzyme immunoassay. Am J Clin Pathol. 1996;105:468-73.
14.
Kang I, Siperstein R, Quan T, Breitenstein ML. Utility of age, gender and the ANA titer and pattern as the predictors of the anti-ENA and the ds-DNA antibodies. Clin Rheumatol. 2004;23:509-15.
15.
Froelich CJ, Wallman J, Skosey JL, Teodorescu M. Clinical evaluation of an integrated ELISA system for the detection of 6 autoantibodies. J Rheumatol. 1090;17:192-200.
16.
Dahle C, Skogh T, Aberg AK, Jalal A, Olcen P. Methods of choice for diagnostic antinuclear antibody (ANA) screening. The benefit of adding antigen-specific assays to immunofluorescence microscopy. J. Autoimmun. 2004;22:241-48.
17.
Bossuyt X, Luyckx A. Antibodies to the extractable nuclear antigens in antinuclear antibody-negative samples. Clin Chem. 2005;51:12-13.
18.
Hanly JG, Thompson K, McCurdy G, Fougere L, Theriault C, Wilston K. Measurement of autoantibodies by using the multiplex methodology in patients with systemic lupus erythematosus. J. Immunol Methods. 2010;352:147-52.
19.
Maguire GA, Ginawi A, Lee J, Lim AY, Wood G, Houghton S, et al. Clinical utility of ANA which was measured by ELISA as compared to ANA which was measured by immunofluorescence. Rheumatol. 2009;48:1013-14.
20.
Binder SR. Autoantibody detection by using multiplex technologies. Lupus. 2006;15:412-21.
21.
Nifli A-P, Notas G, Mamoulaki M, Niniraki M, Ampartzaki V, Theodoropoulos PA, et al. Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of the ANA and ANCA autoantibodies in human serum. J. Immunol Methods. 2006;311:189-97
22.
Hanly JG, Su L, Farewell V, Fritzler MJ. Comparison between the multiplex assays for autoantibody detection in systemic lupus erythematosus. J. Immunol Methods. 2010;358:75-80.

DOI and Others

DOI: JCDR/3954:1943

DISCLOSURE STATEMENT:
The Authors are Employees of Pictor Limited.


Date Of Submission: Oct 09, 2011
Date Of Peer Review: Jan 05, 2012
Date Of Acceptance: Jan 12, 2012
Date Of Publishing: Apr 15, 2012

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • Embase
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)
  • www.omnimedicalsearch.com