WO2021145757A1

WO2021145757A1 - A biomarker for detecting strongyloides, methods of detecting strongyloides and diagnosing strongyloidiasis in a biological sample, and a diagnostic kit comprising thereof

Info

Publication number: WO2021145757A1
Application number: PCT/MY2020/050044
Authority: WO
Inventors: Rahmah Binti Noordin; Norsyahida Binti ARIFIN; Hussain Ahmad
Original assignee: Universiti Sains Malaysia
Priority date: 2020-01-17
Filing date: 2020-06-19
Publication date: 2021-07-22

Abstract

The present invention relates to a biomarker for detecting Strongyloides, characterized by a Strongyloides stercoralis protein comprising an amino acid sequence set forth in SEQ ID NO: 2, in which the Strongyloides stercoralis protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence set forth in SEQ ID NO: 1. The present invention also relates to a method (100) of detecting Strongyloides in a biological sample and a method (200) of diagnosing Strongyloidiasis in a subject comprising the biomarker as an antigen via an immunoglobulin E, IgE, immunoassay. More specifically, the method (200) is configured to detect an acute Strongyloidiasis in the subject. Further, the present invention provides a diagnostic kit for Strongyloides comprising the biomarker for detecting Strongyloides.

Description

A BIOMARKER FOR DETECTING STRONG YLOIDES, METHODS OF DETECTING STRONGYLOIDES AND DIAGNOSING STRONGYLOIDIASIS IN A BIOLOGICAL SAMPLE, AND A DIAGNOSTIC KIT COMPRISING THEREOF

TECHNICAL FIELD

The present invention relates to a biomarker for detecting Strongyloides and uses thereof, more particularly to methods of detecting Strongyloides and strongyloidiasis in a biological sample from human and non-human species. Further, the present invention relates to a diagnostic kit comprising the biomarker.

BACKGROUND ART

Strongyloidiasis is one of the neglected tropical diseases. It is caused by soil- transmitted helminth, specifically Strongyloides stercoralis. However, in some cases, it may be caused by Strongyloides fuelleborni and Strongyloides fuelleborni kellyi. The most common route of infection is skin penetration, but it may be transmitted via oral route and transmission after solid organs transplant.

Although strongyloidiasis is prevalent in tropical, subtropics and temperate regions, other factors may facilitate its transmission in other regions such as poor sanitation, especially in poor countries. While S. stercoralis generally causes clinically asymptomatic infection and chronic infection in an immunocompetent patient, it may cause a life threatening hyper-infection and dissemination in an immunocompromised patient. Many patients are asymptomatic or show mild acute symptoms of cutaneous irritation at the site of skin penetration. In chronic cases, the intestinal tract and skin are clinically affected and frequently goes unnoticed. On the other hand, immunosuppression may lead to severe manifestation of the hyper-infection syndrome and disseminated strongyloidiasis.

Strongyloidiasis patient secretes different classes of antibodies such as immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin M (IgM), immunoglobulin G (IgG) and subclasses of IgG to combat the disease. More specifically, the presence of IgE antibody indicates an acute infection of strongyloidiasis, whereas the presence of lgG4 indicates to chronic infection of strongyloidiasis. In addition, IgE antibodies are important as defence against parasites and have been long known to be induced during helminthic infection. Further, there are significant positive correlations between specific lgG4 and IgE, where a single-positive lgE+/lgG4- may indicates a more recent infection of strongyloidiasis, while a single-positive lgE-/lgG4+ may indicates a chronic infection of strongyloidiasis.

Diagnosis of helminthic infection including S. stercoralis infection remains a challenge to date. The conventional diagnosis by observing the larvae directly or through culture is laborious, has low sensitivity and provides a high index of the false-negative result due to the intermittent larvae release. Promising result can be obtained by molecular diagnosis through real-time polymerase chain reaction (RT- PCR). However, RT-PCR is not widely available, costly and requires technological support, hence does not provide the best solution for diagnosis of helminthic infection, especially in endemic areas which are mostly resource-limited. On the other hand, enzyme-linked immunosorbent assay (ELISA) significantly increases the sensitivity and negative predictive value; however, its general reliance on a crude antigen that requires larvae culture makes it difficult to use.

Apart from that, the use of native Strongyloides protein may cause potential cross-reactivity and standardization issues. Further, diagnosis of acute strongyloidiasis is challenging to ascertain and active and past infections are hard to differentiate due to unreliable indicator. For example, IgG-based immunoassay may detect past infection and hence resulting in inaccurate diagnosis.

Hence, there is a need to develop alternative diagnostic methods that include the development of reliable immunological assays and the use of a recombinant protein which reduces time, cost and cross-reactivity. There have been a number of solutions that provide alternative diagnostic methods for helminthic infections and few of them are discussed below: WO 2017091059 disclosed methods of screening biological samples for the presence of Strongyloides spp. More particularly, the prior art relates to a sensitive and specific screening test for the presence of anti -Strongyloides spp antibodies, protein or nucleic acid in a subject by using a Strongyloides spp L3 stage antigen. In addition, the Strongyloides spp L3 stage antigen is an isolated or a recombinant S. stercoralis protein comprising an amino acid sequence and a nucleic acids sequence that encodes the Strongyloides spp L3 stage antigen. However, the anti -Strongyloides spp antibodies detected are IgG antibodies. Hence, the prior art does not provide a means to detect an acute strongyloidiasis.

CA3051466A1 relates to generation of human allergen and helminth-specific IgE monoclonal antibodies for diagnostic and therapeutic use. The prior art also relates to a method of detecting an IgE antibody with binding affinity or specificity for an antigen such as a dust mite antigen in a subject. The helminth infections diagnosed by the prior art are Wuchereria bancrofti or S. stercoralis, which serve as antigens in the prior art. The prior art comprises a test antibody or a fragment having complementarity-determining region (CDR) of the heavy chain and its corresponding nucleic acid sequence. However, the antibody or antibody fragment comprises heavy and light chain variable sequences in the prior art only have 70 %, 80 % or 90 % identity to clone paired heavy and light chain variable sequences. Hence, the prior art has lower sensitivity and specificity against helminth antigen such as S. stercoralis antigen as it is desirable to have sensitivity and specificity higher than 95 %. Hence, the prior art still needs to be improved to provide a more reliable diagnosis result.

US20120308599 A1 disclosed a vaccine and method of use against S. stercoralis infection. The prior art comprises an isolated S. stercoralis immunoreactive antigen (SSIR), more particularly to an antigen from S. stercoralis stage L3, which is mixed with an adjuvant to immunize a subject. Further, the prior art applies immunoassay, more particularly to ELISA for quantifying antibodies generated to the S. stercoralis immunoreactive antigen. However, the prior art relates more to a therapeutic composition against S. stercoralis rather than detection of acute or chronic strongyloidiasis. Accordingly, it can be seen in the prior arts that there exists a need for a biomarker as an antigen that provides high specificity and selectivity against Strongyloides, specifically S. stercoralis. In addition, there exists a need to utilize the biomarker in both diagnostic and therapeutic applications. There also exists a need to apply the biomarker in producing a diagnostic kit for strongyloidiasis, especially acute strongyloidiasis, with a diagnostic sensitivity and specificity higher than 95 %. SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

An objective of the present invention is to provide a biomarker for detecting Strongyloides, preferably Strongyloides stercoralis. Preferably, the biomarker may be used for diagnosis, epidemiological studies and research.

It is also an objective of this invention to provide a biomarker for detecting Strongyloides, in which the biomarker is an isolated S. stercoralis protein or a recombinant S. stercoralis protein comprising an amino acid sequence. It is also an objective of this invention to provide a biomarker for detecting

Strongyloides that is encoded by a nucleic acid molecule comprising a nucleic acid sequence.

A further objective of the present invention is to provide a method of detecting Strongyloides in a biological sample from a human or a non-human species. More particularly, the method is using the biomarker for detecting Strongyloides as an antigen. Another objective of the present invention is to provide a method of diagnosing strongyloidiasis in a subject via a biological sample taken from the subject, more particularly to a method of diagnosing acute strongyloidiasis in the subject using the biomarker for detecting Strongyloides as an antigen.

Further, another objective of the present invention is to provide a method of detecting Strongyloides in a biological sample and a method of diagnosing strongyloidiasis in a subject using an immunoassay, more particularly to an immunoassay for the measurement of immunoglobulin E (IgE) to diagnose an active infection, including an acute infection.

It is a further objective of the present invention to provide a method of detecting Strongyloides in a biological sample and a method of diagnosing strongyloidiasis using an immunoassay, which provide sensitivity and specificity higher than 95 % against Strongyloides and strongyloidiasis, respectively.

It is a further objective of the present invention to provide a diagnostic kit comprising the biomarker for detecting Strongyloides, more particularly to S. stercoralis.

Accordingly, these objectives may be achieved by following the teachings of the present invention. The present invention relates to a biomarker for detecting Strongyloides, characterized by a S. stercoralis protein comprising an amino acid sequence set forth in SEQ ID NO: 2, in which the S. stercoralis protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence set forth in SEQ ID NO: 1 . The present invention also relates to a method of detecting Strongyloides in a biological sample and a method of diagnosing strongyloidiasis in a subject comprising the biomarker as an antigen via an immunoglobulin E, IgE, immunoassay. More specifically, the method is configured to detect an acute strongyloidiasis in the subject. Further, the present invention provides a diagnostic kit for Strongyloides comprising the biomarker for detecting Strongyloides. The foregoing and other objects, features, aspects and advantages of the present invention will become better understood from a careful reading of a detailed description provided herein below with appropriate reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may have been referred by embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawing illustrates only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments. These and other features, benefits, and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:

Fig. 1 is a flowchart illustrating a method of detecting Strongyloides in a biological sample in accordance with an embodiment of the present invention. Fig. 2 is a flowchart illustrating a method of diagnosing strongyloidiasis in a subject in accordance with an embodiment of the present invention.

Fig. 3 shows a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a western blot analysis of (A) a purified biomarker, (B) probed with anti-His horseradish peroxidase (H RP) antibody, and (C) probed with positive biological samples.

Fig. 4 is a column chart showing a mean value with a standard deviation of immunoglobulin E-enzyme-linked immunosorbent assay (IgE-ELISA) optical density (OD) readings from different biological sample groups.

Fig. 5 illustrates column charts showing comparison of a mean value with a standard deviation of IgE-ELISA OD readings from the different biological sample groups, and the T-test analysis.

Fig. 6 is a receiver operator characteristic (ROC) curve analysis of the IgE- ELISA cut-off value (COV) 0.22 for determination of strongyloidiasis with the area under the ROC curve (AUC) 1.000 (95% confidence interval=0.999 to 1.000, P>0.0001).

Fig. 7 shows OD readings of IgE-ELISA using positive and control biological samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described, and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles and the like is included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention. In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.

The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.

The present invention relates to a biomarker for detecting Strongyloides, the biomarker is characterized by: a Strongyloides stercoralis protein comprising an amino acid sequence set forth in SEQ ID NO: 2; wherein the Strongyloides stercoralis protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence set forth in SEQ ID NO: 1 .

The present invention also relates to a method (100) of detecting Strongyloides in a biological sample, the method comprising the steps of: contacting a biological sample with an antigen; and detecting a specific and selective antigen-antibody binding of the antigen and a first antibody present in the biological sample via immunoassay; characterized in that, the antigen is the biomarker for detecting Strongyloides comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the first antibody is a Strongyloides stercoralis- specific antibody; and wherein presence of Strongyloides in the biological sample is detected via presence of the specific and selective antigen-antibody binding of the antigen and the first antibody in the biological sample.

In addition, the present invention relates to a method (200) of diagnosing strongyloidiasis in a subject, the method comprising the steps of: collecting a biological sample from a subject; contacting the biological sample with an antigen; detecting presence of Strongyloides protein in the biological sample via an immunoassay using a first antibody; contacting the biological sample with a second antibody; and determining presence of strongyloidiasis in the subject; characterized in that, the antigen is the isolated Strongyloides stercoralis protein comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the first antibody is a Strongyloides stercoralis- specific antibody; and wherein the second antibody is an antibody that specifically binds to the first antibody.

Further, the present invention relates to a diagnostic kit for Strongyloides, the diagnostic kit comprising: the biomarker for detecting Strongyloides ; an immunoassay reagent; and an immunoassay device configured to detect presence of Strongyloides.

Referring to the drawings as shown in Fig. 1 to Fig. 6, the invention will now be described in more detail. The biomarker for detecting Strongyloides comprises the amino acid sequence set forth in SEQ ID NO: 2. More particularly, the S. stercoralis protein is encoded by the nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1 . Preferably, the biomarker may be used for, but not limited to, diagnosis, epidemiological studies and research.

In accordance with an embodiment of the present invention, the S. stercoralis protein is an isolated S. stercoralis protein or a recombinant S. stercoralis protein. Preferably, the S. stercoralis protein is produced from an isolated complementary deoxyribonucleic acid (cDNA) clone from immunoscreening of S. stercoralis cDN A library.

In accordance with an embodiment of the present invention, the amino acid sequence set forth in SEQ ID NO: 2 preferably including a complete amino acid sequence, a fragment of the amino acid sequence or a variant of the amino acid sequence.

In accordance with an embodiment of the present invention, the nucleic acid molecule is preferably an isolated nucleic acid molecule or a recombinant nucleic acid molecule.

In accordance with an embodiment of the present invention, the biomarker including, but not limited to, a Strongyloides- specific antigen.

Fig. 1 is a flowchart illustrating the method (100) of detecting Strongyloides in the biological sample in accordance with an embodiment of the present invention. The method (100) begins with contacting the biological sample with the antigen, followed by detecting the specific and selective antigen-antibody binding of the antigen and the first antibody present in the biological sample via immunoassay. Presence of Strongyloides in the biological sample is detected via presence of the specific and selective antigen-antibody binding of the antigen with the first antibody in the biological sample. More particularly, the antigen is the biomarker for detecting Strongyloides comprising the amino acid sequence set forth in SEQ ID NO: 2, while the first antibody is a S. stercoralis- specific antibody.

In accordance with an embodiment of the present invention, the first antibody is preferably an immunoglobulin E, IgE, antibody. In accordance with an embodiment of the present invention, the immunoassay including, but not limited to, an enzyme-linked immunosorbent assay (ELISA), a dot-blot, a Western blot, a vertical flow test, a lateral flow assay, and a biosensor. In accordance with an embodiment of the present invention, the biological sample is from a human or non-human species. Fig. 2 is a flowchart illustrating the method (200) of diagnosing strongyloidiasis in the subject in accordance with an embodiment of the present invention. The method (200) begins with collecting the biological sample from the subject, contacting the biological sample with the antigen, and detecting presence of Strongyloides protein in the biological sample via the immunoassay using the first antibody. The method (200) is then followed by contacting the biological sample with the second antibody, and finally determining presence of strongyloidiasis in the subject.

Specifically, the antigen is the isolated S. stercoralis protein comprising the amino acid sequence set forth in SEQ ID NO: 2. Further, the first antibody is a S. stercoralis- specific antibody, whereas the second antibody is the antibody that specifically binds to the first antibody.

In accordance with an embodiment of the present invention, the second antibody is preferably conjugated to a detector molecule selected from, but not limited to, an enzyme, a fluorescence molecule or a colloidal gold.

In accordance with an embodiment of the present invention, each of the first antibody and the second antibody is an immunoglobulin E, IgE, antibody.

In accordance with an embodiment of the present invention, the method (200) is used for determining acute strongyloidiasis in the subject via detection of the first antibody and the second antibody. Further, the diagnostic kit for Strongyloides is also provided, in which the diagnostic kit comprises the biomarker for detecting Strongyloides , the immunoassay reagent and the immunoassay device configured to detect presence of Strongyloides In accordance with an embodiment of the present invention, the diagnostic kit is configured to detect presence of Strongyloides antibody or Strongyloides protein.

In accordance with an embodiment of the present invention, the diagnostic kit including, but not limited to, an enzyme-linked immunosorbent assay, a dot blot assay, a Western blot assay, a vertical flow assay, a lateral flow assay, and a biosensor.

Hereinafter, example of the present invention will be provided for more detailed explanation. The advantages of the present invention may be more readily understood and put into practical effect from these examples. However, it is to be understood that the following examples are not intended to limit the scope of the present invention in any way.

Examples Example 1 Biological Samples

(1) Selection of biological samples

Biological samples, namely serum samples of patients, were divided into two groups; Group 1A and Group 1 B. Group 1A had biological samples from strongyloidiasis patients that were positive by both of polymerase chain reaction (PCR)/microscopy and serology (n=20), whereas Group 1 B had biological samples from strongyloidiasis patients that were positive by stool-PCR but negative by serology (n=11). The initial conclusion are Group 1 B is more likely to have an acute infection, while Group 1A samples is more likely to have a chronic infection.

The diagnostic specificity was evaluated using 69 serum samples from other parasitoses, specifically Group 2 that comprises patients who had amebiasis (n=4), ascariasis (n=4), echinococcosis (n=4), filariasis (n=9), giardiasis (n=1) hookworm (n=6), schistosomiasis (n=1 1 ), taeniasis (n=2), toxocariasis (n=18), toxoplasmosis (n=5), trichuriasis (n=2) and mixed infection (n=3). In addition, Group 3 comprised 25 biological samples from healthy donors. (2) Pre-adsorption of biological samples for immunoscreening

Prior to immunoscreening, the biological samples, namely serum samples, were pre-adsorbed against two kinds of Escherichia coli L1 -Blue antigen, which are E. coli whole-cell pellet at 100 mg per tube and E.coli whole cell lysate 250 mI_ lysate per 10Oul of 0.5-miti microsphere beads. A volume of 30 mI of biological sample was added to XL1 -Blue whole-cell pellet and the mixture was mixed thoroughly and incubated at 4°C on rotator overnight. Two rounds of overnight incubation of serum were performed with both pellet and beads coated with E. coli lysate. The final pre-adsorbed serum was then stored at -20 °C. Further, the efficiency of serum pre-adsorption was determined using immunoglobulin G- enzyme-linked immunosorbent assay (IgG-ELISA).

Example 2

Immunoscreening of Strongyloides stercoralis

(1) Preparation for immunoscreening of Strongyloides stercoralis A complementary deoxyribonucleic acid (cDNA) library was constructed in a

ATriplEx2 vector by Clontech Laboratories Inc. from a mixture of L3 and adult worms of S. stercoralis. Prior to immunoscreening, the titration of the phage cDNA library with lambda (A) dilution buffer was performed to produce approximately 300 to 500 well-separated plaques per plate. 20 pL of the phage at dilution of 10³ was added to 600 ul of diluted xl-1 blue cells of optical density (OD) of 0.5 at 600 nm. The tubes were then incubated for 15 min at 37 °C to allow attachment and transduction. 7 ml of melted soft top agarose was added to the mixture and then poured to pre-warmed Luria-Bertani/rmagnesium sulfate (LB/MgS04) agar plates, followed by 6 hours incubation at 37 °C for plaque formation.

A nitrocellulose membrane (NC) soaked with 10 mM of isopropyl B-D-1 - thiogalactopyranoside (IPTG) was overlaid on top of the plate containing visible plaques and incubated for 4 hours at 37 °C. The NC membrane was washed 3 times for 5 min each using Tris-buffered saline having 0.05% Tween 20 (TBS-T), namely 20 rmM of Tris and 150 mM of sodium chloride (NaCI), followed by 1 hour blocking with diluted blocking buffer. The NC membrane was then washed and incubated with a serum that had been pre-adsorbed with E. coli antigen earlier, in a total dilution of 1 :100 in TBS having pH 7.5 and incubated overnight at 4 °C. After the overnight incubation, the blot was washed and incubated with secondary antibodies, namely monoclonal mouse anti-human immunoglobulin E-horseradish peroxidase (IgE-HRP) at 1 :2,000 dilution in TBS-T for 2 hours at room temperature. The signal of the reaction was detected using chemiluminescent substrate, which developed an X-ray film. Dark spots on the film represented reactive phage clones. The reactive clones were cored out from the plate and allowed to diffuse in 200 pl_ in l-dilution buffer overnight at 4°C, recovered on the following day by centrifugation at 15,000 c g for 5 min. The procedure for immunoscreening of S. stercoralis cDNA library was performed according to the procedure by Sambrook et al. (Sambrook J, Russell DW, Russell DW. 2001. Molecular cloning: a laboratory manual (3-volume set). Cold spring harbor laboratory press New York.). Primary and secondary immunoscreenings of the library were performed using the pre-absorbed pooled biological samples from Strongyloides patients. The selected clones was further immuno-screened using pooled positive pre-absorbed biological samples from Strongyloides patients, pooled negative serum samples from patients with other parasitoses, specifically soil-transmitted helminths, and healthy donor’s biological samples. Finally, only the clones that react with the biological samples from pooled positive and not reactive with pooled negative biological samples were chosen for a tertiary immunoscreening. In the tertiary immunoscreening, individual pre-adsorbed biological samples were used to determine the diagnostic sensitivity and specificity of the selected clones. (2) Results of immunoscreening of Strongyloides stercoralis

S. stercoralis cDNA library was primary immuno-screened with pooled positive serum in 11 series and a total of 122 IgE cDNA clones were isolated. 27 out of the 122 isolated clones from the primary immunoscreening having good intensity were chosen for the secondary and tertiary immunoscreening. Secondary immunoscreening was performed with pooled positive and pooled negative biological sample, while tertiary immunoscreening was performed with individual positive and negative biological sample. 6 clones were found to have good diagnostic potential ranging from 70 to 100 %. The highest potential clone was identified as A133, corresponding to the biomarker, where its sensitivity and specificity was 100 % and 92.85 %, respectively.

(3) Sequence analysis of biomarker A133 The recombinant clone was identified through immunoscreening of cDNA library of S. stercoralis, in which a novel recombinant protein antigen A133 was produced from the cloned cDNA of S. stercoralis and used for detecting parasite- specific IgE antibodies in strongyloidiasis patients. The gene insert sequence of A133 revealed 100% similarity with S. stercoralis genome assembly S_stercoralis_PV0001 , scaffold SSTP_contig0000002 Sequence ID: LL999051.1 , having Identities = 723/723 [100%], and gaps = 0/723 [0%]. Further, the translated protein analysis of A133 showed highest similarity to Strongyloides ratti PDZ signaling domain protein (GFI21964p), having identities = 207/237 [87%%], positives = 225/237 [94%], and gaps = 0/237 [0%]) and Sequence ID: CEF67580.1. PDZ domain, also known as discs-large homologous regions (DFIR) or GLGF, is one of the most important domains for protein-protein interaction and considered as a novel target of drug discovery. Further, PDZ domain is one of the important modules reserved in the parasite to mediate multiple biological processes.

Example 3

In-vivo Excision of Potential Clones

The highly sensitive and most specific selected Immunoglobulin E- complementary deoxyribonucleic acid (IgE-cDNA) clones were cored out and excised in-vivo from the l vector to form phagemids containing the cloned inserts, allowing conversion of cDNA clones in the form of phagemids into plasmids. 150ul of the eluted plaque was combined with a 200 ul overnight culture of E. coli BM25.8 and incubated at 31 °C for 30 min. 400 uL of LB broth was added to the cell suspension and incubated for 1 hour at 31 °C with shaking at 225 rpm. 5 ul of cell suspension was spread on LB/armpicillin agar plate using a sterile glass spreader and incubated overnight at 37 °C. The isolated colony was cultured and the plasmid was then purified by using QIAprep Spin Miniprep Kit according to the manufacturer’s instructions. DNA sequences were performed and sequences were examined for similarity to the already available sequences in the GenBank non- redundant nucleic acid database using BLAST analysis and other specific public databases for nematodes such as nematode.net and Wellcome Trust Sanger Institute. In addition, Open Reading Frame Finder (ORF finder) was used to analyze the presence of start and stop codons in nucleotide sequence. The translation of the nucleic acid sequence to amino acid sequence was performed using the Expasy translates tool software. Example 4

Preparation of Recombinant Proteins

(1) Custom synthesis into a pET32a expression vector

The IgE reactive clone with the best potential diagnostic value obtained from the IgE-phage immunoblots was selected for production of recombinant protein. The nucleic acid sequence of the clone was sent to a EPOCH Life Science Inc. for codon optimization, DNA synthesis, and cloning into a pET32a expression system.

(2) Expression and purification of recombinant proteins

The custom-cloned recombinant plasmid was transformed into E. coli host cells C41 (DE3) and protein expression was performed in 2 L of terrific broth supplemented with 100 pg/mL of ampicillin at 37 °C with shaking at 200 rpm until the OD600 reached to 0.6. The culture was then induced by adding 1 mM of IPTG and further incubated for 4 hours at 30 °C. The cells were harvested by centrifugation, specifically 10,000 c g for 10 min at 4 °C, and the cell pellet was then resuspended in cold lysis buffer having 50 mM of sodium dihydrogen phosphate (NaH₂P0₄), 300 mM of NaCI, 10 mM of imidazole and pH 8.0, with lysozymes at a final concentration of 0.5 mg/mL and protease inhibitors. After 30 min of incubation on ice, the cell lysate was disrupted by a French press and centrifuged. The obtained supernatant was treated with 0.5 pg/mL of deoxyribonuclease I (DNasel), incubated on ice for 30 min and then centrifuged at 10,000 x g for 30 min. The lysate obtained was filtered and incubated for 1 hour with nickel nitrile tri acetic acid resin slurry on a rotator.

Further, a gradient washing was performed using phosphate buffers having 50 mM of NaH2P04, 300 mM of NaCI and pH 7.4, with imidazole concentration of 20, 30 and 40 mM, and the target protein was eluted with phosphate buffer containing 250 mM of imidazole. Eluted fractions (having 500 mI_ each were collected and then purity of protein in each fraction was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions with high-purity proteins were pooled and concentrated using a protein concentrator spin column. The protein concentration was determined using Bio-Rad Protein assay reagent and then stored at -20 °C.

Example 5

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis

(1) Preparation of SDS-PAGE and Western Blot 10 ug/well of purified protein electrophoresed in 10 % of SDS-PAGE and transferred to NC membranes. The blot was washed 3 times for 5 minutes each using TBS-T having 0.05 % Tween 20, and was subsequently blocked for 1 hour with 5 % of skimmed milk, followed by washing with TBS-T and incubated overnight at 4 °C with biological sample. After the overnight incubation, the membrane was washed and incubated with monoclonal mouse anti-human IgE- HRP at 1 :1000 for 2 hours at room temperature. The signal of the reaction was detected using chemiluminescent substrate and developing an X-ray film. The potential diagnostic value was evaluated using several biological samples from Strongyloides patients with other parasitoses and also healthy donors.

(2) SDS-PAGE and Western Blot Analysis

Fig. 3 shows SDS-PAGE and western blot analysis of (a) a purified biomarker, (b) probed with anti-His horseradish peroxidase (HRP) antibody, and (c) a sandwich format with an aptamer as a capturing element. More specifically, Fig. 3 (C) represents purified biomarker probed with positive biological samples from Group 1A on lane 1 and 2, probed with positive biological samples from Group 1 B on lane 3 and 4, and control biological samples from Group 2 on lane 5 to 19, Group 3 of healthy individuals on lane 20 to 22, and an M lane that represents the Precision Plus Protein Unstained Standard Marker. Further, Group 2 comprising control biological samples of ascariasis on lane 5 to 8, filariasis on lane 9 to 14, mixed hookworm infection and trichuriasis on lane 15 and 16, amoebiasis on lane 17, toxocariasis on lane 18, and giardiasis on lane 19.

SDS-PAGE analysis showed that only the thickest band of approximately 40 kDa showed reactivity with anti-His-HRP western blot. The A133 IgE-western blot using biological sample showed 100 % sensitivity for Group 1A, 100 % sensitivity for Group 1 B. The specificity for Group 3 and Group 2 was 100% and 93.7%, respectively, as one of the filariasis serum was cross-reactive.

Example 6

Immunoglobulin E-Enzyme-Linked Immunosorbent Assay (IgE-ELISA) of the Recombinant Protein (1) Determination of the diagnostic sensitivity of the recombinant protein

Diagnostic sensitivity was determined using the biological samples from Group 1 A and Group 1 B, whereas the diagnostic specificity was evaluated using Group 2 and Group 3. The ELISA was performed as described earlier, except 10ug/ml of target recombinant protein A133 was used for coating the plate well and the IgE-HRP at 1 :750 was used as the secondary antibodies with 2 hours incubation. The cut-off value (COV) of the test was determined by the receiver operator characteristic curve analysis of the ELISA data obtained from testing of all serum samples (n =125).

(2) Evaluation of the diagnostic value of the recombinant protein by IgE-ELISA IgE-ELISA of the recombinant protein, namely A133 IgE-ELISA, was performed with biological sample groups described earlier and statistical analysis was performed. P-value less than 0.05 was considered significant and all the statistical analysis was performed by using Graph Pad Prism version 8.0.2. Referring to Fig. 4, the analysis of variance (ANOVA) results of all the four-group showed that it is statically significant with P-value < 0.0001 . The t-test performed for Group 1 A and Group 1 B, Group 1 B and Group 2, Group 1 B and Group 3 have P-values < 0.0001. In addition, receiver operator characteristic (ROC) curve analysis of the IgE-ELISA (n=125) shown COV 0.22 produced the highest sensitivity of 100 % and specificity of 98 %, with the area under the ROC curve (AUC) is 1 .000, more particularly to 95 % confidence interval of 0.999 to 1 .000 and P-value > 0.0001. Antigenicity evaluation produced the diagnostic sensitivity of 100 % (n=31) and diagnostic specificity of 98 % (n=94) as shown in Fig. 5. Further, the distribution of OD of positive and negative biological sample is shown in Fig. 6.

The purified A133 was used in IgE western blot and IgE-ELISA and showed good diagnostic potential with high sensitivity and specificity. A significant difference was observed between biological samples of Group 1A and Group 1 B, and also between the negative control biological samples of Group 2 and 3 with Group 1 B. These results implied that A133 has a good potential to detect acute Strongyloides infection.

The above-mentioned biomarker overcomes the problems and shortcomings of the existing biomarker for Strongyloides, specifically S. stercoralis. The biomarker showed high potential for diagnosis, epidemiological studies and research, especially for diagnosing acute infection of strongyloidiasis. In addition, IgE-ELISA is applied in the methods of detecting Strongyloides (100) and diagnosing strongyloidiasis (200) in a subject. In addition, the present invention detects IgE in order to detect presence of active or acute infection of

Strongyloides. The biomarker as antigen also showed high diagnostic sensitivity and specificity of higher than 95 % due to the biomarker being an isolated or recombinant S. stercoralis protein. Due to its high sensitivity and specificity, the biomarker is utilized in preparing an improved diagnostic kit for Strongyloides.

The exemplary implementation described above is illustrated with specific shapes, dimensions, and other characteristics, but the scope of the invention also includes various other shapes, dimensions, and characteristics. Also, the components as described above could be manufactured in various other ways and could include various other materials. Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be providing broadest scope of consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims. Although the present invention has been described with reference to specific embodiments, also shown in the appended figures, it will be apparent for those skilled in the art that many variations and modifications can be done within the scope of the invention as described in the specification and defined in the following claims.

SEQUENCE LISTING

<110> UNIVERSITI SAINS MALAYSIA <120> A BIOMARKER FOR DETECTING Strongyloides, METHODS OF

DETECTING Strongyloides AND DIAGNOSING STRONGYLOIDIASIS IN A BIOLOGICAL SAMPLE, AND A DIAGNOSTIC KIT COMPRISING THEREOF

<130> 2019/PT/F4.97/OP

<160> 2

<170> BiSSAP 1.3.6 <210> 1

<211 > 723 <212> DNA

<213> Strongyloides stercoralis

<220>

<223> Biomarker

<400> 1 atgaaaattg agccgagcac ctttattacc aataatgtta atttcggcga cattatttac 60 gcacatatta aaggtcagcg taaagaagtg cagatgacca aaaccagtga tgccctgggc 120 attaccatta ccgataatgg tgccggctat tgttttgtga aaaaaattaa accgggcagc 180 accagcgcac tggcctgccc tgcaattctg attggtgatc atattgaaaa agtgaatggc 240 aatagtatgg tgggcatgac ccattgcagt gtggcccgcg tgctgcgcaa tattagtgtg 300 ggtgaaacct ttattatccg tctggttgaa ccgtataaaa ccggctttag ccatattagc 360 agccgcggca gccgtattcc gaataaaacc accaccgata ttaccagtgg caccggcacc 420 ctgcgcttta aagccaatgg tgatgttgtt attcaggaag caccggataa aatgattatt 480 agcgccatga atgatatctt tgatagctat ctgggcctgc atgatgatga actggccctg 540 agcatttggg aagttggttg taattgtcag gatgtgatgg aactgaccaa aaaaattaat 600 gaaagcgaaa tgggcacctt tgaatttccg gatgaactga tttttgatat gtggggtgtg 660 attgatgatt ttcgcaaaag ccgtctgagt ggtcagagca ccaaaaatga tagcaataaa 720 taa 723

<210> 2 <211 > 240

<212> PRT

<213> Strongyloides stercoralis <400> 2

Met Lys lie Glu Pro Ser Thr Phe lie Thr Asn Asn Val Asn Phe Gly

1 5 10 15

Asp lie lie Tyr Ala His lie Lys Gly Gin Arg Lys Glu Val Gin Met

20 25 30 Thr Lys Thr Ser Asp Ala Leu Gly lie Thr lie Thr Asp Asn Gly Ala

35 40 45

Gly Tyr Cys Phe Val Lys Lys lie Lys Pro Gly Ser Thr Ser Ala Leu

50 55 60

Ala Cys Pro Ala lie Leu lie Gly Asp His lie Glu Lys Val Asn Gly 65 70 75 80

Asn Ser Met Val Gly Met Thr His Cys Ser Val Ala Arg Val Leu Arg

85 90 95

Asn lie Ser Val Gly Glu Thr Phe lie lie Arg Leu Val Glu Pro Tyr

100 105 110 Lys Thr Gly Phe Ser His lie Ser Ser Arg Gly Ser Arg lie Pro Asn

115 120 125 Lys Thr Thr Thr Asp lie Thr Ser Gly Thr Gly Thr Leu Arg Phe Lys

130 135 140

Ala Asn Gly Asp Val Val lie Gin Glu Ala Pro Asp Lys Met lie lie

145 150 155 160 Ser Ala Met Asn Asp lie Phe Asp Ser Tyr Leu Gly Leu His Asp Asp

165 170 175

Glu Leu Ala Leu Ser lie Trp Glu Val Gly Cys Asn Cys Gin Asp Val

180 185 190

Met Glu Leu Thr Lys Lys lie Asn Glu Ser Glu Met Gly Thr Phe Glu 195 200 205

Phe Pro Asp Glu Leu lie Phe Asp Met Trp Gly Val lie Asp Asp Phe

210 215 220

Arg Lys Ser Arg Leu Ser Gly Gin Ser Thr Lys Asn Asp Ser Asn Lys

225 230 235 240

Claims

Claims:

1 . A biomarker for detecting Strongyloides, said biomarker is characterized by: a Strongyloides stercoralis protein comprising an amino acid sequence set forth in SEQ ID NO: 2; wherein said Strongyloides stercoralis protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence set forth in SEQ ID NO: 1 .

2. The biomarker as claimed in claim 1 , wherein said nucleic acid sequence set forth in SEQ ID NO: 1 is introduced into a plasmid to produce an expression construct configured to produce said Strongyloides stercoralis protein encoded by said nucleic acid sequence set forth in SEQ ID NO: 1 .

3. The biomarker as claimed in claim 1 , wherein said Strongyloides stercoralis protein is an isolated Strongyloides stercoralis protein or a recombinant

Strongyloides stercoralis protein.

4. The biomarker as claimed in claim 1 , wherein said amino acid sequence set forth in SEQ ID NO: 2 including a complete amino acid sequence, a fragment of said amino acid sequence or a variant of said amino acid sequence.

5. The biomarker as claimed in claim 1 , wherein said nucleic acid molecule is an isolated nucleic acid molecule or a recombinant nucleic acid molecule.

6. The biomarker as claimed in claim 1 , wherein said biomarker including a

Strongyloides specific antigen.

7. A method (100) of detecting Strongyloides in a biological sample, said method comprising the steps of: contacting a biological sample with an antigen; and detecting a specific and selective antigen-antibody binding of said antigen and a first antibody present in said biological sample via an immunoassay; characterized in that, said antigen is said biomarker for detecting Strongyloides of Claim 1 comprising said amino acid sequence set forth in SEQ ID NO: 2; wherein said first antibody is a Strongyloides stercoralis- specific antibody; and wherein presence of Strongyloides in said biological sample is detected via presence of said specific and selective antigen-antibody binding of said antigen and said first antibody in said biological sample.

8. The method (100) as claimed in claim 7, wherein said first antibody is an immunoglobulin E, IgE, antibody.

9. The method (100) as claimed in claim 7, wherein said immunoassay including an enzyme-linked immunosorbent assay, a dot-blot, a Western blot, a vertical flow test, a lateral flow assay, and a biosensor.

10. The method (100) as claimed in claim 7, wherein said biological sample is from a human or non-human species.

11 . A method (200) of diagnosing Strongyloidiasis in a subject, said method comprising the steps of: collecting a biological sample from a subject; contacting said biological sample with an antigen; detecting presence of Strongyloides protein in said biological sample via an immunoassay using a first antibody; contacting said biological sample with a second antibody; and determining presence of Strongyloidiasis in said subject; characterized in that, said antigen is said isolated Strongyloides stercoralis protein of Claim 1 comprising said amino acid sequence set forth in SEQ ID NO: 2; wherein said first antibody is a Strongyloides stercoralis- specific antibody; and wherein said second antibody is an antibody that specifically binds to said first antibody.

12. The method (200) as claimed in claim 11 , wherein each of said first antibody and said second antibody is an immunoglobulin E, IgE, antibody.

13. The method (200) as claimed in claims 11 and 12, wherein said second antibody is conjugated with a detector molecule selected from an enzyme, a fluorescence molecule or a colloidal gold.

14. The method (200) as claimed in claims 11 , wherein said method is used for determining acute strongyloidiasis in said subject via detection of said first antibody and said second antibody.

15. A diagnostic kit for Strongyloides, said diagnostic kit comprising: said biomarker for detecting Strongyloides of claim 1 ; an immunoassay reagent; and an immunoassay device configured to detect presence of

Strongyloides.

16. The diagnostic kit as claimed in claim 15, wherein said diagnostic kit is configured to detect presence of Strongyloides antibody or Strongyloides protein.

17. The diagnostic kit as claimed in claim 16, wherein said diagnostic kit including an enzyme-linked immunosorbent assay, a dot blot assay, a Western blot assay, a vertical flow assay, a lateral flow assay, and a biosensor.