DESC:-: Complete Specification :-

Lateral flow assay method, and device implementing the same, for detection of glycoproteins

Cross-reference to related applications: This complete specification is filed further to application for patent No. 202121004327 filed on 01/02/2021 with provisional specification, the contents of which are incorporated herein in their entirety, by reference.

Field of the invention
This invention belongs to the field of molecular biology and in that, relates generally to standardized assay systems for determination of chemical / biological target(s) of interest in a test sample. Specifically, the present invention is concerned with a specific detection system, and method employing the same, for determination of an endoglin, in a test sample.

Definitions
Before undertaking the description of the invention below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document. Also, as some technical terms are not used uniformly in the field of the instant invention, a few definitions are given in the following to clarify the meaning of terms as they are used in this paper. Accordingly, the term “Analyte of Interest” (abbreviated as AoI) shall refer any one among biological / chemical products, fragments or whole target(s), e.g. nucleic acid sequences, cells, viruses, pathogens, chemicals or their equivalents and their combinations; the term “LFD” refers a Lateral Flow Device; the term “LFA” refers a Lateral Flow Assay; the term “MNPs” refers magnetic nanoparticles; the term “GcMNPs” refers gold-coated magnetic nanoparticles.

Background of the invention and description of related art
Molecular technologies have been instrumental in the establishment of highly accurate as well as precise methodologies for qualitative and / or quantitative determination of AoI(s) in test samples.

As readily appreciable, the following two aspects are critical for any assay system-
1) Background interference – detection of the AoI(s) may be masked by an interfering consortium of non-target entities including chemicals, metabolites, macromolecules, cells, virions, organisms, and nucleic acid sequences and other noise that may be incidental to the test samples which makes detection of the AoI(s) difficult to ascertain; and
2) Limit of detection – the AoI(s) may be present in a very small amount, often with a large load of background interference, in test samples which further makes detection of the AoI(s) difficult to ascertain.

Naturally, it is required that the assay systems are sensitive enough and have necessary resolution for discerning the AoI(s) to thus have adequate resolution (ability to distinguish between target and non-target entities). Usually, this mandates the implementation of highly sophisticated instruments and specialty detection systems which are not only marred by technical complexities, but also impeded from mass utilization due to inherent high costs and overall inapplicability without the involvement of highly skilled personnel, access to high-tech laboratory setups which makes their utility for on-field analytics an impossible proposition.

Beyond accuracy and precision, it would be advantageous and hence desirable for any ideal assay system to have the following considerations -
1) Ability to satisfactorily work with native samples by having an adequate resolution, thus negating the need for pre-processes for purifying the sample in order to reduce background interference;
2) Ability to satisfactorily work with native samples by having an adequate limit of detection which can ably detect the AoI(s) at concentrations, even if minimal, as may be present naturally in the test sample;
3) Ability of presenting the assay results visibly to the naked eye, to thereby obviate the need for microscopy, imaging or other non-visual systems as well as make the system amenable to even a layman;
4) Ability of quantizing the AoI(s) determined, in form of a gradable result, for helping an analyst to acknowledge the presence as well as apportion and / or correlate significance of the measured level of said AoI(s);
5) Ability of being mass produced, distributed, and used at low costs;
6) Long shelf life, ease of on-field implementation and / or disposal, the disposal being typified in being non-deleterious to the environment.

Currently available and popularly practiced methods commonly rely upon removal of background interference and amplification of signal (that is, some marker correlated to the AoI(s)). Thus, these methods and their implementing apparatuses have not filled the need for a robust, cost effective, rapid, easy to use detection method for target AoI(s), which is compatible in resource limited environments.

Presence of specific proteins in test samples is required for myriad purposes ranging from laboratory assays to clinical trials, diagnostics, prognostics, detection of chemical contamination, food safety, to production in-line quality monitoring / control / assurance applications in industries including pharmaceuticals, environmental testing, animal health, food and feed testing, and plant and crop health. LFAs, and their implementing LFDs are advocated for on-field rapid detection of AoI(s) in such use-cases, however their applicability for detection of specific AoI(s), proteins in particular, in test samples is by and large unattended till date and / or continue to be riddled by the aforementioned lacunae.

While there were many common art references researched by the inventor(s) in ensuring that the present invention is novel, the following patent prior art was identified as related to the present invention, and thus worthwhile to discuss in more detail in context of the present invention. One example is US10598656B2 (assigned to Credo Biomedical Pte Ltd) which discloses an enzyme-based lateral flow assay device for detecting the presence of an analyte within a test sample. Here, a reporting carrier is involved which is capable of forming a complex with the analyte, and wherein said reporting carrier comprises a carrier and at least one proficient enzyme. The reporting carrier is either between an antibody and a nucleic acid which is covalently conjugated to the proficient enzyme.

Another example is US9989527B2 (assigned to AlverixInc) which discloses a diagnostic test strip comprising multiple localized regions containing an immobilized substance configured to bind a target analyte – determination of the captured analyte is made via optical inspection via automated readers.

It is categorically evident that prior art, to the limited extent presently surveyed, lists various LFA methods and their implementing LFDs, but however which unequivocally do not list a single effective solution embracing all advantageous and hence desirable considerations mentioned hereinabove, thus preserving an acute necessity-to-invent for the present inventor who, as result of his focused research, has come up with novel solutions for resolving all needs of the art once and for all.

On the backdrop of the foregoing narrative, the establishment of a rapid and field-deployable LFA method and its implementing LFD for detection of glycoprotein, endoglin in particular, in a test sample is an immediate focus area of research undertaken by the applicant named herein. A better understanding of underlying principles of this invention will be obtained from the following narration which sets forth an illustrative yet-preferred embodiment.

Objectives of the present invention
The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:

It is a primary objective hereof to provide an on-field deployable lateral flow device to detect the presence, and if present the gradation of content, of an analyte of interest in a test sample.

It is another objective hereof to provision an embodiment of the on-field deployable lateral flow device which is directed for detection of the presence, and if present the gradation of content, of endoglin in a test sample.

It is another objective hereof to provision another embodiment of the on-field deployable lateral flow device which is directed for detection of the presence, and if present the gradation of content, of insulin-like growth factor binding protein 1 in a test sample.

It is another objective hereof to provision a reference / lookup table for quantization of visual results obtained using the on-field deployable lateral flow device.

It is another objective hereof, that the on-field deployable lateral flow device established herein is amenable as well as cost-effective to manufacture on an industrial scale.

It is another objective hereof, that the on-field deployable lateral flow device established herein is easy to use and within ambit of even a layman to implement.
The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claims. Other advantageous embodiments of the invention are specified in the dependent claims.

Brief description of drawings
The present invention is explained herein under with reference to the following drawings, in which,

FIG. 1 illustrates the lateral flow immunoassay assembly included in the present invention.

FIG. 2A is an external schematic view of the housing used to enclose the lateral flow immunoassay assembly shown in FIG. 1.

FIG. 2B illustrates the housing shown in FIG. 2A, with its base and top in opened configuration.

FIG. 3A is an external view of the LFD of the present invention, in a state before being used for the LFA according to the disclosures herein.

FIG. 3B is an external view of the LFD shown in FIG. 3A when utilized, and showing the first test band and the control band being developed.

FIG. 3C is an external view of the LFD shown in FIG. 3A when utilized, and showing the first and second test bands and the control band being developed.

FIG. 3D is an external view of the LFD shown in FIG. 3A when utilized, and showing the first, second and third test bands and the control band being developed.

FIG. 4A is an external view of the LFD shown in FIG. 3A when utilized, and showing the first test band developed but the control band being not developed.

FIG. 4B is an external view of the LFD shown in FIG. 3A when utilized, and showing the first and second test bands developed but the control band being not developed.
FIG. 4C is an external view of the LFD shown in FIG. 3A when utilized, and showing the first, second and third test bands developed but the control band being not developed.

The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. The drawings are not to scale (unless so stated) and are intended for use solely in conjunction with their explanations in the following detailed description. In above drawings, wherever possible, the same references and symbols have been used throughout to refer to the same or similar parts, as under-
(01) - sample pad made of glassfiber
(02) - conjugation pad made of polyester
(03) - chromatography membrane
(04) - absorbent pad made of cellulose fiber
(05) - First printed line of detection antibody
(06) - Second printed line of detection antibody
(07) - Third printed line of detection antibody
(08) - Control line printed with control antibody
(09) - Reference marking on housing, aligning and corresponding to position of line (05) on chromatography membrane (03)
(10) - Reference marking on housing, aligning and corresponding to position of line (06) on chromatography membrane (03)
(11) - Reference marking on housing, aligning and corresponding to position of line (07) on chromatography membrane (03)
(12) - Reference marking on housing, aligning and corresponding to position of control line (08) on chromatography membrane (03)
(13) - Sample well
(14) - Observation window
(15) - Housing

The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. The drawings are not to scale (unless so stated) and are intended for use solely in conjunction with their explanations in the following detailed description.

In above drawings, wherever possible, the same references and symbols have been used throughout to refer to the same or similar parts. Though numbering has been introduced to demarcate reference to specific components in relation to such references being made in different sections of this specification, all components are not shown or numbered in each drawing to avoid obscuring the invention proposed

Summary / Statement of the invention
A low cost, point-of-care, complex equipment-free, semi-quantitative, field-deployable LFA method and its implementing LFD platform for detection of glycoprotein, endoglin in particular, in a test sample are disclosed herein which are typified by the ability to detect the presence, and if present the gradation of content in reference to a simple lookup table, of endoglin in a test sample, by virtue of a plurality of printed test lines embedded / impregnated with different amount of detection antibody specific to endoglin, use of gold-coated magnetic nanoparticles conjugated with capture antibody specific to endoglin, and presence of a printed control line embedded / impregnated with a control antibody, specific to endoglin but being from a different system than the detection antibody.

Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

Detailed description
The present invention is directed at absorbing all advantages of prior art while overcoming, and not imbibing, any of its shortfalls, to thereby establish a rapid and field-deployable LFA method and its implementing LFD for detection of an analyte of interest, being either between a glycoprotein, endoglin in particular, in a test sample.

Reference is hereinafter made to certain embodiments which illustrate, in a non-limiting manner, an illustrious preferred way in which the present invention may be implemented.

Construction of the lateral flow immunoassay assembly of the LFD:-
As seen in the accompanying FIG. 1, FIG. 2A and FIG. 2B, the LFD proposed herein can be understood as a visual lateral flow assay system provided in a sandwich format in a compact and portable cuboid hollow housing (15) made of plastic or polyvinyl chloride. The housing (15) is hollow, and has one face incorporating a sample well (13) and an observation window (14). Dimensions of said housing include a length of 7cm, breadth of 2cm and a height of 0.5cm.

With continued reference to the accompanying FIG. 1, it can be seen that within the hollow space of the housing, and specifically therein on the floor of said housing, are assembled, in sequence with each succeeding component positioned downstream and in contact with the preceding component, among-
a) A sample pad (01) made of glassfiber for allowing a user to load the sample to be assayed for presence of an analyte of interest;
b) A conjugation pad (02) made of polyester and pre-impregnated, via passive adsorption, with gold-coated magnetic nanoparticles conjugated with capture antibodies specific to endoglin, for binding to endoglin if present in the sample being assayed,
c) A chromatography membrane (03) for allowing the sample being assayed to flow under capillary action. The chromatography membrane (03) used is a 26 mm strip of nitrocellulose (10µm-CNPF, MDI); and
d) An absorbent pad (04) made of cellulose fiber for absorbing / resting the amount of sample which flows out of the chromatography membrane (03) before the LFD is discarded.

The aforementioned assembly makes it possible for the test sample, when loaded with help of a dropper or equivalent device, into the LFD proposed herein through the sample well (13), to get absorbed into the sample pad (01) and thereafter flow, under capillary action, in a direction from the sample pad (01), through the conjugation pad (02) and then the chromatography membrane (03) to finally rest / get absorbed within the absorbent pad (04).

As seen in the FIG. 1, the sample pad (01) is juxtaposed just below the sample well (13) of the housing (15) and the chromatography membrane (03) runs under the observation window (14) of said housing. The conjugation pad (02) and the absorbent pad (04) are hidden from external view once the housing (15) is closed.

As per another aspect hereof, three test lines (05, 06 and 07) of the detection antibody and 1 control line (08) of the control antibody are printed, using an Easy printer LPM -02, onto the nitrocellulose membrane (03) at a distance of 19 mm from the start of the nitrocellulose membrane (03). The three test lines (05, 06 and 07) are arranged to be printed with the detection antibody at concentrations of 5mg/ml, 2.8 mg/ml, and 1.4 mg/ml respectively. Control antibody is printed at a concentration of 0.5 mg/ml. In corresponding alignment as seen in FIG. 2, four markings (09, 10, 11, and 12) are printed onto the observation window (14) which align correspondingly with the test lines (05, 06, and 07) and the control line (08) respectively.

It shall be understood that the test lines (05, 06 and 07) and control line (08) are otherwise invisible to the naked eye, and can be seen only when the LFD is used successfully, with a positive determination of endoglin being present in the test sample.

On the other hand, the markings (09, 10, and 11) are always visible, and serve to indicate the positions on the nitrocellulose membrane (03), where the LFA outcome, i.e. lines, should develop / appear when the LFD is used successfully, with a positive determination of endoglin being present in the test sample.

According to another aspect hereof, the distance between test lines (05 and 06) is 4mm and (06 and 07) is 3mm, the distance between third test line (07) and the control line (08) is 8mm, which as per combination developed / appeared, indicate the manner in which results of the LFA are to be visually read / interpreted by the user. The user may thus visually determine the result as per the following proforma (provided along with the LFD in form of a lookup table, described in the later part of this disclosure) as under-
a) As seen in FIG. 3B, appearance / Development of 1 test and 1 control line indicates normal concentration of the AoI.
b) As seen in FIG. 3C, appearance / Development of 2 test and 1 control line indicates mild concentration of the AoI.
c) As seen in FIG. 3D, appearance / Development of 3 test and 1 control line indicates high concentration of the AoI.
d) As seen in FIG. 4A, 4B and / or 4C, appearance of any number of test lines without control line being developed indicates an inconclusive result, suggesting that the LFD should be discarded and replaced with another LFD prepared in accordance of this invention.

Preparation of reagents:-
According to a core aspect of the present invention, the conjugate pad (02) is coated with GcMNPs conjugated to capture antibody using passive adsorption method. The preparation of said GcMNPs is carried out as per the process outlined below-

a) Synthesis of MNPs – MNPs are synthesized using FeCl3 and FeSO4 salts. These salts are weighed and dissolved, at a ratio of 2:1, in distilled water. The solution is stirred at 1500 RPM while increasing the temperature from 80°C to 100°C on magnetic stirrer for 15 minutes. A 10% solution of NaOH is then added drop-wise into this solution over a period of 15 to 20 minutes till formation of a black precipitate at the end of the reaction. The resulting solution is stirred for another 10 to 15 minutes keeping the temperature and stirring speed constant. The black precipitate is indicative of formation of iron oxide nanoparticles. MNPs are separated out using a magnet and the supernatant is discarded.

b) Surface modification – The MNPs obtained as per the foregoing disclosure are treated with a 50% solution of citric acid to modify the surface charge of the nanoparticles. The resulting mixture is stirred at 90°C for 60 min with stirring (1500rpm).

c) Characterization of the MNPs – The MNPs so formed were characterized via dynamic light scattering and charge analysis, in which the MNPs were observed to have an average size of 358.46 ± 5.17 nm and charge of -31.4 ± 0.1 while the GcMNPs were observed to have an average size of 358.46 ± 5.17 nm and charge of -31.4 ± 0.1.

d) Gold coating of MNPs – The MNPs synthesized in step a) above were coated with gold using citrate reduction method. The pH of the GcMNPs is adjusted to 8 - 8.5 by using 0.2M K2CO3.

LFA, intended for implementation using the LFD disclosed above, is arranged for detection and grading the content, if present, of the AoI in test samples by involving the following selection of commercially-available antibodies-
a) Capture antibody - Polyclonal Human Endoglin Antibody
b) Detection antibody - Polyclonal Human Endoglin Antibody
c) Control antibody - Anti-Mouse IgG (whole molecule) antibody produced in goat
Reference is now made to certain non-limiting examples which showcase the manner in which principles of the present invention may be employed.

Example 1: Detection of Endoglin in-vitro conditions
The expression of endoglin is elevated on the endothelial cells of healing wounds, developing embryos, inflammatory tissues, and solid tumors. Endoglin is a marker of activated endothelium, and its vascular expression is limited to proliferating cells.

LFD of the present invention is intended to be employed for critical time-points based measurements of / from a continuous cell culture systems to avoid contamination and enable rapid monitoring to prevent loss of biomarker / AoI concentration.

Accordingly, the LFD proposed herein is intended to be used in in-vitro conditions for assaying the expression of endoglin on tumor endothelium in monolayers, co-culture and 3D cell culture models to determine its diagnostic value for studies in its role in tumor growth, metastasis and endothelial marker of angiogenesis in cancers.

According to an exemplary protocol of implementation standardized by the applicant named herein, samples (50µl to 100 µl) from cell culture supernatants (viz. fibroblasts, choriocarcinoma, fibrosarcoma, glioblastoma and breast carcinoma cell line / cell –free broth / supernatant samples) are used. Measurement of endoglin secretion from in-vitro trophoblast cultures, placental explants from preeclamptic pregnancies, and endothelial cells (HUVECs) is also intended.

The gradation or scale corresponding to the normal, mild or high concentration of the AoI in the present in-vitro studies may be linked / charted to a quantitative estimation of the concentration or titer of said AoI for further elucidative studies as per lookup tables illustrated below in Table 1.

Control 1st Line 2nd Line 3rd Line Concentration of Endoglin (ng/ml)
Yes Yes No No = 30
Yes Yes Yes No 30 to 100
Yes Yes Yes Yes > 100
No Yes No No Inconclusive result. Discard LFD
No Yes Yes No Inconclusive result. Discard LFD
No Yes Yes Yes Inconclusive result. Discard LFD

Table 1: Lookup table for Endoglin

The results of these in-vitro analyses are intended to lay the foundation of future therapeutic modalities. For example, vascular-targeting antiangiogenic therapy of cancer can be advantageous over conventional tumor cell targeted cancer therapy and endoglin is such a target. Therapeutic modalities to control endoglin secretion can help regulate endothelial dysfunction. In complicated pregnancies, this can potentially dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

Experimental validation-
The LFD proposed herein has been reduced to practice and experimentally validated by the Applicant in studies requiring to monitor endoglin secretion, ex-vivo, by different cell-lines.

According to an exemplary protocol of implementation standardized by the applicant named herein, a 50µl to 100 µl sample of supernatant of cell cultures is taken for evaluation of endoglin expression.

Presence and gradation of content of Endoglin levels, as determined by implementing the LFA using the LFD proposed herein, is thus determined for fundamental research / academic purposes. Sample results of these studies, in average values representing the data set captured, are presented in Table 2 below.

# Control Line 1 Line 2 Line 3 Level of endoglin determined
(ng/ml) Inference Confirmation by ELISA
(ng/ml)
(+/- 5)
1) MDA-MB-231
Reading A Y Y N N = 30 Non- invasive phenotype 15.2
Reading B Y Y Y Y 30 to 100 Invasive phenotype 47.2
2) HUVEC
Reading A Y Y N N = 30 Without hypoxia 23.5
Reading B Y Y Y N 30 to 100 Under hypoxia 47.2
3) T98G glioma cells
Reading A Y Y N N = 30 Non- invasive phenotype 9.6
Reading B Y Y Y N 30 to 100 Invasive phenotype 34.7
4) Saline control
Saline control Y N N N Control validated LFD is performing correctly 0.0

Table 2
Legend-
Y ? line observed
N ? No line observed
O ? Normal determination. No further investigation needed.
M ? Further differential / conclusive investigation needed
N ? Further differential / conclusive investigation critically needed

Example 2: Detection of Endoglin in body fluids (viz. plasma, serum, saliva, urine etc) for early detection of placental dysfunction in pregnant subjects for providing therapeutic targets and diagnostic tools.

Therapeutic modalities to control endoglin secretion can help regulate endothelial dysfunction. In complicated pregnancies, this can potentially dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

The gradation or scale corresponding to the mild, moderate or high concentration of the AoI may be linked / charted to a quantitative estimation of the concentration or titer of said AoI for further elucidative studies as per lookup table shown in Table 1 in the foregoing part of this description.

Experimental validation-
The LFD proposed herein has been reduced to practice and experimentally validated by the Applicant in studies requiring to monitor endoglin secretion in pregnant human subjects.

According to an exemplary protocol of implementation standardized by the applicant named herein, a 50µl to 100 µl sample of body fluid (viz. plasma, serum, saliva, urine etc) from pregnant women is taken for evaluation of degree of placental dysfunction using the LFD of the present invention.

Presence and gradation of content of Endoglin levels, as determined by implementing the LFA using the LFD proposed herein, is thus determined corresponding to advancement of disease causing faulty placental growth/damage/ developmental issues with the placenta.

Sample results of these studies, in average values representing the data set captured, are presented in Table 2 below.
Subject Control Line 1 Line 2 Line 3 Level of endoglin determined
(ng/ml) Inference Confirmation by ELISA
(ng/ml)
(+/- 5)
A. Y Y N N = 30 O 24.2
B. Y Y N N = 30 O 18.4
C. Y Y Y N 30 to 100 M 38.6
D. Y Y Y N 30 to 100 M 51.2
E. Y Y Y Y > 100 N 125.7
F. Y Y Y N 30 to 100 M 62.4
G. Y Y Y Y 30 to 100 N 110.3
H. Y Y N N = 30 O 22.3
I. Y Y Y N 30 to 100 M 77.8
J. Y Y Y Y > 100 N 114.3
K. N Y Y N Inconsequential No inference ---
Saline control Y N N N Control validated LFD is performing correctly 0.0

Legend-
Y ? line observed
N ? No line observed
O ? Normal determination. No further investigation needed.
M ? Further differential / conclusive investigation needed
N ? Further differential / conclusive investigation critically needed

Table 4

Overall, the present invention is differentiated and scores above the teachings of prior art in having technical advancements identified in-
a) Modifications in the synthesis of MNPs as compared to conventional methods;
b) Synthesis of GcMNPs- anti-endoglin antibody conjugate and its use for detection of AoI, being endoglin, in the LFA, as discussed herein, being reported for the first time; and
c) Detection of endoglin in a barcode LFA format, wherein varying concentrations of the same AoI is visually possible in a graded format, as evidenced above, being reported for the first time.

From the above narration, a low cost, point-of-care, complex equipment-free, semi-quantitative, field-deployable LFA method and its implementing LFD platform for detection of glycoprotein, endoglin in particular, in a test sample is thus provided which finds specific utility in qualitative / semi-quantitative (or both) use-cases including –
a) in-vitro diagnosis / in-vitro cell cultures studies aimed at determining the presence (and if possible objectifying the role of endoglins) in-
1) Cancer metastasis
2) Tumor growth
3) Vascular-targeting antiangiogenic therapy for tumors

b) Early detection of placental dysfunction in pregnant subjects.
1) Trophoblast invasion
2) Early onset of preeclampsia
3) Conditions related to vascular endothelium

It shall be appreciated that the present invention embraces all advantageous and hence desirable considerations mentioned in the background section of this specification, and moreover is significantly improved over teachings of prior art by having the following virtues-
1) Non-dependency on costly analytical instruments and specialized laboratory set-ups;
2) Critical time points based measurements of from a continuous cell culture system to avoid contamination
3) Rapid (under 15 minutes) and accurate assessments to prevent loss of biomarker/AoI concentration, hence enabling faster decision making;
4) Point of care utility usable in low resource settings;
5) Requirement of very low volume (between 50µl to 100µl) of test sample
6) Testing can be performed at room temperature (Range- 15oC to 50oC) and normal atmospheric pressure
7) Visual test which negates the need for costly / complex ancillary equipment.

It shall be generally understood by the reader that although the present invention is described herein using specific terms, these are used in a generic and descriptive sense only and are not intended to be limiting. Additionally, non-pharmaceutical uses of the composition identified in the foregoing narration are also intended, which shall form part of disclosures in the non-provisional specification, and / or child patent applications intended to be filed pursuant to this provisional patent application.

As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention.

Accordingly, the foregoing description will be regarded as illustrative in nature and not as restrictive in any form whatsoever. Modifications and variations of the system and apparatus described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within ambit of the present invention, which is limited only by the appended claims. ,CLAIMS:1) A semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample, consisting of-
a) a lateral flow immunoassay assembly, being an arrangement in particular, consisting of-
? A sample pad (01) for allowing a user to load the test sample;
? A conjugation pad (02) positioned downstream in contact with the sample pad (01) and pre-impregnated, via passive adsorption, with gold-coated magnetic nanoparticles conjugated with capture antibodies specific to endoglin, for binding to endoglin if present in the test sample being assayed;
? A chromatography membrane (03) positioned downstream in contact with the conjugation pad (02) for allowing the test sample being assayed to flow under capillary action;
? three test lines (05, 06, 07) impregnated with a detection antibody specific to endoglin and 1 control line (08) impregnated with a control antibody specific to endoglin printed, using an Easy printer LPM -02, on the chromatography membrane (03) to allow the user to visualize the presence, and if present the gradation of content, of endoglin in the assayed sample; and
? An absorbent pad (04) positioned downstream in contact with the chromatography membrane (03) for absorbing the test sample after it has flowed through said chromatography membrane (03).
b) a cuboid hollow housing (15) made of a formable material selected between plastic and polyvinyl chloride for containing the lateral flow immunoassay assembly, said housing having-
? Dimensions admeasuring a length of 7cm, breadth of 2cm and a height of 0.5cm;
? A sample well (13) traversing the body of said housing and positioned overhead the sample pad (01), for allowing the user to load the test sample; and
? An observation window (14) traversing the body of said housing (15) on the same face as the sample well (13), and positioned overhead the chromatography membrane (03) and having reference markings (09, 10, 11, and 12) to denote the positions for visualization of the lines (05, 06, 07 and 09) respectively, to thereby allow the user to visualize the presence, and if present the gradation of content, of endoglin in the assayed sample.

c) A lookup table for referencing the concentration of endoglin in the assayed sample as corresponding to the visualization of the number of the test lines (05, 06, and 07) and the control line (08).

2) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 1, wherein the chromatography membrane (03) is made of a 26 mm strip of polyester backed nitrocellulose membrane of pore size 10 µm.

3) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 1, wherein the three test lines (05, 06, and 07) to allow the user to visualize the presence, and if present the gradation of content, of endoglin in the assayed sample are indicative of-
a) A first test line (05) at a position nearest in direction of the conjugation pad (02) at 19 mm from the start of the nitrocellulose membrane (03), serving to indicate if developed, a normal concentration being =30ng/ml in particular, of endoglin in the test sample;
b) A second test line (06) at a position downstream of the first test line (05) and at a distance of 4mm from said first test line (05) to indicate if developed in addition to the first test line (05), a mild concentration being <30-100 ng/ml in particular, of endoglin in the test sample; and
c) A third test line (07) at a position downstream of the second line (06) and at a distance of 3mm from said second test line (06) to indicate if developed in addition to the first test line (05) and the second test line (06), a high concentration, being <100 ng/ml in particular, of endoglin in the test sample;

4) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 3, wherein the first test line (05) is printed by impregnation at a concentration of 5mg/ml of the detection antibody, being polyclonal human endoglin antibody in particular.

5) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 3, wherein the second test line (06) is printed by impregnation, at a concentration of 2.8 mg/ml, of the detection antibody, being polyclonal human endoglin antibody in particular.

6) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 3, wherein the third test line (07) is printed by impregnation, at a concentration of 1.4 mg/ml, of the detection antibody, being polyclonal human endoglin antibody in particular.

7) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 1, wherein the 1 control line (08) for confirming ability of the lateral flow device to detect endoglin if present in the test sample being assayed, is printed by impregnation, at a concentration of 0.5 mg/ml, of the control antibody, being anti-mouse Immunoglobulin G produced in goat in particular.

8) The semi-quantitative, field-deployable lateral flow device to detect the presence, and if present the gradation of content, of endoglin in a test sample as claimed in claim 1, wherein the capture antibody is Polyclonal Human Endoglin Antibody.

9) A method for detection of the presence, and if present the gradation of content, of endoglin in an test sample, implementable at temperatures below 50oC, comprising the use of the on-field deployable lateral flow device of claim 1 in an in-vitro environment, said method consisting of-
a) sampling 50µl to 100 µl of cell culture supernatant to serve as a test sample;
b) loading the test sample into the semi-quantitative, field-deployable lateral flow device of claim 1, to allow implementation of the lateral flow immunoassay;
c) Within a time of 15 minutes, visualizing the development of test lines and control lines to thereby determine, in reference to the lookup table, the presence, and if present the gradation of content, of endoglin in the assayed test sample.

10) A method for early detection of placental dysfunction in pregnant subjects, implementable at temperatures below 50oC, comprising the use of the on-field deployable lateral flow device of claim 1 in a point of care environment, said method consisting of-
a) sampling 50µl to 100 µl of a body fluid selected among the plasma, serum, urine, and saliva of the pregnant subject to serve as a test sample;
b) loading the test sample into the semi-quantitative, field-deployable lateral flow device of claim 1, to allow implementation of the lateral flow immunoassay;
c) Within a time of 15 minutes, visualizing the development of test lines and control lines to thereby determine, in reference to the lookup table, as to the presence, and if present the gradation of content, of endoglin in the assayed test sample and therefore in correlation the diagnosis and severity of conditions related to placental dysfunction in said pregnant subject.