WO2019138430A1

WO2019138430A1 - A time-based semi-quantitative and quantitative lateral flow assay (lfa) for early detection of renal injury

Info

Publication number: WO2019138430A1
Application number: PCT/IN2019/050033
Authority: WO
Inventors: Rashmi SHUKLA; Kunal Shukla; Santosh GURRAM; Mohan Singh
Original assignee: Shukla Rashmi
Priority date: 2018-01-15
Filing date: 2019-01-14
Publication date: 2019-07-18

Abstract

The present invention provides a point of care test which works on a gold colloidal lateral flow assay (LFA) that detects the concentration of the analyte such as NGAL using gold-conjugated anti-analyte antibody. The LFA of the present invention detects the renal injury at early stages. This analysis is time-based, as well as multi-test line or multi-concentration based or color intensity-time relation feature based, and does not use any specific make analyzer/reader instrument for its working.

Description

A TIME-BASED SEMI-QUANTITATIVE AND QUANTITATIVE LATERAL FLOW ASSAY (LFA) FOR EARLY DETECTION OF RENAL INJURY FIELD OF THE INVENTION

[0001] The present invention relates to detection of renal injury.

Particularly, the present invention relates to a time-based and/or multi-test line gold colloidal lateral flow assay (LFA) method and a kit for early detection of renal injury.

[0002] More, particularly, the present invention relates to a time -based and/or multi-test line gold colloidal lateral flow assay (LFA) method and a kit for detection of renal injury by means of NGAL.

INTRODUCTION AND BACKGROUND

[0003] Kidney injury and disease embody a substantial and serious health problem which may result into mortality. AKI involves increase in serum creatinine (SCr) levels, which takes several hours to days. Acute kidney injury (AKI) is often asymptomatic and its detection is currently based on functional biomarkers such as serum creatinine. Various AKI biomarkers such as NGAL, KIM-1, L-FABP and IL-18 are comparatively more sensitive and can be used for detection of renal injury. In particular, NGAL (Neutrophil Gelatinase- Associated Lipocalin) is emerging as an excellent biomarker in the urine and

l plasma, for the early prediction of AKI. NGAL is a member of the lipocalin family of proteins, is expressed and secreted from renal tubular cells at low concentrations in the case of healthy individuals. [0004] CKD is abnormality of kidney structure or function with significant health implications.

[0005] There are few diagnostic kits for AKI available in the market. Most of the available test for the analyte (NGAL), are way too expensive, need a well- trained operator, a laboratory to perform test and an expensive and bulky electronic reader/analyzer (mostly unmovable) for the read out. Even the point- of-care (POC) tests by Alere and Getein need an analyzer for the read out.

[0006] Accordingly, it is envisaged to provide a time-based and/or multi-test line gold colloidal lateral flow assay (LFA) and a kit/device for early detection of renal injury.

OBJECTS OF THE INVENTION

[0007] It is an object of the present invention to provide cost effective, simple yet sensitive time-based and/or multi-test line gold colloidal lateral flow assay (LFA) for early detection of renal injury. [0008] It is another object of the present invention to provide a simple and cost-effective kit or device for early detection of renal injury.

[0009] It is still another object of the present invention to provide a visual or electronically or software or mobile application or recording device aided quantitative / semi-quantitative assay for detection of the analyte which includes, however is not limited to NGAL protein concentration in the urine sample which provides estimation in a quick time. SUMMARY OF THE INVENTION

[00010] Accordingly, the present invention provides a method of determining the concentration of analyte; the method comprises detecting the concentration of the analyte using at least one technique selected from the group consisting of time of intensity matching (TOM) and time of appearance (TO A), gold nanoparticle labeled anti-analyte antibody, immobilized test line antibody, and a device comprising a cassette a conjugate pad, sample application pad, and absorbent pad,

wherein the time of intensity matching comprises determining the time, visually or electronically, at which colour and intensity of test line matches to that of guiding lines present on either side of a cassette; wherein the time of appearance (TOA) comprises determining the time of appearance of test and control lines in the device, visually or electronically. [00011] In one embodiment, the device comprises a sample application pad, a conjugate pad, a nitrocellulose membrane, an adsorbent pad and a plastic backing card with guiding lines printed over it.

[00012] In one embodiment, the gold-conjugated anti-analyte antibody is anti-NGAL antibody.

[00013] In one embodiment, the method comprises wherein the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein the immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, wherein the time taken by the line to appear represents a concentration range of analyte.

[00014] In another embodiment, the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein the immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, and observing the color and intensity of the test line appears in comparison with the color and intensity of the guiding lines present on the cassette of device, wherein the time required for matching the color and intensity of the test line to the color and intensity of the guiding lines represents a concentration range of analyte. [00015] In one embodiment, the analyte is NGAL and the the sample is a urine sample.

[00016] In one embodiment, the method comprises the following steps:

- adding at least one drop of a urine sample to be analyzed onto a sample application pad;

- observing the time at which the control line and the test line appears respectively; and

- calculating the difference between the time of appearance of the test and control lines, wherein a predetermined value corresponds to a particular concentration range of analyte in the urine sample, which is being used to interpret the renal condition. [00017] In another embodiment, the method comprises the following steps: adding at least one drop of a urine sample to be analyzed onto a sample application pad;

observing the color and intensity of the test line appears in comparison with the color intensity of guiding lines present on the device; and

determining the time required for matching the color and intensity of the test line the color intensity of guiding lines, wherein the time is used to interpret the concentration of analyte in the sample.

[00018] In one embodiment, the color intensity is observed manually or using an electronic device or a software or a mobile application or a recording device.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[00019] The invention will now be described in relation to the accompanying drawings, in which: [00020] FIG. 1. illustrates device with guiding lines; and

[00021] FIG. 2. illustrates Logarithmic relation between Time versus NGAL concentration using TOM(Y) and TOM(E). DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[00022] The present invention provides a point of care test which works on a gold colloidal lateral flow assay (LFA) that detects the concentration of the analyte using gold-conjugated anti-analyte antibody. The LFA of the present invention detects renal injury at early stage. This analysis is time-based and colour intensity-time relation feature based, and does not use any specific make analyzer/reader instrument for its working. Importantly, the antibody concentration in the present LFA is standardized in such a way that it allows the operator to perform the time-based and/or multi-test line assay and detect the severity of condition that the patient suffers from.

[00023] Definitions:

[00024] The term“Control line” refers to a line above the test line wherein anti-anti antalyte conjugated antibody is immobilized and is used to confirm the test has operated correctly.

[00025] The term“Guiding lines” refers to a colored lines present on the either side of the test area where the test line appears on the plastic cassettes. [00026] Accordingly, in one embodiment, there is provided a method of determining the concentration of analyte. The method involves detecting the concentration of the analyte using at least one technique selected from the group consisting of time of intensity matching (TOM) and time of appearance (TO A), gold nanoparticle labeled anti-analyte antibody, immobilized test line antibody, and a device comprising a cassette a conjugate pad, sample application pad, and absorbent pad. In one embodiment, the time of intensity matching comprises determining the time at which colour and intensity of test line matches to that of guiding lines present on either side of a cassette. In one embodiment, the time of appearance (TOA) comprises determining the time of appearance of test and control lines in the device. [00027] In one embodiment, the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein an immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, wherein the time taken by the line to appear represents a concentration range of analyte.

[00028] In another embodiment, the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein the immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, and observing the color and intensity of the test line appears in comparison with the color and intensity of the guiding lines present on the cassette of device, wherein the time required for matching the color and intensity of the test line to the color and intensity of the guiding lines represents a concentration range of analyte.

[00029] In accordance with another aspect of the present invention there is provided a device or kit of the present invention for performing a gold colloidal lateral flow assay or detecting the concentration of an analyte such as NGAL. In one embodiment, the kit comprises: a) a device comprising a sample application pad, a conjugate pad, a nitrocellulose membrane, an adsorbent pad and a backing card with guiding lines; b) gold nanoparticle labeled anti-analyte antibody; c) immobilized test line antibody; and an instruction leaflet.

[00030] The method of the present invention includes a gold nanoparticle labeled anti-analyte antibody. In accordance with one of the preferred embodiments the anti-analyte antibody is anti-NGAL antibody. This labeled antibody is adsorbed on conjugate pad. As soon as the sample is applied to the sample application pad, the labeled antibody captures the analyte present in the sample. This complex moves due to capillary action of the nitrocellulose membrane and reaches the test line. At the test line, another monoclonal/polyclonal anti-analyte antibody is immobilized which is a primary antibody against the analyte. This immobilized antibody captures the analyte- antibody complex. After a certain amount of complex has been captured, the threshold of the labeled antibody is crossed making a visual line (pink in color) to appear. The time taken by this pink line or test line to appear represents a certain concentration range of analyte such as NGAL.

[00031] In one embodiment of the present invention the method comprises adding at least one drop of a urine sample to be analyzed onto a sample application pad; and observing the appearance of the control line and the test line.

[00032] In another embodiment of the present invention the method comprises the following steps:

• adding at least one drop of a urine sample to be analyzed onto a sample application pad;

• observing the time at which the control line and the test line appears respectively; and

• calculating the difference between the time of appearance of the test and control lines,

wherein a predetermined value corresponds to a particular concentration range of analyte in urine sample, which is being used to interpret the renal condition. [00033] In one illustrative embodiment, the concentration of NGAL in the sample (urine) is determined using the graph of time Ys. NGAL concentration shown in Figure 2. The time in which a test line appears could help in concluding the NGAL concentration and in turn the patient’s condition.

[00034] In another embodiment, the method involves Time Of intensity Matching (TOM) technique. This technique is used to interpret results on the device. The device of plastic cassette has guiding lines (pink coloured lines) present on the either side of the test area where the test line appears (Figure 1). After addition of the sample, the time at which the intensity of test line exactly matches the intensity of color of guiding lines is called time of intensity matching or TOM. TOM is then used to interpret the concentration of NGAL in the patient’s sample using an algorithm. In one embodiment, the method comprises the following steps:

- observing the color intensity of the test line appears in comparison with the color intensity of guiding lines present on the device; and

- determining the time required for matching the color intensity of the test line the color intensity of guiding lines,

wherein the time is used to interpret the concentration of analyte in the sample. [00035] In one embodiment, the colour intensity is observed manually or using an electronic device or software or mobile application or any recording device. In illustrative embodiments, two types of assays are carried out, semi- quantitative and quantitative. Semi-quantitative works on the feature called TOM(Y), wherein Y represents visual method . On the other hand, quantitative assay uses the feature TOM(E), where E stands for any electronic device or software or mobile application or any recording device.

[00036] Working of device, quantitative by TOM (E) and semi-quantitative by TOM(V) is provided herein below:

1. Start video mode in a mobile phone or mobile application or software in mobile phone any recording device.

2. Place the mobile on a stand such that the distance between mobile and the kit/device is enough for good resolved video or image to capture. -10 cm.

3. Bring the kit to room temperature. Cut open the pouch, take out a dropper and the kit.

4. Place kit below and focus such that the result window and the pink guiding lines can be viewed very clearly.

5. Bring the sample to room temperature. Add 50 pl or 2 drops of urine or calibrator to the sample well and immediately start recording video for around 5-10 minutes or else record till the application gives the TOM(E) The analysis can be performed through naked eyes as well as using the video recording using a camera / or application from a mobile phone or software or any other recording device.

Analysing the result:

a. After addition of the sample, a test line will develop after sometime between the two pink guiding lines in the test area and another line will develop 0.5 cm above the test line that is called the control line.

b. For quantitative analysis one has to note the time TOM(E) at which test line intensity matches exactly with the color intensity of the guiding lines and a continuous straight pink line is formed by the guiding lines and the test line. For semi-quantitative analysis, same analysis is to be done using naked eye. Quantitative assay gives exact concentration of NGAL in sample, while semi-quantitative assay gives a range for NGAL concentration.

The time of intensity matching noted by naked eye is called TOM(Y) and time of intensity matching using an electronic device or software or mobile application or any recording device TOM(E). Standardized TOM(E) and TOM(Y) for different NGAL calibrators are shown in Table

1. 9. Table 1: Standardized TOM(E) and TOM(Y) for different NGAL calibrator concentrations, with 15% ± variation and should give RSQ value greater than 0.95.

The Logarithmic relation between Time versus NGAL concentration using TOM(E) and TOM(Y) is shown in figure 2

[00037] The invention is now illustrated with the help of non-limiting examples. The examples provided herein below are for illustration purpose and should not be construed as limitation to scope of the present invention.

[00038] Example 1: [00039] A gold colloidal Lateral flow assay with two test lines (multi-test line assay) to distinguish between Acute Kidney Injury and Chronic Kidney Disease was performed using Neutrophils Gelatinase Associated Lipocalin (NGAL) as an analyte. Test materials:

Antibody (A): of 0.5-2.0 mg/ml or 1-20 pg/ml concentration

Antibody (D): of 1-20 pg/ml or 0.5-2.0 mg/ml concentration

Controls/calibrators: 100 to 700 ng/ml of NGAL

The kit mainly comprises of cassettes, sample application pad, conjugate pad, nitrocellulose membrane and adsorbent pad. Process:

A kit and urine sample were brought to room temperature. The kit was opened to take out Gold colloidal LFA cassette. At least one drop of urine sample was added into the sample port of cassette and the appearance of control line and the test line was observed.

Interpretation of Results:

If only control line appears then the patient’ s kidney is functioning normally.

If test line 2 appears along with control line, then the patient is likely to have NGAL concentration just above the normal levels which can be a case of chronic kidney disease.

If test line 1 and 2 both appear along with control line, then the patient is likely to have concentration very high from the normal which is generally found the acute kidney patients.

[00040] Example 2:

A gold colloidal Lateral flow assay to semi quantitatively estimate the concentration of NGAL in urine for early estimation of renal injury was performed. Test materials:

Antibody (A): of 0.5-2.0 mg/ml or 1-20 pg/ml concentration

Antibody (D): of 1-20 pg/ml or 0.5-2.0 mg/ml concentration

Controls/Calibrators: 100 to 700 ng/ml of NGAL

The kit mainly comprises of cassettes, sample application pad, conjugate pad, nitrocellulose membrane and adsorbent pad.

Process:

A kit and urine sample were brought to room temperature. The kit was opened to take out Gold colloidal LFA cassette. At least one drop of urine sample was added into the sample port of cassette and the time required for the appearance of control line and the test line was noted. Interpretation of results:

- The Control line generally takes around 30 seconds to develop.

- If the time difference between control and test line to appear is 45-50 seconds then the concentration of NGAL is at higher side and the patient is likely to be suffering from AKI.

- If the time difference between control and test line to appear is 75-140 seconds then the concentration of NGAL above the normal range and patient is likely to be suffering from CKD.

- If the time difference between control and test line to appear is 150-183 seconds, then the concentration of NGAL is normal The patient’s kidneys are functioning normally.

[00041] Example 3:

A gold colloidal Lateral flow assay to semi quantitatively or quantitatively estimate the concentration of NGAL in urine to know the concentration of NGAL in sample was performed.

Test materials:

Antibody (A): of 0.5-2.0 mg/ml or 1-20 pg/ml concentration Antibody (D): of 1-20 pg/ml or 0.5-2.0 mg/ml concentration Controls/Calibrators: 100 to 700 ng/ml of NGAL

The kit mainly comprises of plastic cassettes with guiding lines printed on either side of cassette along the side of test line, sample application pad, conjugate pad, nitrocellulose membrane and adsorbent pad.

Process:

A kit and urine sample were brought to room temperature. The kit was opened to take out Gold colloidal LFA cassette. At least one drop of urine sample was added into the sample port of cassette and the time of intensity matching observed by naked eye is called TOM(Y) and time of intensity matching using an electronic device or software or mobile application or any other recording device called TOM(E) was noted.

Interpretation of results:

The TOM(E) or TOM(Y) was then put into algorithm which will be provided along with device to calculate the exact concentration of NGAL in the urine sample. The higher concentration of NGAL shows patient is likely to be suffering from renal injury. [00042] Advantages of the present device/kit/assay method:

The device is a simple, point-of-care or on-site gold colloidal- based LFA kit that does not require any specific make reader/analyzer or any specialized equipment(s) for its working. The device use does not demand any special training/skills.

The assay method is semi-quantitative in nature, and is time-based and/or multi-test line-based which will be able to diagnose renal injury at early stages. The device/method gives results within 2-10 minutes.

The device is cost effective, thereby can be affordable to poor and lower middle-class population.

The present LFA uses a non-invasive sample type (urine) for testing.

[00043] The use of the expression“at least” or“at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.

[00044] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[00045] The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary. [00046] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims

CLAIMS:

1. A method of determining the concentration of an analyte; the method comprises detecting the concentration of the analyte using at least one technique selected from the group consisting of time of intensity matching (TOM) and time of appearance (TO A), gold nanoparticle labeled anti-analyte antibody, immobilized test line antibody, and a device comprising a cassette a conjugate pad, sample application pad, and absorbent pad,

wherein the time of intensity matching (TOM) comprises determining the time at which colour and intensity of test line matches to that of guiding lines present on either side of a cassette;

wherein the time of appearance (TOA) comprises determining the time of appearance of test and control lines in the device.

2. The method as claimed in claim 1, wherein the gold-conjugated anti- analyte antibody is anti-NGAL antibody.

3. The method as claimed in claims 1 to 2, wherein the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein the immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, wherein the time taken by the line to appear represents a concentration range of analyte.

4. The method as claimed in claims 1 to 2, wherein the method comprises adsorbing labeled antibody on the conjugate pad and applying a sample to the sample application pad, wherein the labeled antibody captures the analyte present in the sample and forms a complex which moves due to capillary action of the nitrocellulose membrane and reaches a test line, wherein the immobilized monoclonal/polyclonal anti-analyte antibody present at the test line captures the analyte-antibody complex and develops a visual line, and observing the color and intensity of the test line appears in comparison with the color and intensity of the guiding lines present on the cassette of device, wherein the time required for matching the color and intensity of the test line to the color and intensity of the guiding lines represents a concentration range of analyte.

5. The method as claimed in claims 1 to 4, wherein the analyte is NGAL.

6. The method as claimed in claims 1 to 5, wherein the sample is a urine sample.

7. The method as claimed in claims 1 to 6, wherein the method comprises adding at least one drop of a urine sample to be analyzed onto the sample application pad; and observing the appearance of a control line and a test line.

8. The method as claimed in claims 1 to 7, wherein the presence of only control line / appearance of test line 1 or 2 along with the control line is being utilized to determine whether the patient’s kidney is functioning normally or the patient is likely to be suffering from chronic kidney disease or acute kidney injury.

9. The method as claimed in claim 1, wherein the method comprises the following steps:

- calculating the difference between the time of appearance of the test and control lines, wherein a predetermined value corresponds to a particular concentration range of analyte in the urine sample, which is being used to interpret the renal condition.

10. The method as claimed in claim 1, wherein the method comprises the following steps:

- observing the color and intensity of the test line appears in comparison with the color and intensity of the guiding lines present on the cassette of device; and

- determining the time required for matching the color and intensity of the test line to the color and intensity of the guiding lines, wherein the time is used to interpret the concentration of analyte in the sample.

11. The method as claimed in claim 10, wherein the color intensity is observed manually (TOM(V)) or using an electronic device or a software or a mobile app or any other recording device (TOM(E)).

12. The method as claimed in claim 9 and 11, wherein the observed TOM(E) or TOM(Y) is used in an algorithm to calculate the concentration of analyte in the sample.

13. A kit for determining the concentration of an analyte; said kit comprising: a) a device comprising a sample application pad, a conjugate pad, a nitrocellulose membrane, an adsorbent pad and a backing card with guiding lines; b) gold nanoparticle labeled anti-analyte antibody; c) immobilized test line antibody; and an instruction leaflet.