FERRITIN ANALYSIS VIA LATERAL FLOW IMMUNOASSAY

Abstract

A lateral flow immunoassay (LFIA) strip comprises a sample pad (2) for receiving a sample and at least one conjugate pad (3) in operable contact with the sample pad. The at least one conjugate pad is loaded with a first antibody specific against ferritin and tagged with detectable label. A flow-through assay device for detecting ferritin levels in a sample is also provided comprising a sample pad (10) for receiving a sample and at least one conjugate pad (11) in operable contact with the sample pad. The at least one conjugate pad is loaded with a first antibody specific against ferritin and tagged with detectable label. An assay platform (12) is in operable contact with the at least one conjugate pad. The assay platform comprises a substrate having a test region onto which the first test antibody to the ferritin is immobilized. A method for measuring the level of ferritin in a sample is also provided. The sample may be a blood or saliva sample.

Claims

1. A lateral flow immunoassay (LFIA) strip, comprising: a sample pad for receiving a sample; at least one conjugate pad in operable contact with the sample pad, the at least one conjugate pad loaded with a first antibody specific against ferritin and tagged with detectable label; wherein the conjugate pad optionally contains a dried format of bioactive molecules in a salt sugar-matrix; an assay platform in operable contact with the at least one conjugate pad, the assay platform comprising a substrate having a test region onto which the first test antibody to ferritin is immobilized and optionally a control region onto which a control antibody directed to the first antibody is immobilized; and optionally, an absorbent pad in operable contact with the assay platform.

2. A flow-through assay device for detecting ferritin levels in a sample, the device comprising: a sample pad for receiving a sample; at least one conjugate pad in operable contact with the sample pad, wherein the at least one conjugate pad is loaded with a first antibody specific against ferritin and tagged with detectable label; an assay platform in operable contact with the at least one conjugate pad, the assay platform comprising a substrate having a test region onto which the first test antibody to the ferritin is immobilized and optionally a control region onto which a control antibody directed to the first antibody is immobilized; optionally, an absorbent pad in operable contact with the assay platform.

3. A method for measuring the level of ferritin in a sample, said method comprising: i) providing a flow-through assay device that comprises a sample pad for receiving a sample, at least one conjugate pad in operable contact with the sample pad, the at least one conjugate pad loaded with a first antibody specific against the ferritin and tagged with detectable label, an assay platform in operable contact with the at least one conjugate pad, the assay platform comprising a substrate having a test region on to which the first test antibody to the ferritin is immobilized and optionally a control region onto which a control antibody directed to the first antibody is immobilized; and optionally, an absorbent pad in operable contact with the assay platform; (ii) contacting the conjugate pad with the sample and allowing the particles to flow to the test region, iii) using a mobile test reader to scan the results and detect and quantify the level of ferritin; and sending the results to a database.

4. A method for measuring the level of ferritin in a sample comprising; i) providing a lateral flow immunoassay strip or device according to the invention; ii) applying a sample to the sample pad, preferably a saliva sample; iii) determining the intensity of labelled antibody bound to the test region/line; iv) determining from the intensity of the label in iii) the level of ferritin in the sample.

5. A method according to claim 3, wherein the sample is a blood or saliva sample.

6. A method according to claim 3, wherein the sample is a saliva sample.

7. (canceled)

8. A method of monitoring and/or diagnosing an iron disorder in a subject, the method comprising; i) providing a sample of saliva from the subject; ii) performing the method of claim 4; iii) determining from the level of ferritin observed in the sample if the subject has an iron disorder or monitoring the progression of a subject with an iron disorder.

9. The method of claim 8 wherein in step iii) the level of ferritin observed is compared to the level observed in a previous sample from the same subject.

10. The method of claim 8 wherein the iron disorder is hemochromatosis, anaemia or an iron overload.

11. A lateral flow immunoassay strip according to claim 1, wherein the sample is a blood or saliva sample.

12. A kit for determining the level of ferritin in a sample, the kit comprising a lateral flow immunoassay strip according to claim 1, instructions to apply a sample of saliva to the sample pad and instructions to determine the level of ferritin from the intensity of label observed on the test line.

13. A flow-through assay device according to claim 2, wherein the sample is a blood or saliva sample.

14. A kit for determining the level of ferritin in a sample, the kit comprising a flow-through assay device according to claim 2, instructions to apply a sample of saliva to the sample pad and instructions to determine the level of ferritin from the intensity of label observed on the test line.

15. A method according to claim 3, wherein the sample is a blood or saliva sample.

Description

[0071] Embodiments of the present invention will now be further described, purely by way of example, with reference to the accompanying drawings, in which

[0072] FIG. 1 shows a schematic diagram of a test strip based on lateral flow immunoassay according to the invention; and

[0073] FIG. 2 shows a schematic diagram of a flow-through assay device according to the invention for detecting ferritin levels in a sample.

[0074] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purpose of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention in anyway.

[0075] The method of the present disclosure uses lateral-flow assay to provide a sensitive tool for detecting ferritin in a sample.

[0076] The sample 1 containing the analyte (ferritin) is applied to the sample pad 2.

[0077] From the sample pad 2, the sample travels to the conjugate pad 3, where the sample mixes with a first antibody specific against ferritin to form analyte complexes. Because the conjugate pad 3 is in fluid communication with the membrane 4, the analyte complexes then migrate from the conjugate pad 4 to the test line (region) 5 and control line (region) 6.

[0078] The first test antibody to the ferritin is immobilized at the test line (region) 5 and optionally the control antibody directed to the first antibody is immobilized at the control line (region) 6.

[0079] The sample then optionally enters the wicking pad 7 which acts as a waste container. The flow-through assay device containing the sample pad 10 receives the sample containing the analyte.

[0080] From the sample pad 10, the sample travels to the conjugate pad 11 which is in operable contact with the sample pad 10. The conjugate pad 11 is loaded with a first antibody specific against the ferritin and tagged with a detectable label. The assay platform 12 is in operable contact with the conjugate pad 11 and the assay platform 12 comprises a substrate having a test line (region) 13 onto which the first test antibody to the ferritin is immobilized and optionally a control line (region) 14 on to which a control antibody directed to the first antibody is immobilized.

[0081] Wherein a number of embodiments of the present invention have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skill in the art without materially departing from the invention herein.

[0082] To summarise, the invention provides a lateral flow device for detecting the level of ferritin in a sample. Preferably the sample is saliva.

[0083] In an embodiment the invention provides a lateral flow device comprising a sample pad 2, a conjugate pad 3, a test line 5, a control line 6 and optionally a wicking pad 6. The sample pad 2, onto which a sample to be tested is placed, is in fluid communication with the conjugate pad 3. The conjugate pad contains first antibodies to ferritin, the antibodies are free in the conjugate pad, that is they are not bound to the device. The first antibodies are labelled, for example with gold nanoparticles. The conjugate pad 3 is in fluid communication with the test line 5 and/or the control line 6. The test line 5 comprises antibodies bound to the lateral flow device which are directed to ferritin bound to the first antibodies. The antibodies bound to the test line may be the same as the first antibodies without the label. The test line is in fluid communication with the conjugate pad and/or the control line. The control line 6 comprises antibodies bound to the lateral flow device which are directed to the first antibody, preferably to a part of the first antibody that is masked if the first antibody is bound to ferritin. The control line is in fluid communication with the test line and/or the conjugate pad. The test line and control line may be referred to together as the assay platform. The wicking pad 6 is configured to absorb any sample that is not bound to the test or control line, and is in fluid communication with the control line and/or the test line.

[0084] In use a sample, preferably a saliva sample is placed on the sample pad 2 on the lateral flow device of the invention. The sample then moves through the device to the conjugate pad 3 wherein ferritin in the sample is bound by the first antibodies. The sample and antibodies from the conjugate pad then move to the test and/or control line. Ferritin bound to the first antibodies bind to antibodies in the test line and the first antibodies are immobilised at the test line. Unbound first antibodies (that is, not bound to ferritin) bind to antibodies in the control line and are immobilised there. Antibodies immobilised to the control and test line are visualised using the label on the first antibodies. Binding to the control line shows the lateral device is working. Binding to the test line shows the presence, and preferably the level, of ferritin in the sample. Any unbound sample may move through the device to the wicking pad. The sample moves through the device by wicking.

[0085] Aspects/embodiments of the invention may be implemented in a hand-held point of care (POC) device. The device may comprise a casing to support the lateral flow assay and analyte. This can be converted to a quantatative reading of ferratin, e.g. when combined with using an electronic or mobile device. Once the saliva/blood is placed on the analyte and passes through the lateral flow assay, the ferratin level can be converted to a reading, e.g. by a processor, which is viewable via a mobile device.

[0086] The advantages of the invention include the following: there is currently no hand-held point of care (POC) device to establish ferratin levels outside of a laboratory environment, and as such no method for a Hemochromatosis patient and/or iron disorder patient such as; anaemia or iron overload to monitor their own ferratin levels. The provision of a POC device will allow a patient with an iron disorder to monitor their own ferratin levels, allowing them to either; donate to a local blood bank and/or adjust their food intake to maintain a healthy level. In addition to this; removal of the patient from the norms of the NHS protocols will free up valuable time to be utilised elsewhere, notwithstanding the savings to the NHS in time, equipment; together with the cost to the patient, parking, time off work, and the employer to assist with their daily homecare outside of the scope of the NHS care system. Further, enhancing the quality of life for the patient, assisting GPs with a ferratin result reading in a timely manner (e.g. within about 3 mins), without the need for routine blood tests and laboratory testing.