Method for detection of CD16b
The invention provides methods of determining the neutrophil level in a sample by determining the level of CD16b, including the intracellular form of CD16b in a sample, such as a blood sample. The methods may further comprise the determination of lactate levels Also provided are associated diagnostic and therapeutic methods. Further provided are kits and devices for performing the methods, particularly lateral flow kits and devices.
1 . A method of determining a neutrophil level in a sample from a subject, the method comprising:
determining the level of CD16b in the sample, wherein the CD16b comprises the intracellular form of CD16b, wherein
(i) the method further comprises a step of removing at least a proportion, most, or all of the soluble form of CD16b from the sample prior to determining the level of CD16b; or
(ii) the method is performed on a sample from the subject from which at least a proportion, most, or all of the soluble form of CD16b has previously been removed, and
wherein the step of removing at least a proportion, most, or all of the soluble form of CD16b is a filtration step, wherein the filtration step yields:
(a) a filtered sample comprising white blood cells, wherein the filtered sample is depleted of the soluble components of the sample; and
(b) a solution comprising the soluble form of CD16b, and
wherein determining the level of CD16b in the sample comprises assaying the filtered sample comprising white blood cells for CD16b.
2 . The method according to claim 1 , wherein the CD16b comprises, consists essentially of, or consists of the membrane-anchored form of CD16b and the intracellular form of CD16b.
3 . The method of claim 1 , wherein the step of removing at least a proportion of the soluble form of CD16b from the sample is carried out under conditions that keep at least a large proportion, or substantially all of the neutrophils present in the sample intact.
4 . The method according to claim 1 , wherein the level of CD16b is determined using one or more CD16b-binding antibodies.
5 . The method according to claim 1 , wherein the sample is from a subject who (i) has been exposed to radiation; (ii) has received or is receiving a drug capable of causing neutropenia; (iii) is suffering from human immunodeficiency virus (HIV), hepatitis and/or an autoimmune disorder; (iv) has cancer and/or has received or is receiving chemotherapy and/or radiotherapy; (v) is displaying or experiencing symptoms of an infection; or (vi) has recently undergone surgery.
6 . The method according to claim 1 , wherein the method comprises a step of cell lysis prior to determining the level of CD16b in the sample, wherein the step of cell lysis is carried out:
(i) after a step of removing at least a proportion of the soluble CD16b; or
(ii) on a sample from which at least a proportion of the soluble CD16b has previously been removed.
7 . The method of claim 1 , wherein the level of CD16b in the sample is determined via a lateral flow assay.
8 . The method according to claim 1 , wherein the method further comprises detecting one or more different markers indicative of the presence of sepsis in the sample.
9 . The method according to claim 1 , wherein at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more samples are taken from the subject at different times and the levels of CD16b and optionally one or more further markers are determined and wherein the samples are taken every 6 to 24 hours, daily, or every 2, 3, 4, 5, 6, 7 or 14 days.
10 . A method of selecting a subject for treatment with an antibiotic comprising performing the method of claim 1 and selecting the subject for treatment on the basis of the determined neutrophil level.
11 . The method according to claim 1 , comprising the steps of:
i. adding the sample to a first sample application zone of a first test strip to trap any neutrophils on a filter comprised within the first test strip;
ii. transferring the filter with trapped neutrophils to a second sample application zone of a second test strip;
iii. adding to the filter a lysis reagent to lyse the neutrophils; and
iv. determining the level of CD16b.
12 . The method of claim 11 , wherein the step of determining the level of CD16b comprises:
i. contacting a solution of the lysed neutrophils with a conjugate zone comprising at least one labelled detection moiety which binds to CD16b;
ii. contacting the mixture of the lysed neutrophils and the at least one labelled detection moiety which binds to CD16b with:
(i) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that also binds to CD16b thereby immobilising the CD16b at the test line to produce a signal via the labelled detection moiety also bound to the CD16b; or
(ii) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that binds to the CD16b-bound labelled detection moiety of step ii. thereby immobilising it at the test line producing a signal.
13 . The method of claim 12 wherein the labelled detection moiety which binds to CD16b is an anti-CD16b antibody conjugated to a gold particle.
14 . The method of claim 12 , wherein the further detection moiety is an anti-CD16b antibody.
15 . The method of claim 11 , wherein the lysis reagent is a surfactant, which may be ionic or non-ionic.
16 . The method of claim 11 , wherein the step of adding the sample to the first application zone is followed by a step of cell wash.
17 . The method of claim 11 , wherein the filter is a membrane suitable for trapping neutrophils.