Method for the qualitative and quantitative detection of alginate oligomers in body fluids

Abstract

Qualitative and quantitative methods are for the detection of alginate oligomers in body fluids based on analyzing the Fourier transform infrared spectroscopy (FTIR) spectrum of a body fluid sample, for example a sputum sample, at a specific wave number range and, more particularly, certain specific characteristic wavenumbers.

Claims

1. A method for determining the concentration of alginate oligomer in a sample of body fluid, said method comprising (i) obtaining for the sample of body fluid one or more normalized IR absorbance values, one or more normalized IR transmittance values, one or more second derivative IR absorbance values, or one or more second derivative IR transmittance values, at one or more wavenumbers selected from one or more of the following wavenumber ranges: (a) 1596 cm.sup.−1 to 1606 cm.sup.−1, (b) 1407 cm.sup.−1 to 1417 cm.sup.−1, (c) 1120 cm.sup.−1 to 1130 cm.sup.−1, (d) 1081 cm.sup.−1 to 1091 cm.sup.−1, (e) 1023 cm.sup.−1 to 1033 cm.sup.−1, and (f) 943 cm.sup.−1 to 953 cm.sup.−1; (ii) for one or more of the wavenumbers selected in step (i) providing corresponding IR absorbance values, IR transmittance values, second derivative IR absorbance values, or second derivative IR transmittance values, for a plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer, and (iii) determining the concentration of the alginate oligomer in the sample of body fluid by determining the relative position of the values obtained in step (i) amongst the values provided in step (ii).

2. The method of claim 1, wherein: (i) the wavenumber range of (a) is 1597 cm.sup.−1 to 1605 cm.sup.−1, (ii) the wavenumber range of (b) is 1408 cm.sup.−1 to 1416 cm.sup.−1, (iii) the wavenumber range of (c) is 1121 cm.sup.−1 to 1129 cm.sup.−1, (iv) the wavenumber range of (d) is 1082 cm.sup.−1 to 1090 cm.sup.−1, (v) the wavenumber range of (e) is 1024 cm.sup.−1 to 1032 cm.sup.−1, and/or (vi) the wavenumber range of (f) is 944 cm.sup.−1 to 952 cm.sup.−1.

3. The method of claim 1, wherein the corresponding IR absorbance values, IR transmittance values, second derivative IR absorbance values, or second derivative IR transmittance values for a plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are conveniently arranged in a tabulated form, or as a data matrix, or as Cartesian coordinates in a two dimensional Cartesian coordinate system, in which the various alginate oligomer concentrations of each of said plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are arranged in one dimension.

4. The method of claim 3 wherein the corresponding IR absorbance values, IR transmittance values, second derivative IR absorbance values, or second derivative IR transmittance values for a plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are provided as a standard curve in which the alginate oligomer concentrations of each of said plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are arranged on one axis.

5. The method of claim 1, wherein one or more of the IR absorbance, IR transmittance values, second derivative IR absorbance values or second derivative IR transmittance values are provided as part of a continuous IR spectrum, or portions thereof, covering one or more of said wavenumber ranges.

6. The method of claim 1, wherein the sample is selected from the group consisting of blood, cerebrospinal fluid, faeces, gastric juice, lymph, mucus, plasma, pus, saliva, serum, semen, sweat, tears, vaginal secretion, vomit, urine and wound exudate and sputum.

7. The method of claim 1, wherein the IR absorbance or IR transmittance values or spectra are prepared by a Fourier transform infrared (FTIR) spectrometer.

8. The method of claim 1, wherein the normalized IR absorbance or IR transmittance values or spectra are normalized by Min-Max or vector normalization.

9. The method of claim 8, wherein normalization is based on absorbance or transmittance at the Amide I wavenumber.

10. The method of claim 1, wherein the sample is from a subject with an infection, bacteraemia, sepsis, septic shock; a burn, an acute wound, a chronic wound, reduced or abrogated epithelial or endothelial secretion or secretion clearance, COPD, COAD, COAP, bronchitis, cystic fibrosis, CFTR gene mutation heterozygosity, emphysema, bronchiectasis, lung cancer, asthma, pneumonia or sinusitis, a subject fitted with a medical device, a subject in need of anticoagulation therapy, or a smoker.

11. The method of claim 1, wherein the alginate oligomer has: (i) an average molecular weight of less than 35,000 Daltons; or (ii) a degree of polymerization (DP), or a number average degree of polymerization (DPn), of 4 to 100.

12. The method of claim 1, wherein the alginate oligomer has: (i) at least 70% G residues, (ii) a number average degree of polymerization in the range 5 to 20, a guluronate fraction (FG) of at least 0.85 and a mannuronate fraction (FM) of no more than 0.15; or (iii) at least 70% M residues.

13. An infrared spectrometer configured to perform a method as claimed in claim 1.

14. A method for determining the concentration of alginate oligomer in a sample of body fluid, said method comprising (a)(i) obtaining a first IR spectrum for the sample of body fluid, wherein the first IR spectrum for the sample of body fluid is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (a)(ii) providing a first IR spectrum for the alginate oligomer, wherein the first IR spectrum for the alginate oligomer is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first spectrum for the sample of body fluid is an absorbance spectrum, or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first IR spectrum for the sample of body fluid is a transmittance spectrum, (a)(iii) performing a non-parametric correlation analysis on the first IR spectrum for the sample of body fluid and the first IR spectrum for the alginate oligomer thereby producing a first correlation coefficient, (a)(iv) providing corresponding correlation coefficients for a plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer, and (a)(v) determining the concentration of the alginate oligomer in the sample of body fluid by determining the relative position of the first correlation coefficient obtained in step (a)(iii) amongst the correlation coefficients of step (a)(iv); or (b)(i) obtaining a second IR spectrum for the sample of body fluid, wherein the second IR spectrum for the sample of body fluid is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (b)(ii) providing a second IR spectrum for the alginate oligomer, wherein the second IR spectrum for the alginate oligomer is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is an absorbance spectrum, or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is a transmittance spectrum, (b)(iii) performing a non-parametric correlation analysis on the second IR spectrum for the sample of body fluid and the second IR spectrum for the alginate oligomer thereby producing a second correlation coefficient, (b)(iv) providing corresponding correlation coefficients for a plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer, and (b)(v) determining the concentration of the alginate oligomer in the sample of body fluid by determining the relative position of the second correlation coefficient obtained in step (b)(iii) amongst the correlation coefficients of step (b)(iv).

15. The method of claim 14, wherein the corresponding correlation coefficients of step (a)(iv) or (b)(iv) are arranged in a tabulated form, as a data matrix, or as Cartesian coordinates in a two dimensional Cartesian coordinate system, in which the alginate oligomer concentrations of each of said plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are arranged in one dimension.

16. The method of claim 15 wherein the corresponding correlation coefficients of step (a)(iv) or (b)(iv) are provided as a standard curve in which the alginate oligomer concentrations of each of said plurality of samples of said body fluid which contain a range of known amounts of the alginate oligomer are arranged on one axis.

17. The method of claim 14, wherein the non-parametric correlation analysis is Spearman's Rank, Kendall's Tau and/or point biserial correlation analysis.

18. A method for determining the concentration of alginate oligomer in a sample of body fluid, said method comprising (i) obtaining an first IR spectrum for the sample of body fluid, wherein the first IR spectrum for the sample of body fluid is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (ii) obtaining a second IR spectrum for the sample of body fluid, wherein the second IR spectrum for the sample of body fluid is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (iii) providing a first IR spectrum for the alginate oligomer, wherein the first IR spectrum for the alginate oligomer is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first IR spectrum for the sample of body fluid is an absorbance spectrum, or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first IR spectrum for the sample of body fluid is a transmittance spectrum, (iv) providing a second IR spectrum for the alginate oligomer, wherein the second IR spectrum for the alginate oligomer is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second IR spectrum for the sample of body fluid is an IR absorbance spectrum, or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second IR spectrum for the sample of body fluid is an IR transmittance spectrum, (v) performing a non-parametric correlation analysis on the first IR spectrum for the sample of body fluid and the first IR spectrum for the alginate oligomer thereby producing a first correlation coefficient, (vi) performing a non-parametric correlation analysis on the second IR spectrum for the sample of body fluid and the second IR spectrum for the alginate oligomer thereby producing a second correlation coefficient, (vii) providing corresponding first correlation coefficients and second correlation coefficients for a plurality of samples of said body fluid which contain a range of known concentrations of the alginate oligomer as Cartesian coordinates in a two or three dimensional Cartesian coordinate system, and (viii) determining the concentration of the alginate oligomer in the sample of body fluid by determining the relative position of a Cartesian coordinate comprising the first and second correlation coefficients obtained in steps (v) and (vi) in the Cartesian coordinate system of step (vii).

19. A method for determining the presence or absence of an alginate oligomer in a sample of body fluid, said method comprising (i) obtaining an first IR spectrum for the sample of body fluid, wherein the first IR spectrum for the sample of body fluid is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (ii) obtaining a second IR spectrum for the sample of body fluid, wherein the second IR spectrum for the sample of body fluid is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (iii) providing a first IR spectrum for the alginate oligomer, wherein the first IR spectrum for the alginate oligomer is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first spectrum for the sample of body fluid is an absorbance spectrum, or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first IR spectrum for the sample of body fluid is a transmittance spectrum, (iv) providing a second IR spectrum for the alginate oligomer, wherein the second IR spectrum for the alginate oligomer is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is an absorbance spectrum, or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is a transmittance spectrum, (v) performing a non-parametric correlation analysis on the first IR spectrum for the sample of body fluid and the first IR spectrum for the alginate oligomer thereby producing a first correlation coefficient, (vi) performing a non-parametric correlation analysis on the second IR spectrum for the sample of body fluid and the second IR spectrum for the alginate oligomer thereby producing a second correlation coefficient, (vii) providing corresponding first correlation coefficients and second correlation coefficients for a plurality of samples of said body fluid which contain an alginate oligomer and corresponding first correlation coefficients and second correlation coefficients for a plurality of samples of said body fluid which do not contain an alginate oligomer as Cartesian coordinates in a two or three dimensional Cartesian coordinate system, and (viii) determining the presence or absence of an alginate oligomer in the sample of body fluid by determining the relative position of a Cartesian coordinate comprising the first and second correlation coefficients obtained in steps (v) and (vi) in the Cartesian coordinate system of step (vii), wherein (a) collocation of said Cartesian coordinate comprising the first and second correlation coefficients obtained in steps (v) and (vi) with the Cartesian coordinates of the samples of said body fluid which do not contain an alginate oligomer is indicative that the sample does not contain an alginate oligomer, and (b) collocation of said Cartesian coordinate comprising the first and second correlation coefficients obtained in steps (v) and (vi) with the Cartesian coordinates of the samples of said body fluid which do contain an alginate oligomer is indicative that the sample does contain an alginate oligomer.

20. The method for determining the presence or absence of an alginate oligomer in a sample of body fluid of claim 19, said method comprising: (i) obtaining an first IR spectrum for the sample of body fluid, wherein the first IR spectrum for the sample of body fluid is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (ii) obtaining a second IR spectrum for the sample of body fluid, wherein the second IR spectrum for the sample of body fluid is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (iii) providing a first IR spectrum for the alginate oligomer, wherein the first IR spectrum for the alginate oligomer is a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first spectrum for the sample of body fluid is an absorbance spectrum, or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the first IR spectrum for the sample of body fluid is a transmittance spectrum, (iv) providing a second IR spectrum for the alginate oligomer, wherein the second IR spectrum for the alginate oligomer is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is an IR absorbance spectrum, or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the second spectrum for the sample of body fluid is a transmittance spectrum, (v) performing a non-parametric correlation analysis on the first IR spectrum for the sample of body fluid and the first IR spectrum for the alginate oligomer thereby producing a first correlation coefficient, (vi) performing a non-parametric correlation analysis on the second IR spectrum for the sample of body fluid and the second IR spectrum for the alginate oligomer thereby producing a second correlation coefficient, (vii) inputting the first correlation coefficient of step (v) into the linear prediction model Formula I
Y=nX+m  Formula I as X and determining a value for Y, (viii) wherein a value for Y which is equal to or less than the second correlation coefficient of step (vi) is an indication that the alginate oligomer is present in the sample of body fluid, and a value for Y which is greater than the second correlation coefficient of step (vi) is an indication that the alginate oligomer is absent from the sample of body fluid, and wherein the linear regression model and has been prepared by (a) obtaining one or more normalized IR absorbance spectra or one or more normalized IR transmittance spectra from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for a plurality of samples of said body fluid which contain the alginate oligomer and a plurality of samples of said body fluid which do not contain the alginate oligomer, (b) providing a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for the alginate oligomer when the spectra of step (a) are IR absorbance spectra, or a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for the alginate oligomer when the spectra of step (a) are IR transmittance spectra, (c) performing a non-parametric correlation analysis on the IR spectra of step (a) and the IR spectrum for the alginate oligomer of step (b) thereby producing at least one correlation coefficient for each of the samples of step (a); and (d) obtaining the second derivatives of one or more IR absorbance spectra or the second derivatives of one or more IR transmittance spectra from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for the plurality of samples of said body fluid which contain the alginate oligomer and the plurality of samples of said body fluid which do not contain the alginate oligomer of step (a), (e) providing the second derivative of a normalized IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for the alginate oligomer when the spectra of step (d) are the second derivatives of IR absorbance spectra, or the second derivative of a normalized IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 for the alginate oligomer when the spectra of step (d) are the second derivatives of IR transmittance spectra, (f) performing a non-parametric correlation analysis on the second derivatives of the IR spectra of step (d) and the second derivative of the IR spectrum for the alginate oligomer of step (e) thereby producing at least one further correlation coefficient for each of the samples of step (a); and (g) for each of the plurality of samples of step (a) plotting the further correlation coefficient produced in step (f) and the correlation coefficient produced in step (c) as a Cartesian coordinates in a two dimensional Cartesian coordinate system, and (h) determining the optimum separation of the Cartesian coordinates of the plurality of samples of said body fluid which contain the alginate oligomer and the Cartesian coordinates of the plurality of samples of said body fluid which do not contain the alginate oligomer to yield a separation threshold defined by the linear regression model Y=nX+m, wherein n and m are numerical constants, and X is a correlation coefficient of step (c) for a sample of step (a).

21. A method for determining the presence or absence of an alginate oligomer in a sample of body fluid, said method comprising: (i) obtaining an infrared (IR) spectrum for the sample of body fluid, wherein the IR spectrum for the sample of body fluid is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 or the second derivative of an IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1, (ii) providing an IR spectrum for the alginate oligomer, wherein the IR spectrum for the alginate oligomer is the second derivative of an IR absorbance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the IR spectrum for the sample of body fluid is an absorbance spectrum, or a second derivative IR transmittance spectrum from the wavenumber range of about 1200 cm.sup.−1 to about 900 cm.sup.−1 when the IR spectrum for the sample of body fluid is a transmittance spectrum, and (iii) comparing the IR spectrum for the sample of body fluid to the IR spectrum for the alginate oligomer, wherein (a) an IR spectrum for the sample of body fluid which corresponds to the IR spectrum for the alginate oligomer is an indication that the alginate oligomer is present in the sample of body fluid, and (b) an IR spectrum for the sample of body fluid which does not correspond to the IR spectrum for the alginate oligomer is an indication that the alginate oligomer is absent from the sample of body fluid.

Description

(1) FIG. 1 shows second derivative FTIR absorbance spectra (1200 cm.sup.−1 to 950 cm.sup.−1) for patient 20801001 at visit 4 (V4, placebo) and visit 7 (V7, treatment) aligned to second derivative OligoG spectrum (1200 cm.sup.−1 to 950 cm.sup.−1). The top image shows second derivative spectra for OligoG treated (solid line) and placebo treated (dashed line) phases. The bottom image shows the second derivative spectrum for OligoG with good correspondence of peaks to the treated spectrum in the top image.

(2) FIG. 2 shows the distribution of correlation coefficients between raw, processed FTIR absorbance spectra for patient samples and raw, processed FTIR absorbance spectra for OligoG. Screening samples (downwards left to right diagonal); placebo samples (hatched); OligoG treated samples (upwards left to right diagonal).

(3) FIG. 3 shows the distribution of correlation coefficients between second derivative FTIR absorbance spectra for patient samples and between second derivative FTIR absorbance spectra for OligoG. Screening samples (downwards left to right diagonal); placebo samples (hatched); OligoG treated samples (upwards left to right diagonal).

(4) FIG. 4 shows a scatterplot of all correlation coefficients between sputum sample and OligoG for raw as well as second derivative FTIR absorbance spectra simultaneously. The black line optimally separates samples from treatment phase (black diamonds) from samples from placebo or screening/day 0 phases (white diamonds and white circles, respectively) and gives rise to the linear prediction model of the invention that predicts the presence or absence of OligoG (Y=−1.9643X+1.7286).

(5) FIG. 5 shows vector-normalised, baseline corrected FTIR absorbance spectra from 1800-900 cm.sup.−1 for OligoG (grey), control CF sputum (black), and OligoG-incubated CF sputum in progressively increasing concentrations from 0.02% to 20% (short dash 0.02%; long dash single dot 0.05%; medium dash 0.1%; long dash double dot 0.2%, dotted 0.5%; long dash 1.0%; medium dash double dot 1.5%; extra-long dash 2.0%).

(6) FIG. 6 shows vector-normalised, baseline corrected FTIR absorbance spectra from 1200-900 cm.sup.−1 for OligoG (grey), control CF sputum (black), and OligoG-incubated CF sputum in progressively increasing concentrations from 0.02% to 20% (short dash 0.02%; long dash single dot 0.05%; medium dash 0.1%; long dash double dot 0.2%, dotted 0.5%; long dash 1.0%; medium dash double dot 1.5%; extra-long dash 2.0%).

(7) FIG. 7 shows second derivative FTIR absorbance spectra from 1200-900 cm.sup.−1 for OligoG (grey), control CF sputum (black), and OligoG-incubated CF sputum in progressively increasing concentrations from 0.02% to 20% (short dash 0.02%; long dash single dot 0.05%; medium dash 0.1%; long dash double dot 0.2%, dotted 0.5%; long dash 1.0%; medium dash double dot 1.5%; extra-long dash 2.0%).

(8) FIG. 8 shows FTIR absorbance values at 1601 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(9) FIG. 9 shows FTIR absorbance values at 1412 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(10) FIG. 10 shows FTIR absorbance values at 1125 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(11) FIG. 11 shows FTIR absorbance values at 1086 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(12) FIG. 12 shows FTIR absorbance values at 1028 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(13) FIG. 13 shows FTIR absorbance values at 948 cm.sup.−1 for CF sputum samples of increasing OligoG concentration.

(14) FIG. 14 shows Spearman's Rho values for correlations between the raw FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for CF sputum samples of increasing OligoG concentration and the raw FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for OligoG.

(15) FIG. 15 shows Spearman's Rho values for correlations between the second derivative FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for CF sputum samples of increasing OligoG concentration and the second derivative FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for OligoG.

(16) FIG. 16 shows Spearman's Rho values for correlations between the second derivative FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for CF sputum samples of increasing OligoG concentration and the second derivative FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for OligoG plotted over Spearman's Rho values for correlations between the raw FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for CF sputum samples of increasing OligoG concentration and the raw FTIR absorbance spectra at wavenumbers 1200-900 cm.sup.−1 for OligoG. Control sputum (X); 0% OligoG (black diamond); 0.02% OligoG (white square); 0.05% OligoG (white diamond); 0.1% OligoG (circle); 0.2% OligoG (black triangle), 0.5% OligoG (white triangle); 1.0% OligoG (inverted triangle); 1.5% OligoG (black square); 2.0% OligoG (black circle). The data points cluster according to OligoG concentration.

EXAMPLES

Example 1—Detection of OligoG by FTIR in Sputum of Cystic Fibrosis Patients

(17) Fourier Transform Infrared Spectroscopy (FTIR)

(18) IR spectra were obtained with a Bruker Vertex Fourier Transform IR (FTIR) instrument equipped with a high-throughput system (HTS) module (Bruker Optics). FTIR transmission mode was used for spectral generation. For all samples, 3 μL of sputum was carefully spotted directly onto a 96-well silicon plate using a pipette tip to ensure that the entire volume covered spot with no spillage. Each sample was slowly evaporated at room temperature. Infrared spectra were collected as an average of 24 scans per sample between the wavenumber range of 4000-450 cm.sup.−1 at a resolution of 4 cm.sup.−1, controlled by Optics User Software (OPUS) version 7.0 (Bruker Optics). The process was repeated until six replicates of set reproducibility were generated per sample. An infrared spectrum was generated for an alginate oligomer of DP 5 to 20 (weight average molecular weight 3200 Da) and 90-95% G residues (OligoG) by following the same procedure with an 0.2% (w/v) solution of the oligomer in distilled water.

(19) Prior to the acquisition of each sample spectrum, the silicon plates were thoroughly cleaned using isopropyl alcohol in de-ionized water and dried. A background spectrum was automatically measured for each sample using 24 scans and subtracted from the sample spectrum acquired immediately after.

(20) Data Processing

(21) The resulting sputum spectra were pre-processed using OPUS software version 7 (Bruker Optics) by subtracting a baseline between 1800 and 950 cm.sup.−1. The level of reproducibility among the six replicates within each sample was calculated for the 1800-950 cm.sup.−1 region using the spectral distance (D), a dissimilarity measure, where: D is equal to (1−r)×1000 and r is Pearson's correlation coefficient. A spectrum having a D value greater than 10 when compared to any another spectrum would be considered an inadequate replicate and rejected. All spectral comparisons had a D value less than 10 showing high reproducibility. An average spectrum was then calculated for each sample based on the six replicates. Average spectra were normalized using vector normalization. This had the effect of absorbance at the Amide 1 peak at approximately 1640 cm.sup.−1 to be equal across spectra allowing all spectra to be compared. Second derivative spectra were calculated for all processed raw spectra using the OPUS software with 9 point Savitzky-Golay smoothing.

(22) Statistical Analysis

(23) All data analysis was performed using the R Statistical Programming Environment. Correlation analysis was applied to sputum raw or second derivative spectra and OligoG spectra using Spearman's correlation within the ‘cor.test’. All data were initially assessed for normality using the Shapiro-Wilk test.

(24) Patients

(25) Sputum samples from 15 patients involved in a phase IIb trial involving OligoG were obtained. All patients included a sample at screening/day 0 (pre-treatment/pre-placebo) and at least one sample during placebo or OligoG treatment phase.

(26) Infrared Spectral Data

(27) Visit dependent variation in infrared absorbance was observed at various regions across the spectra for all patients. It was also seen that OligoG absorbs strongly between infrared wave numbers 1200 cm.sup.−1 and 950 cm.sup.−1 and so it was decided to assess this region further for OligoG detection during treatment phase. We refer to this region of the spectrum hereafter as the ‘OligoG region’.

(28) Examples of second derivative infrared spectral data within the OligoG region for patient 20801001 as well as an OligoG standard infrared spectrum is shown in FIG. 1. Graphical analysis of treated phase spectra aligned with OligoG spectra showed that OligoG peaks could be detected by eye.

(29) To maximise the ability to differentiate a treatment spectrum from a placebo or untreated spectrum (i.e. determine if OligoG was present in a sample) it was decided to apply correlation analysis to both raw and second derivative spectra and use the correlation coefficients in a two dimensional Cartesian plot to separate samples according to how similar their infrared profiles were to a standard OligoG spectrum. This two-dimensional ‘map’ of similarity could then be used to develop a simple linear model that could predict if a sample contained OligoG or not.

(30) Correlation Analysis of Treated and Untreated Spectra.

(31) Within the ‘OligoG region’, we compared each sample spectrum with the OligoG standard spectrum for both raw absorbance spectra and second derivative spectra. We calculated Spearman's correlation coefficients in each case which showed a degree of similarity between 0 and 1 (where a coefficient of 1 would mean identical). Both sets of correlation coefficients for each sample are shown in columns 5 and 6 in Table 1.

(32) TABLE-US-00001 5 6 1 2 3 4 Abs 2D 7 8 9 Patient Visit Sample Treatment Spearman's Spearman's OligoG Correct? Correct? ID No. ID Phase Rho Rho Predicted? 2A 2B* 20801001 V2 AP103 Screening/Day 0 0.85 0.09 Y N Y 20801001 V3 AP105 Placebo 0.81 0.11 N Y Y 20801001 V4 AP114 Placebo 0.84 0.05 N Y Y 20801001 V5 AP127 Wash out 0.90 0.86 Y N N 20801001 V7 AP148 Treated 0.93 0.90 Y Y Y 20801001 V7 AP149 Treated 0.88 0.82 Y Y Y 20801001 V7 AP148 + Treated 0.92 0.88 Y Y Y 149 27601001 V2 AP112 Screening/Day 0 0.79 0.09 N Y Y 27601001 V3 AP120 Placebo 0.80 0.09 N Y Y 27601001 V4 AP123 Placebo 0.80 0.07 N Y Y 27601001 V5 AP140 Washout 0.80 0.07 N Y Y 27601001 V6 AP154 Treated 0.82 0.40 Y Y Y 27601001 V7 AP170 Treated 0.82 0.14 Y Y Y 27601002 V2 AP116 Screening/Day 0 0.83 0.02 N Y Y 27601002 V3 AP119 Treated 0.84 0.30 Y Y Y 27601002 V4 AP122 Treated 0.80 0.25 Y Y Y 27601002 V6 AP155 Placebo 0.85 0.11 Y N Y 27601002 V7 AP171 Placebo 0.79 0.05 N Y Y 27605001 V1 AP190 Screening/Day 0 0.81 0.08 N Y Y 27605001 V2 AP200 Screening/Day 0 0.83 0.00 N Y Y 27605001 V3 AP219 Placebo 0.85 0.02 N Y Y 27605001 V4 AP233 Placebo 0.78 0.09 N Y Y 27605001 V6 AP271 Treated 0.86 0.04 Y Y Y 27605001 V7 AP292 Treated 0.84 0.18 Y Y Y 27606002 V1 AP178 Screening/Day 0 0.79 0.10 N Y Y 27606002 V3 AP192 Placebo 0.78 0.15 N Y Y 27606002 V4 AP202 Placebo 0.77 0.04 N Y Y 27606002 V6 AP263 Treated 0.81 0.18 Y Y Y 27606002 V7 AP287 Treated 0.81 0.09 N N N 27606004 V1 AP203 Screening/Day 0 0.86 0.24 Y N Y 27606004 V2 AP217 Screening/Day 0 0.85 0.07 Y N Y 27606004 V3 AP235 Placebo 0.80 0.07 N Y Y 27606004 V4 AP248 Placebo 0.80 0.06 N Y Y 27606004 V6 AP303 Treated 0.92 0.89 Y Y Y 27606004 V7 AP330 Treated 0.80 0.31 Y Y Y 27606005 V1 AP218 Screening/Day 0 0.68 0.03 N Y Y 27606005 V3 AP247 Treated 0.80 0.35 Y Y Y 27606005 V4 AP262 Treated 0.82 0.12 Y Y Y 27606005 V6 AP312 Placebo 0.81 0.06 N Y Y 27606005 V7 AP337 Placebo 0.85 0.10 Y N N 27607001 V1 AP229 Screening/Day 0 0.79 0.06 N Y Y 27607001 V3 AP245 Placebo 0.84 0.09 Y N N 27607001 V4 AP266 Placebo 0.86 0.01 N Y Y 27607001 V7 AP339 Treated 0.85 0.38 Y Y Y 75201001 V1 AP104 Screening/Day 0 0.84 0.08 Y N Y 75201001 V2 AP110 Screening/Day 0 0.81 0.08 N Y Y 75201001 V3 AP117 Treated 0.86 0.39 Y Y Y 75201001 V4 AP121 Treated 0.88 0.57 Y Y Y 75201001 V6 AP150 Placebo 0.84 0.09 Y N Y 75201001 V7 AP161 Placebo 0.84 0.12 Y N N 82603001 V1 AP164 Screening/Day 0 0.83 0.09 N Y Y 82603001 V2 AP165 Screening/Day 0 0.81 0.09 N Y Y 82603001 V3 AP166 Placebo 0.84 0.01 N Y Y 82603001 V4 AP205 Placebo 0.85 0.08 Y N N 82603001 V6 AP227 Treated 0.86 0.13 Y Y Y 82603001 V7 AP242 Treated 0.85 0.07 Y Y Y 82603002 V1 AP201 Screening/Day 0 0.16 0.05 N Y Y 82603002 V2 AP226 Screening/Day 0 0.82 0.05 N Y Y 82603002 V3 AP241 Treated 0.83 0.11 Y Y Y 82603002 V4 AP267 Treated 0.83 0.07 N N N 82603002 V6 AP332 Placebo 0.84 0.09 N Y Y 82604004 V1 AP230 Screening/Day 0 0.88 0.78 Y N Y 82604004 V2 AP261 Screening/Day 0 0.83 0.07 N Y Y 82604004 V4 AP307 Placebo 0.86 0.00 N Y Y 82604004 V6 AP354 Treated 0.85 0.63 Y Y Y 82604004 V7 AP381 Treated 0.86 −0.07 N N N 82606001 V1 AP199 Screening/Day 0 0.84 0.09 Y N Y 82606001 V2 AP222 Screening/Day 0 0.88 0.79 Y N Y 82606001 V3 AP221 Treated 0.86 0.05 Y Y Y 82606001 V6 AP294 Placebo 0.85 0.06 N Y Y 82606001 V7 AP300 Placebo 0.84 0.07 N Y Y 82606003 V1 AP274 Screening/Day 0 0.84 0.09 Y N Y 82606003 V2 AP295 Screening/Day 0 0.83 0.10 N Y Y 82606003 V3 AP323 Placebo 0.86 0.01 N Y Y 82606003 V4 AP336 Placebo 0.85 0.07 Y N N 82606003 V6 AP390 Treated 0.84 0.55 Y Y Y 82606003 V7 AP401 Treated 0.86 0.66 Y Y Y 82606004 V2 AP301 Screening/Day 0 0.79 0.12 N Y Y 82606004 V3 AP319 Treated 0.85 0.48 Y Y Y 82606004 V4 AP350 Treated 0.81 0.08 N N N 82606004 V6 AP392 Placebo 0.80 0.07 N Y Y *revised predictions based on influence of test inhalation at screening and potential carry-over of OligoG from first treatment period.

(33) The distributions of the Spearman's Rho coefficients for samples at treated, placebo and screening phases are shown for raw (FIG. 2) and second derivative (FIG. 3) spectra. It is clear that the second derivative spectra are much more discriminatory between phases.

(34) For each sample, scatter plots for the raw versus second derivative spectra correlation coefficients were constructed to visualise how the treated phase samples compared to the placebo phase and screening/day 0 samples. These scatterplots represents ‘maps’ of how similar or different the sample spectra are to a standard OligoG spectrum. It was generally observed that the second derivative spectra from treatment samples had higher correlation than raw spectra but both might be useful to generate a predictive model.

(35) To develop a predictive model, a single scatter plot was generated to show all correlation coefficients from Table 1 mapped simultaneously (FIG. 4). A linear model was then developed to optimise the separation of treated and placebo samples, using both raw and second derivative correlation coefficients. Visually, the model is represented as a line in FIG. 4. Any point on or above the line is predicted as being a sample containing OligoG and any point below the line is predicted as not containing OligoG. The model is based on the following formula:
Y=−1.9643X+1.7286

(36) Therefore, for any sample, to predict if OligoG is present, the correlation coefficient between the raw absorbance spectrum and OligoG standard is inputted as ‘X’ into the equation. The resulting ‘Y’ value is then used to determine if OligoG is present. If the sample's correlation coefficient between the second derivative spectrum and OligoG standard spectrum is greater than or equal to the predicted Y the model predicts OligoG is present.

(37) The sensitivity and specificity for the model were then determined by inputting the absorbance spectra correlation coefficients (column 5 in Table 1) into the equation for each sample. Predicted Y values were then compared to the second derivative correlation coefficients (column 6 in Table 1) and checked if the prediction was correct or not.

(38) The prediction in each case is shown in column 7 in Table 1 and the accuracy of prediction in column 8. Those cases where the model incorrectly predicted that OligoG was not present were always for the second treatment visit in the time series.

(39) Table 2A shows the sensitivity and specificity of the prediction model. The sensitivity of detecting OligoG was 0.86 whereas the overall specificity of the model (i.e. determining that OligoG was not present) was 0.70. The sensitivity as calculated using just placebo samples was 0.74. However, when allowance is made for the OligoG test inhalation in all subjects at screening, combined with the carry-over effects of OligoG when administered in the first treatment period, the overall test specificity increased to 0.90 (Table 2B).

(40) TABLE-US-00002 TABLE 2A Sensitivity and specificity calculations for the OligoG predictive model. The table shows a breakdown of samples accurately predicted to contain or not contain OligoG according to whether the samples were taken at treatment, placebo or Screening/Day 0 phase (or time point). The placebo or Screening/Day 0 phase predictions were also combined for an overall estimate of specificity. Placebo & Screening/ Screening/ Treated Placebo Day 0 Day 0 Total Samples 29 27 23 50 Correctly 25 20 15 35 Predicted Incorrectly 4 7 8 15 Predicted Sensitivity 0.86 Specificity 0.74 0.65 0.70

(41) TABLE-US-00003 TABLE 2B Revised sensitivity and specificity calculations based on the revised predictions (Table 1, “Correct? 2B”) in view of influence of test inhalation at screening and potential carry-over of OligoG from first treatment period. The placebo or Screening/Day 0 phase predictions were also combined for an overall estimate of specificity. Placebo & Screening/ Screening/ Treated Placebo Day 0 Day 0 Total Samples 29 27 23 50 Correctly 25 22 23 45 Predicted Incorrectly 4 5 0 5 Predicted Sensitivity 0.86 Specificity 0.81 1.0 0.90

Discussion

(42) The results of this study show that FTIR was able to identify alginate oligomers (OligoG) in sputum from cystic fibrosis patients. At its simplest this may be by comparing a second derivative FTIR spectrum of a test sample with a second derivative FTIR spectrum of the alginate oligomer. Alginate oligomers can also be detected by simply calculating the correlations between raw and second derivative FTIR absorbance spectra for a test sample and an alginate oligomer standard and applying said coefficients to a simple linear formula. The procedure is simple and rapid and all data are generated from a single sample and a reference alginate spectrum in a library. The method is non-invasive, reproducible and cost-effective per sample.

(43) The calculated sensitivity value for the proposed prediction model suggests that 80% of samples tested would correctly show if an alginate oligomer spectrum is present. However, some notable exceptions suggest that the sensitivity and specificity could be significantly higher: the high rate of apparently false positive predictions for the V2 screening samples is likely due to patients having already received a single dose of alginate oligomer as a test inhalation at the V2 screening visit. Although not all screening samples showed presence of OligoG this may have been due to how effective the test inhalation was and whether the sputum sample was taken before or after the inhalation. In addition, the wider clinical findings of the clinical trial also indicated that the OligoG effects are sustained well after the cessation of OligoG treatment, suggesting that OligoG may still be present in the sputum and sputum plugs yet to be expectorated. That OligoG could still be present in the sputum at the end of the wash-out period would therefore result in a true positive detection for OligoG in those V6 samples at the start of the placebo period. Taking these effects into consideration, it is likely that the true specificity exceeds 80%.

(44) Additional factors influencing the detection of OligoG, include patient compliance (ie., patients not inhaling medication correctly or missing doses), interference from other ‘foreign’ molecules (e.g. food) providing dominant infrared peaks in the spectrum that could lower the correlation between sample and OligoG standard. False positive results might also occur due to ‘foreign’ conflicting sugars in sputum that yield a similar infrared profile to OligoG.

Example 2—Determining the Concentration of OligoG in Sputum of Cystic Fibrosis Patients by FTIR

(45) The aim of this experiment was to incubate a CF-patient sputum sample with increasing concentrations of OligoG with the objective of measuring absorbance at OligoG-specific wavenumbers. Standard curves of absorption at each OligoG-specific wavenumber were then calculated. Correlation analysis between each spectrum and a reference OligoG spectrum was also performed. Standard curves of absorption and correlation are shown below with polynomial trendlines showing R.sup.2 values in excess of 0.95, indicating that FTIR analysis is capable of accurately estimating OligoG concentration in sputum.

(46) Method

(47) OligoG stock solutions from 0% w/v up to 20% w/v were prepared for incubation with CF-patient sputum at a 1:10 (OG:Sputum) dilution. Table 1 shows all the stock concentrations and final OG in sputum concentrations. OG was incubated with sputum at 37° C. for 30 minutes.

(48) TABLE-US-00004 TABLE 3 OligoG stock solution concentrations and final concentration of OligoG in sputum after incubation Stock conc. Dilution factor Final OligoG conc. OligoG (% w/v) (OligoG:Sputum) in Sputum (% w/v) 0 1:10 0 0.2 0.02 0.5 0.05 1 0.1 2 0.2 5 0.5 10 1 15 1.5 20 2

(49) FTIR analysis was performed, as described in Example 1, on the incubated CF sputum samples, and non-incubated CF sputum was used as a control sample. Replicate spectra (n=3) were acquired using the Bruker Alpha ATR-FTIR spectrometer. Spectra were processed using the in-built OPUS spectral processing tools for normalization, baseline correction, and second derivative calculation.

(50) Absorption Curves

(51) OligoG-incubated sputum IR-spectra were generated and the absorption at peak positions corresponding to major OligoG absorption peaks were plotted against the corresponding concentration of OligoG in sputum (FIGS. 8-13). Table 4 shows the wavenumbers of interest which were examined.

(52) TABLE-US-00005 TABLE 4 Oligo G absorption peak positions, intensities and widths Peak Absorption Positon Intensity Width 1601.522 0.417 55.0644 1412.537 0.256 36.4529 1124.84 0.187 13.1034 1086.016 0.272 19.9577 1028.324 0.374 35.0735 949.3862 0.129 19.5884
Correlation Analysis

(53) Each IR-spectrum of OligoG-incubated and control CF-sputum was correlated to a reference OligoG spectrum using a non-parametric correlation test (Spearman's). Both absorbance and second derivative spectra were correlated to the corresponding OligoG absorbance or second derivative spectrum. The most distinct peaks found in the OligoG spectrum are found within the glycogen-rich region from 1200-900 cm.sup.−1. For this reason, the correlation analysis was focused within this region.

(54) Results and Discussion

(55) ATR-FTIR spectroscopy has highlighted the relationship between OligoG-concentration in CF sputum and absorption by OligoG-specific IR wavenumbers. In this way, FTIR can be used to estimate the OligoG concentration in unknown CF-patient sputum samples. The IR-spectra shown in FIGS. 5, 6 and 7 demonstrate the strong affects OligoG has on sputum FTIR spectra, with increases in absorption observed across wavenumbers 1800-900 cm.sup.−1, including the glycogen-rich region (1200-900 cm.sup.−1), especially near wavenumbers associated with OligoG (Table 4). An increase in peaks in this region can also be observed in OligoG-incubated sputum spectra, compared to non-incubated and 0% OligoG-incubated spectra.

(56) FIGS. 8-13 show that at each wavenumber of Table 4, the relative absorbance measured is proportional to the concentration of OligoG in the sputum sample. Thus, for sputum samples with an unknown concentration of OligoG it will be possible to measure IR absorbance at these wavenumbers and use the appropriate chart to determine the OligoG concentration in that sample.

(57) Correlation analysis of FTIR absorbance spectra in the wavenumber range 1200-900 cm.sup.−1 shows how an increasing concentration of OligoG corresponds to an increase in Spearman's Rho coefficient when comparing the raw and second derivative sputum spectra to OligoG reference spectra (FIGS. 14 and 15). An increase in correlation can be observed even at 0.02% OligoG, which was found to be statistically significant at the 95% confidence level for both the absorbance spectra (one-tailed p=0.0142), and second derivative spectra (one-tailed p=0.0278). Correlation coefficients for all other concentrations were also found to be statistically significantly different from the non-incubated control and 0%-OligoG incubated sputum (p<0.05). The correlation coefficients for the control and 0%-OligoG incubated sputum were not found to be statistically significantly different from each other at the 95% confidence level, for either the absorbance or second derivative spectra (absorbance p=0.2261, second derivative p=0.6585), showing that the differences observed were not due to the experimental conditions.

(58) Thus, for sputum samples with an unknown concentration of OligoG it will be possible to measure IR absorbance at these wavenumbers, correlate the raw FTIR absorbance spectra and/or the second derivative thereof with corresponding FTIR absorbance spectra from OligoG and use an appropriate standard curve to determine the OligoG concentration in that sample.

(59) FIG. 16 further shows that when the correlation coefficients for second derivative FTIR absorbance spectra from OligoG containing sputum and second derivative FTIR absorbance spectra from OligoG are plotted over the correlation coefficients for raw FTIR absorbance spectra from OligoG containing sputum and raw FTIR absorbance spectra from OligoG, the data points cluster in distinct groups according to OligoG concentration. Thus for sputum samples with an unknown concentration of OligoG it will be possible to measure IR absorbance at these wavenumbers, correlate both the raw FTIR absorbance spectra and the second derivative thereof with corresponding FTIR absorbance spectra from OligoG and use both of the correlation coefficients so obtained to determine OligoG concentration by determining to which cluster the test data point associates in such a chart.