Methods, assays and kits for detecting exposure to cyanotoxins

Abstract

Methods and kits for the detection of toxic cyanobacteria in a sample by analyzing the sample for the presence antibodies raised in a host, where the presence of antibodies is indicative of toxic cyanobacteria, are described.

Claims

1. A method for detecting exposure to a cyanotoxin, the method comprising: analyzing a sample from a host for the presence of antibodies raised in the host, wherein the presence of the antibodies is indicative of exposure to a cyanotoxin, and wherein the antibodies are against the ADDA region of a microcystin or a nodularin.

2. A method for detecting exposure to a cyanotoxin in an assay, comprising: analyzing a sample with an ELISA method for the presence of ADDA-specific antibodies complexed to an immobilized microcystin, wherein the sample comprises blood of a subject suspected of being exposed to microcystin, and the presence of the antibodies is indicative of exposure by the subject to microcystin.

3. The method of claim 1, wherein the sample comprises blood of the host, and the host is suspected of being exposed to cyanobacteria.

4. The method of claim 1, wherein the exposure is from salt water or freshwater.

5. The method of claim 1, wherein the exposure is from a blue-green algal bloom.

6. The method of claim 1, wherein the sample is taken days, weeks, or longer after cyanotoxin exposure.

7. The method of claim 1, wherein the cyanotoxin is selected from the group consisting of microcystins, nodularins, anatoxin-a, anatoxin-a(S), aplysiatoxins, cylindrospermopsins, lyngbyatoxin-a, and saxitoxins.

8. The method of claim 1, wherein the cyanotoxin is selected from the group consisting of a microcystin and a nodularin.

9. The method of claim 1, wherein the cyanotoxin is a microcystin.

10. The method of claim 2, wherein the sample is taken days, weeks, or longer after microcystin exposure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees.

(2) FIG. 1: Microcystin structure is a cyclic heptapeptide that contains the amino acid ADDA, a11-S,a11-E)-3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid.

(3) PRIOR ART FIG. 2: Schematic illustration of a current ELISA method to detect MC toxins themselves.

(4) FIG. 3: Schematic illustration of presently described modified ELISA method which detects antibodies to the toxin, not the toxin itself.

(5) FIG. 4: Schematic illustration of a test procedure for performing an in vivo proof-of-concept study of exposure to low-dose microcystin-LR (e.g., measuring “No Observed Adverse Effect Level” (NOAEL)).

(6) FIG. 5: Anti-ADDA antibodies in a plasma sample, where 3% BSA is used as the blocking reagent.

(7) FIG. 6: Anti-ADDA antibodies in a plasma sample, where 5% Skim Milk is used as the blocking reagent.

(8) FIG. 7: Plasma from NAFLD mice exposed with 100 μg/kg of MC-LR showed a much higher signal (Green bars) as compared to control mice exposed to saline (yellow bars) indicating presence of ADDA-specific antibodies.

(9) FIG. 8: Plasma from normal C57Bl/6J MC-LR exposed mice (Green Bars) also showed a much higher signal as compared to saline exposed mice (Yellow bars) indicating presence of ADDA-specific antibodies.

(10) FIG. 9: Anti-ADDA antibodies in a plasma sample, where 5% Skim Milk is used as the blocking reagent after a 2 hour incubation.

(11) FIG. 10: Anti-ADDA antibodies in a plasma sample, where 5% Skim Milk is used as the blocking reagent after an overnight (18 hour) incubation.

(12) FIG. 11: Plasma samples from different saline exposed C57Bl/6J mice (n=4, yellow bars) and MC-LR exposed (n=7, green bars) C57Bl/6J mice were compared to the plasma obtained from C57Bl/6J mice from a separate unexposed colony (purple bars).

DETAILED DESCRIPTION OF THE INVENTION

(13) Described herein is a method for the detection of antibodies against the ADDA region of microcystins/nodularins in blood of exposed individuals.

(14) Also described herein is a method of measuring host antibodies (e.g. immune serum or plasma) against cyanotoxins such as any congener of the cyclic heptapeptide microcystin.

(15) The presently described methods use host antibodies as a way of detecting exposure of the host to cyanotoxins, such as microcystins.

(16) The presently described methods are useful for a diagnostic test of exposure to microcystins.

(17) FIG. 1 shows that the microcystin structure is a cyclic heptapeptide that contains the unusual amino acid ADDA. There are more than 150 identified that vary in positions X and Z, as well by methylation, hydroxylation, or epimerization. The most common is MC-LR. Table 1 below shows certain MC variants:

(18) TABLE-US-00001 TABLE 1 MC variant X Z MC-LR L-Leu R-Arg MC-RR R-Arg R-Arg MC-LA L-Leu A-Ala MC-LF L-Leu F-Phe MC-LW L-Leu W-Trp MC-YR Y-Tyr R-Arg

(19) Described herein is a method of measuring host antibodies (e.g. immune serum or plasma) against cyanotoxins such as any congener of the cyclic heptapeptide microcystin.

(20) One aspect of this method is the ability to use host antibodies as a way of detecting exposure of the host to cyanotoxins (such as microcystins) as a diagnostic test of exposure.

(21) PRIOR ART FIG. 2 is a schematic illustration of a currently available ELISA method to detect MC toxins themselves. The amount of signal generated is inversely proportional to the amount of free microcystin toxin that is present in the sample because it binds to the antibody and is washed away.

(22) In contrast, FIG. 3 shows a schematic illustration of presently described modified ELISA method which detects antibodies to the toxin, not the toxin itself. The amount of signal generated is directly proportional to the amount of ADDA-reactive antibodies present in the sample because it binds to the immobilized ADDA group on the plate and is not washed away.

(23) One method for measuring antibodies for cyanotoxins is to react serial dilutions of serum or plasma with an immobilized cyanotoxin of interest (e.g., microcystin); for example, on a micro-titer plate. After the reaction and washing steps, secondary reagents (such as secondary antibodies and enzyme complexes which aid in visualization of the reaction) which detect the presence of the antibody of interest which is bound in complex to the immobilized cyanotoxin on the micro-titer plate. The reaction in the micro-titer plate is then read by a detector capable of detecting the reaction (e.g. by change in light absorbance).

(24) In one example, plasma from mice exposed to the cyanotoxin microcystin-LR was compared to control mice which had not been exposed to microcystin-LR. The dilution series of plasma from the microcystin treated mice demonstrated higher levels of antibody complexed to the immobilized microcystin-LR vs control plasma, showing that this method detects antibodies against the cyanotoxin microcystin-LR.

(25) The method is suitable for testing plasma or serum samples and can detect exposure to cyanotoxins such as microcystin and is useful in the differential diagnosis of patients with potential exposure to cyanotoxins.

(26) The method measures a specific by-product of cyanotoxins, namely antibodies made against them by the host, which can be present days, weeks, or even longer after cyanotoxin exposure.

(27) In order for a host to generate an immune response to a foreign antigen such as a peptide, it was previously believed that the antigen needs to be at least 9-15 linear amino acids to bind in the MHC cleft of antigen-presenting cells.

(28) It is now shown herein, the contrary where cyanotoxins such as microcystins which only contain 7 modified amino acids in a cyclic, non-linear structure, generate an immune response, which can, in turn, be measured. Thus, it would not be readily obvious that such peptides would permit an immune reaction capable of generating antibodies.

Examples

(29) The presently described method measures a specific by-product of cyanotoxins, namely antibodies made against them by the host, which can be present days, weeks, or even longer after cyanotoxin exposure.

(30) Thus, even if a patient presents to a health care provider after the toxin itself is no longer detectable in biological fluids (as measure by methods such as mass spectrometry or ELISA), the disclosed test is designed to be able to help determine exposure.

(31) Mouse Model of Exposure:

(32) The current No Observed Adverse Effect Level (NOAEL) of 40 μg/Kg (91 days) or 200 ug/Kg (14 days) used to derive safe exposure guidelines for microcystin. The experiments herein used 50 and 100 ug/Kg for 15 doses over 4 weeks—approximately 2.4 to 4.8 times below total NOAEL exposure. See FIG. 4.

(33) The method of testing was as follows:

(34) 1) ELISA strips coated with a protein conjugated with the ADDA region of the Microcystin toxin (from Abraxis anti-ADDA ELISA kit) was blocked with either 3% BSA or 5% Skim Milk overnight;

(35) 2) Appropriate dilutions of plasma from either control or MC-LR exposed mice was added to the respective wells;

(36) 3) Excess plasma was washed off and the bound anti-ADDA antibodies from the plasma were detected using a Universal Ig antibody labelled with HRP (horseradish peroxidase); and,

(37) 4) Streptavidin substrate was added to give a colored product which was quantitated by reading the absorbance at 450 nm.

(38) FIG. 5 is a graph showing the anti-ADDA antibodies in a plasma sample, where 3% BSA is used as the blocking reagent.

(39) FIG. 6 is a graph showing the anti-ADDA antibodies in a plasma sample, where 5% Skim Milk is used as the blocking reagent.

(40) The percent increase in signal over control plasma is shown in Table 2 below:

(41) TABLE-US-00002 TABLE 2 Dilution BSA block Skim Milk block 1:10 7% 72% 1:100 119% 119% 1:1000 302% 105%

(42) These results show that the presence of anti-ADDA antibody is detectable in plasma samples from exposed mice.

(43) FIG. 7 shows that plasma from NAFLD mice exposed with 100 μg/kg of MC-LR showed a much higher signal (Green bars) as compared to control mice exposed to saline (yellow bars) indicating presence of ADDA-specific antibodies. For this experiment, blocking was done overnight with 5% skim milk.

(44) The previous experiments used plasma from mice (exposed with 100 μg/kg MC-LR) that had pre-existing liver disease (AKA Non-Alcoholic Fatty Liver Disease or “NAFLD model”. In order to extend these findings into normal healthy settings, normal mice on the C57Bl/6J background (same background as the NAFLD model but without liver disease), were exposed with 100 μg/kg MC-LR (vs saline control) and the plasma was used for the subsequent experiments.

(45) FIG. 8 shows that plasma from normal C57Bl/6J MC-LR exposed mice (Green Bars) also showed a much higher signal as compared to saline exposed mice (Yellow bars) indicating presence of ADDA-specific antibodies.

(46) Varying the Time Required for Blocking the Plates with 5% Skim Milk

(47) Blocking the plate with 5% Skim milk overnight (FIG. 10) shows a robust increase between Control and Treated samples vs 2 hour incubation. MC-LR exposed mice (Green Bars) vs saline exposed mice (Yellow bars). FIG. 9 shows a 2 hour incubation with 5% milk FIG. 10 shows an overnight (18 hour) incubation with 5% milk.

(48) FIG. 11 shows after method optimization, plasma samples from different saline exposed C57Bl/6J mice (n=4, yellow bars) and MC-LR exposed (n=7, green bars) C57Bl/6J mice were compared to the plasma obtained from C57Bl/6J mice from a separate unexposed colony (purple bars). Data indicates that MC-LR exposed mice develop measurable antibody response which can be distinguished from various untreated control mice.

(49) These results confirm the presence of anti-ADDA antibody can be detectable in plasma samples from exposed mice in both healthy and pre-existing liver disease settings. Blocking the plate overnight (18 hrs) with 5% skim milk showed a robust increase and difference in the signal during optimization vs 2 hour incubation.

(50) Certain embodiments of the present invention are defined in the Examples herein. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

(51) While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

(52) Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.