Methods and kits for eluting analytes from immunoaffinity columns

Abstract

The present technology provides a method of eluting an analyte of interest from an immunoaffinity column. The method of the present technology provides a simplified method for dissociating the bound analyte from the immunoaffinity column and allows direct downstream treatment of the eluent.

Description

[0001] Cross-references to related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 525,813, filed July 10, 2023, entitled “Methods and Kits for ElutingAnalytes From Immunoaffinity Columns”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to methods and kits for eluting analytes from immunoaffinity columns. In particular, this disclosure relates to methods and kits for improving the compatibility of eluents with downstream analyses. Background Technology

[0003] Immunoaffinity columns are used for sample purification and enrichment prior to analytical assays (e.g., prior to HPLC analysis, immunoassay, or LC-MS / MS). Immunoaffinity columns combine the use of liquid chromatography with the specific binding of antibodies or related reagents to capture the analyte of interest. The resulting method can be used in the assay of a specific target or to purify and concentrate the analyte of interest before further examination by another technique. Immunoaffinity columns are particularly useful in methods for detecting the presence of harmful substances (e.g., toxins, analytes of interest) in a variety of complex samples (e.g., food or crop samples, patient samples, biological samples).

[0004] Typically, analytes of interest are eluted from an immunoaffinity column using harsh elution solutions to dissociate the analyte from the stationary phase containing antibodies that capture the analyte. Examples of typical elution solutions include strongly acidic or alkaline buffers, high-salt solutions with strong ionic strength, organic solvents, denaturing agents, and competing ligands or the like. In many cases, the resulting eluted sample (i.e., the eluent) is diluted or buffer-exchanged to ensure compatibility with downstream assays. That is, additional solutions are added to the eluent to condition it for downstream processing. Dilution and / or buffer exchange processes generally reduce the sensitivity and accuracy of results. For example, dilution can affect the sensitivity of diagnostic tests, such as when analyzing purified samples by immunoassays (e.g., ELISA and lateral flow assays), leading to false negatives (i.e., the diluted eluent is too dilute to detect a positive result). Furthermore, buffer exchange processes are cumbersome and can introduce impurities and matrix effects by introducing additional solutions. These additional solutions and processes can affect recovery rates and the accuracy of results. Summary of the Invention

[0005] By using alternative methods for dissociating analytes from the immunoaffinity column, the problems associated with modifying or adjusting the eluent to address its adverse effects are mitigated. This technique provides methods and related kits with improved simplicity and performance compared to conventional elution methods that introduce harsh solutions and / or require adjustment for compatibility with downstream analytical processes. This technique offers simplicity because no special eluent formulation is required for dissociation. Instead, it uses thermal energy (i.e., heat) to dissociate the analyte of interest from the stationary phase. Therefore, the eluent or wash solution can be selected to be directly compatible with downstream processes (e.g., spectroscopic, HPLC, fluorescence assays, immunoaffinity assays) without any modification to the eluent (i.e., no dilution, no buffer exchange). Eliminating dilution not only eliminates processing steps and reduces processing time but also improves the sensitivity of results. Furthermore, eliminating buffer exchange eliminates time-consuming steps that can introduce impurities and matrix effects. Therefore, accuracy and recovery can be improved by using non-chemical dissociation methods and related kits that utilize the analyte of interest with the stationary phase of an immunoaffinity column (i.e., antibody-containing material).

[0006] In one aspect, the present technology relates to a method for eluting bound analytes from an immunoaffinity column. The method includes passing a sample solution containing an analyte of interest through an example immunoaffinity column to associate a portion of the example analyte of interest with a stationary phase material of the example immunoaffinity column; heating the immunoaffinity column at a temperature between about 65°C and about 95°C for a period of time between about 5 minutes and about 30 minutes to dissociate a portion of the analyte of interest from the stationary phase material; and rinsing the immunoaffinity column with a solution to produce an eluent containing the dissociated portion of the analyte of interest.

[0007] The foregoing aspects may include one or more of the following features. In one embodiment, the method may further include injecting the eluent into a downstream processing system without modifying the eluent. Some embodiments are characterized by using phosphate-buffered saline or a running buffer solution for rinsing the immunoaffinity column. Some embodiments are characterized by conditioning the immunoaffinity column before loading it with the sample solution. Other embodiments do not include column conditioning. These methods may feature heating the column at 90°C for 12 minutes, 10 minutes, 7 minutes, or 5 minutes. Some embodiments are characterized by heating the column at 95°C to 105°C for less than 5 minutes (e.g., 3 minutes, 1 minute, 30 seconds). In some embodiments, the eluent is injected into the HPLC column. In some embodiments, the eluent is injected into a sideflow device. A reader can be used to assess the ratio of the peak height of the test line to the peak height of the control line. In some embodiments, the eluent is used for an ELISA assay. In some embodiments, the eluent is analyzed by a fluorometer. In some embodiments, the eluent is injected into an LC-MS system. The sample solution used in these methods may include powdered food samples, such as cocoa powder. To form a solution, the powdered food sample is mixed with water, an organic solvent, or both. In some embodiments, the powdered food sample is mixed with a salt solution to extract or form a sample solution. The sample solution may be filtered before passing through an immunoaffinity column. In some embodiments, the immunoaffinity column is a mycotoxin immunoaffinity column, such as an ochratoxin immunoaffinity column.

[0008] In another aspect, this technology relates to a kit for eluting analytes captured from an immunoaffinity column. The kit includes an immunoaffinity column, a consumable analytical processing device (e.g., a lateral flow assay, a liquid chromatography column) having an inlet for injecting an eluent formed using the method according to this technology; and a rinsing solution in vials compatible with the consumable analytical processing device. This kit can be tailored for detecting target analytes in complex samples. For example, the kit can be used to test the level of ochratoxin in a cocoa powder sample. Attached Figure Description

[0009] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which: Figure 1 It displays images of various immunoaffinity chromatography columns.

[0010] Figure 2 A flowchart illustrating a routine method for eluting analytes from an immunoaffinity column is provided.

[0011] Figure 3 A flowchart illustrating a method for eluting analytes from an immunoaffinity column according to the present technique is provided.

[0012] Figure 4A This is an example Figure 3 An image of the first step of the method shown.

[0013] Figure 4B This is an example Figure 3 Image of the second step of the method shown.

[0014] Figure 4C This is an example Figure 3 Image of the third step of the method shown Figure 5 It is a table that provides peak height results for test lines and control lines using various elution methods. Detailed Implementation

[0015] This technique relates to an improved method for eluting analytes of interest from an immunoaffinity column. This method is particularly suitable for purifying, enriching, and analyzing analytes that specifically bind affinity ligands (i.e., antibodies or antigens).

[0016] As used herein, the terms “analyte” or “analyte of interest” refer to any molecule of interest required for quantification, measurement, or detection using the methods described herein.

[0017] As used herein, the term "sample" means any medium that includes an analyte or analyte of interest (e.g., a fungal toxin) to be quantified using the methods of this disclosure. Samples may be selected from agricultural, environmental, or biological samples. Samples may include, but are not limited to, food ingredients (e.g., poultry, lean meat, milk, yogurt, dairy products, baked goods, beverages, juices, cheeses, vegetables, fruits, fish, etc.), animal feed, water bodies or wastewater (e.g., ponds, lakes, rivers, oceans, sewage channels, drinking water, etc.), clinical specimens (e.g., blood, plasma, serum, saliva, tissue, urine, saliva, samples / fluids from the respiratory tract, etc.), dirt, and cosmetic and pharmaceutical products (e.g., lotions, creams, ointments, solutions, drugs, eye drops, and ear drops, etc.).

[0018] As used herein, the term "immunoaffinity chromatography" refers to a method for separating a target antibody or antigen from a heterogeneous sample solution. It is a column-based method, meaning the sample solution flows through a column. The column contains a stationary material / carrier functionalized with a capture antibody or antigen (also known as an affinity ligand). The target protein (e.g., the analyte of interest) is adsorbed onto the stationary phase / carrier via binding to the capture antibody or antigen and is retained, while the remaining sample solution is washed away. As used herein, the term "affinity ligand" refers to a substance (e.g., a functional group) that selectively captures (binds) a target molecule from a mixture of molecules based on specific intermolecular affinity (e.g., antigen-antibody binding).

[0019] As used in this paper, the term "matrix" refers to the sample components other than the analyte of interest. The matrix has a significant impact on the method of analysis and the quality of the results obtained.

[0020] As used herein, the term "matrix effect" refers to the combined effect of all components of a sample other than the analyte. Components derived from the sample matrix that co-elute with the analyte may interfere with analyte measurements, leading to false negative or false positive results.

[0021] As used herein, the term "analyte standard" refers to material containing an analyte of known concentration.

[0022] As used herein, the term "elution solution" refers to a solution that desorbs (or elutes) an analyte from column material by disrupting the specific binding of the analyte to its affinity ligand. The salt, pH, and ionic conditions required to determine such functionality of an elution solution are well known in the art.

[0023] As used herein, the term "rinse solution" refers to a solution that does not desorb or disrupt the specific binding of the analyte to the affinity ligand. Instead, the rinse solution is used to remove dissociated components from the stationary phase of an immunoaffinity column. Typical rinse solutions include aqueous solutions such as water and phosphate-buffered saline. Lower concentrations of organic solvents, such as methanol, may also be used.

[0024] refer to Figure 1 This technique involves immunoaffinity chromatography columns. Figure 1 This is an image showing a plurality of immunoaffinity columns 100. Each column 100 includes an inlet 105 for adding sample and an outlet 110 for components to leave the column. Figure 1 As shown, a removable cover can be included on both inlet 105 and outlet 110.

[0025] Within the immunoaffinity column 100 (between inlet 105 and outlet 110) is a stationary phase or carrier comprising a capture antibody or antigen. When a sample is added to inlet 105, the sample passes through the column, and any analyte with affinity for the capture antibody or antigen is absorbed (e.g., bound or associated) to the capture antibody or antigen. The stationary phase or carrier of the immunoaffinity column is functionalized to have affinity for a specific analyte. For example, some immunoaffinity columns are functionalized to have affinity for specific mycotoxins. In particular, some immunoaffinity columns are functionalized to have affinity for ochratoxin A; other immunoaffinity columns may be functionalized to have affinity for fumonisins; and other immunoaffinity columns have affinity for aflatoxins. Immunoaffinity columns can be functionalized with any capture antibody or antigen. That is, immunoaffinity columns are not limited to affinity for mycotoxins. Mycotoxins are merely illustrative.

[0026] Immunoaffinity chromatography columns, also known as IAC columns, are used for sample purification or enrichment. That is, by passing a complex sample through an IAC column functionalized to capture a specific analyte, the analyte is captured by the column, while other components and impurities of the complex sample elute from the column. The analyte within the complex sample absorbs or binds to the capture antibody or antigen within the IAC column. During the washing step, the wash solution, which washes away impurities and other components not adsorbed to the capture antibody or antigen, is also released from the column. The captured analyte is then dissociated from the column using an elution solution. That is, the analyte is eluted from the column using a specific elution solution designed to chemically release the bound analyte, forming an eluent containing both the elution solution and the released analyte.

[0027] The problem with conventional methods using IAC columns lies in the elution solution. To release the bound analyte, the elution solution needs to chemically dissociate the analyte from the captured antigen or antibody. This requires strongly acidic, strongly alkaline, strongly salted, or strongly organic solvents (i.e., the elution solution) to desorb the captured analyte. These elution solutions remain in the eluent, which is released from the column and poses challenges for downstream processing. For example, strong salt solutions can interfere with mass spectrometry analysis, and therefore, eluents containing strong salt solutions cannot be directly analyzed by LC-MS.

[0028] Conventional methods address the problems arising from the elution solution by adding a dilution or buffer exchange step after elution from the IAC column to modify the elution solution before any subsequent analytical steps. Figure 2 Typical methodological steps required for elution and subsequent further processing are illustrated. Reference Figure 2 Conventional method 200 includes step 205, in which a sample extract (i.e., a sample solution) is loaded and passed through an IAC column. During the passage of the sample extract through the column, any impurities are washed away using a washing solution. Next, the bound analyte is eluted from the IAC column using an elution solution, step 215. Because the elution solution is specific to the type of IAC column (i.e., specific to the chemical dissociation of the analyte from the column), the elution solution must be obtained or matched to the column. In some cases, it is desirable to prepare the elution solution prior to use (optional step 210). For example, as... Figure 2 As shown, the elution solution can be prepared by mixing two or more solutions together (before using them as the elution solution).

[0029] Once the elution solution is used to form the eluent from the column, it must be modified or adjusted for use in downstream analyses, such as ELISA assays, HPLC analysis, and / or mass spectrometry. Figure 2In step 220, the eluent is diluted before performing the ELISA test or using the side-flow strip (step 225). In other conventional methods, a buffer exchange process may be required instead of dilution step 220 or as a supplement to dilution step 220 to allow for downstream processing, such as HPLC and / or LC-MS analysis.

[0030] While dilution and buffer exchange steps (e.g., step 220) are necessary to address issues arising from the type of elution solution required to dissociate the analyte from the column, they are time-consuming and introduce potential impurities and matrix effects. In addition to these drawbacks, dilution and / or buffer exchange steps add an additional volume of liquid to the analysis, which reduces sensitivity and / or requires a larger volume of the sample extract to be tested.

[0031] This technique provides an elution method that does not require a specific elution solution to dissociate the bound analyte from the IAC column. This technique does not rely on the chemical dissociation of the bound analyte, thus avoiding the use of an elution solution. Figure 3 The method (300) of this technique is illustrated. Method 300 includes loading a sample extract and passing it through an IAC column, step 305. Once the sample extract has passed through the column and the analyte therein has been absorbed into the IAC column, a heat treatment is applied to dissociate the analyte from the captured antibody / antigen, step 315. The heat treatment includes heating the IAC column at a temperature high enough to denature the analyte (e.g., typically greater than 35°C or 40°C), but low enough not to structurally degrade the IAC column (e.g., typically less than 100°C). To dissociate substantially all of the captured analyte from the column, heat should be applied for a period between 3 minutes and 60 minutes. More specifically, at lower applied temperatures (e.g., between 50°C and 70°C), a longer period of heat will be required, such as 35 minutes to an hour or longer. At higher temperatures (e.g., 75°C to 95°C), shorter periods of heat (e.g., 1 minute (or even 30 seconds) to 25 minutes) can be applied to dissociate the analyte from the captured antibody / antigen. One example is heating at 89°C for 5 minutes. Another example is heating at 90°C for 2 minutes. Yet another example is heating at 94°C for 1 minute.

[0032] The final step in elution in Method 300 is to wash the dissociated analyte off the column with any type of solution. Because a chosen solution is used to generate the eluent (i.e., no conventional eluent is required to desorb the analyte), the eluent can be directly injected into or applied to downstream analytical processing techniques (step 325) without any intermediate treatment or modification steps (i.e., no dilution, no buffer exchange). Figure 3In this process, an eluent compatible with lateral strip tests used for direct processing from the IAC column is generated by rinsing with running buffer.

[0033] Figure 4A , Figure 4B and Figure 4C Method 300 is illustrated graphically. Figure 4A Step 305 is illustrated, where the IAC column is loaded with the sample extract. The sample is allowed to flow through the column to allow the analyte within the sample to bind to the capture antibody / antigen. Figure 4B An example is given of a method for applying heat treatment to the column, such as step 315. Figure 4B As shown, the column can be inserted into a heater with a slot designed to receive the column. In this case, a digital dry bath device (trade name MyBlock) available from Benchmark Scientific is used. ™ (Mini version available). However, the column can be heated to a certain temperature and held for a sufficient period of time using any type of applied energy (e.g., RF radiation, microwave radiation). After the desired heat treatment, the dissociated analyte is washed from the column with any solution to form an eluent that can be directly used in downstream analytical processes, such as by injecting into a solution such as... Figure 4C On the sideflow test strip shown.

[0034] This technique offers numerous advantages over conventional elution methods from IAC columns. For example, it allows the eluent to be injected into downstream processing systems without modification. In some embodiments, the eluent can be injected directly into the HPLC or LC-MS system without any modification. That is, the solution used to elute and dissociate the analyte from the column can be any solution and can therefore be selected to be compatible with the mobile phase and / or downstream detector of the HPLC system. Furthermore, there is no need to dilute the eluent for compatibility with side-flow devices, ELISA methods, or any other downstream detector / detection method. As a result, the sensitivity of downstream analysis is improved because the eluent is not diluted. That is, the detection limit of downstream analysis is not challenged by the addition of diluent solutions. In addition, by limiting the amount of solution added to the eluent, a smaller amount of sample extract is required to ensure correct results (i.e., no false negatives due to below the detection limit). This advantage is illustrated, for example, by comparing the amounts of sample extract and solution added in Method 200 (a conventional method using an eluent to dissociate the captured analyte) with those in Method 300 (the technique). Method 300 uses 1 / 10 of the sample and forms an eluent containing at least twice the enrichment of Method 200.

[0035] In biochemistry and other fields, IAC columns can be used to selectively purify target compounds from complex samples. Some compounds that have been isolated using this method include proteins, glycoproteins, carbohydrates, lipids, bacteria, viral particles, drugs, and environmental agents. Furthermore, IAC columns have been used in conjunction with detectors to measure or identify the amount of analytes captured from complex samples. Examples of analytes that have been measured using this method include human serum albumin, recombinant tissue plasminogen activator, recombinant antithrombin III, IgG, *Escherichia coli*, isoproturon, phenylurea herbicide, benzidine, dichlorobenzidine, aminoazidobenzyl, azo dyes, triazine, diethylstilbestrol, acetylcholinesterase, transferrin, and insulin. The methods described in this technique can be used to detect any of these analytes.

[0036] Sample extracts can be prepared from urine, sputum, food, crops, water, and soil extracts. Examples of analytes that have been examined using this method include α1-antitrypsin, atrazine, benzylpenicillin acyl-peptide, bovine serum albumin, carbendazim, chloramphenicol, cortisol, clenbuterol, and phenytoin, among others. Additional analytes include toxins such as fungal toxins (e.g., aflatoxin, fumonisin, zearalenone, ochratoxin, DON, T2 / HT2). For solid samples, such as food, crops, and soil, the solids can be pulverized or obtained in powder form. Water or an organic solvent is added to the powdered sample, and in most cases, filtration is performed using filter paper and, possibly, a glass fiber filter, to form a sample solution (i.e., the sample extract) for loading and passing through an IAC column.

[0037] In one or more embodiments, the analyte to be captured by the IAC column is selected from toxins, allergens, or antibiotics. In one embodiment, the toxin is a fungal toxin and is selected from the group consisting of: aflatoxin, ochratoxin, deoxyfusinol, fusinol, T2 / HT2 toxin, patulin, zearalenone, citrinin, fumonisin, or analogues thereof. In another embodiment, the aflatoxin is selected from the group consisting of: aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, or aflatoxin M2.

[0038] In some implementations, the IAC column is conditioned with a saline solution (e.g., phosphate-buffered saline) before use. However, conditioning is not necessary and is undesirable in some cases.

[0039] In this technique, any type of solution can be used for analytes eluted from the column after heat treatment. Generally, the wash solution can be selected based on its compatibility with downstream analytical processing equipment or steps. For example, aqueous solutions such as water or brine can be used. In other embodiments, organic solvents such as methanol or a combination of water and methanol can be used. In some embodiments, the wash solution can be the same solution used to form the sample solution. For example, if the sample is formed from a powdered food source and a mixture of water and methanol, the wash solution can also be the same water and methanol mixture used to form the sample extract.

[0040] The method of this technique can be used in conjunction with diagnostic or analytical assay kits. Kits based on this technique include an IAC column, a consumable analytical processing device (e.g., a lateral flow strip, an LC column), and a wash solution in vials compatible with the consumable analytical processing device. The kit also includes instructions for performing a heat treatment step on the IAC column after the sample solution has been loaded and passed through it.

[0041] An illustrative example of such a kit is one relating to the testing of ochratoxin in food sources such as cocoa powder. The kit includes an IAC column (e.g., the OchraTest IAC column, commercially available from Vicam (Milford, MA)) functionalized to capture ochratoxin, a side-flow test strip for detecting the presence of ochratoxin (e.g., the Ochra-V test strip, commercially available from Vicam), and a vial of compatible wash solution, such as methanol, water, or a combination thereof. The kit also includes instructions for performing the method and, in some cases, may include a pipette tip, filter paper, sample extraction solution, and a sample reader (such as the Vertu reader, commercially available from Vicam (Milford, MA)). ™ One or more of the TOUCH readers; and / or calibration information or calibration samples used in conjunction with the sample reader.

[0042] The following examples illustrate the above-described methods and kits of this technology.

[0043] Example The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that have proven effective in this technical practice, and therefore can be considered to constitute a preferred mode of practice for the present invention. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the technology, and similar or analogous results can still be obtained.

[0044] Example 1: Testing of elution temperature and time period for aflatoxin recovery To investigate the effect of temperature on aflatoxin B1 elution, seven aflatoxin samples were generated using aflatoxin B1 analyte standard (product model CRM 44647) available from Sigma Aldrich. Specifically, 5 nanograms of aflatoxin B1 standard were incorporated into 10 ml of 1xPBS solution. This solution was then passed through an IAC column functionalized for aflatoxin capture (Aflatest IAC column, lot number P2309DR, available from Vicam).

[0045] Seven aflatoxin samples were loaded and passed through an Aflatest IAC column at a flow rate of approximately 1 drop every 2 seconds. The column was washed with 2 mL of purified water (from a Millipore Mini-Q purification system) and dried. One column was then subjected to a standard elution technique, and the remaining six columns were subjected to various heat treatments. For the columns subjected to the standard elution technique, 1 mL of methanol was used for elution to produce an eluent. The eluent was then diluted with 1 mL of purified water. 10 µL of the diluted eluent was then injected into a UHPLC column for analysis.

[0046] The remaining six columns were heat-treated for 5 or 10 minutes using heating blocks set to 60°C, 70°C, or 90°C. Specifically, after sample loading and passage, the first column was heated for 5 minutes using a heating block set to 60°C; the second column for 10 minutes using a heating block set to 60°C; the third column for 5 minutes using a heating block set to 70°C; the fourth column for 10 minutes using a heating block set to 70°C; the fifth column for 5 minutes using a heating block set to 90°C; and the sixth column for 10 minutes using a heating block set to 90°C. After each heat treatment, the columns were washed with 1 mL of purified water to form an eluent. 1 mL of methanol was added to the eluent and mixed, and then 10 µL of the mixture was injected into the UHPLC column for analysis.

[0047] Table 1 below provides the amount of aflatoxin recovered from seven samples and their recovery percentages. The results presented in Table 1 show that heat treatment at 90°C for 10 minutes disrupts the binding of aflatoxin to the capture antibody, resulting in a recovery rate exceeding 85% for standards injected into the IAC column. The amount recovered for this specific heat treatment (90°C, 10 minutes) is equivalent to the recovery of samples in which the organic solvent (methanol) was used to elute / chemically dissociate aflatoxin from the capture antibody in the IAC column.

[0048] Table 1: Aflatoxin B1 recovered from the IAC column Example 2: Evaluation of the sensitivity of sideflow test results using different elution techniques .

[0049] To illustrate the advantages of this technique, the following example compares test / control responses read from a lateral flow test after applying various elution techniques. Specifically, the cocoa powder sample was adulterated with a known amount of ochratoxin (e.g., less than 0.5 ppb; 2.1 ppb; or 4.3 ppb). A sample solution was prepared from the ochratoxin-adulterated cocoa powder by measuring 2.5 g of the adulterated sample, adding 10 mL of 80% methanol aqueous solution, and mixing for 2 minutes. 40 mL of 1 xPBS was added to the sample solution and mixed, and the extract was filtered through paper (Fisherbrand P8 filter paper, product model 09-795D). The filtered sample solution (10 mL) was then applied (i.e., 1 drop / second) to an IAC column functionalized to have an affinity for ochratoxin (OchraTest IAC column, available from Vicam); eluent: one of the following three elution methods was applied, and the eluent was tested using a lateral flow test strip (Ochra-V strip, available from Vicam). Note that for elution methods A and B, the eluent was conditioned (i.e., diluted) before the lateral flow test; for elution method C using heat treatment, the eluent was not diluted.

[0050] For elution method A, the captured analyte is eluted using an elution solution (elution solution A) containing 0.1 M glycine and 0.1 M HCl (pH 1.8). 500 µL of a solution containing phosphate, saline, and surfactant (Aqua Premix solution available from Vicam) and 100 µL of 0.5 NaOH solution are added to the elution solution to adjust it. The solution is then injected onto a lateral flow test strip designed to detect ochratoxin (Ochra-V strip available from Vicam).

[0051] For elution method B, the captured analyte was eluted using a different elution solution (elution solution B) containing 0.1 M glycine, 0.108 M HCl, 2% Tween-20 (a nonionic surfactant, including polysorbate, commercially available from Thermo Fisher Scientific Inc.), and 1% sodium dodecyl sulfate (detergent). The elution solution was adjusted by adding 500 µL of 0.1 M NaOH, 2.48% NaCl, and 0.56 μL of Na₂HPO₆, and then injected onto a side-flow test strip (Ochra-V strip, commercially available from Vicam).

[0052] For elution method C, the captured analyte is released from the IAC column by incubating the column in a water bath set to 90°C for 10 minutes. The IAC column is then washed with a saline solution containing a surfactant (a running buffer called Aqua Premix solution, available from Vicam) to form the eluent. The eluent is then injected directly onto a side-flow strip (an Ochra-V strip, available from Vicam).

[0053] After being injected onto the sidestream test strip, the test sample was incubated for 5 minutes and then treated with Vertu. ™ Use a side-flow test strip reader (available from Vicam) to read the strips. Figure 5 Three test results are presented for each incorporation concentration level involving each of the three different elution methods. Figure 5 The sample ID designation identifies the concentration of ochratoxin incorporated into the sample (i.e., 0.5 ppb, 2.1 ppb, or 4.3 ppb) and the elution method used (i.e., A, B, or C). For example, observe... Figure 5 As can be seen from the rightmost column of the illustrated table, elution method C (i.e., the method of this technique) provides the highest response (142% and 89%) compared to conventional methods that require dilution before being injected onto the side-flow test strips.

[0054] In view of this specification, further modifications and alternative embodiments of various aspects of the present technology will be apparent to those skilled in the art. Therefore, this specification should be construed as illustrative only and intended to teach those skilled in the art the general manner of implementing the present technology. It should be understood that the forms of the present technology shown and described herein are intended as examples of embodiments. Elements and materials may be substituted for those shown and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all of which will be apparent to those skilled in the art upon obtaining the benefits of this specification of the present technology. Various changes may be made to the elements described herein without departing from the spirit and scope of the present technology as set forth in the following claims.

Claims

1. A method for eluting bound analytes from an immunoaffinity column, the method comprising: A sample solution containing an analyte of interest is passed through the immunoaffinity column so that a portion of the analyte of interest associates with the stationary phase material of the immunoaffinity column; The immunoaffinity column was heated at a temperature between approximately 65°C and approximately 95°C for a period of time between approximately 2 minutes and approximately 30 minutes to allow the portion of the analyte of interest to dissociate from the stationary phase material; and The immunoaffinity column is rinsed with a solution to produce an eluent containing the dissociated portion of the analyte of interest.

2. The method according to claim 1, further comprising: The eluent is injected into the downstream processing system without modifying the eluent.

3. The method of claim 1, wherein the solution used to rinse the immunoaffinity column is an aqueous solution.

4. The method of claim 1, wherein the immunoaffinity column is conditioned with a phosphate-buffered saline solution before the sample solution is passed through the immunoaffinity column.

5. The method of claim 1, wherein the column is heated at a temperature of about 90°C.

6. The method of claim 5, wherein the column is held at the temperature for about 10 minutes.

7. The method of claim 5, wherein the column is held at the temperature for about 7 minutes.

8. The method of claim 5, wherein the column is held at the temperature for about 5 minutes.

9. The method of claim 2, wherein the eluent is injected into an HPLC column.

10. The method of claim 2, wherein the eluent is injected onto the side-flow test strip.

11. The method of claim 10, wherein a reader is used to determine the ratio of the peak height of the test line to the peak height of the control line.

12. The method of claim 2, wherein the elution buffer is used in an ELISA test.

13. The method of claim 2, wherein the eluent is injected into the LC-MS system.

14. The method of claim 1, wherein the sample solution comprises a powdered food sample mixed with water.

15. The method of claim 1, wherein the sample solution comprises a powdered food sample mixed with an organic solvent.

16. The method of claim 14 or 15, wherein the sample solution is filtered before passing it through the immunoaffinity column.

17. The method according to claim 1, wherein the immunoaffinity column is a fungal toxin immunoaffinity column.

18. The method of claim 16, wherein the mycotoxin immunoaffinity column is an ochratoxin immunoaffinity column.

19. A kit for eluting captured analytes from an immunoaffinity column, the kit comprising: Immune affinity column; A consumable analytical processing device having an inlet for injecting the eluent of claim 1; and Rinse solution in vials compatible with the consumable analytical processing device.

20. The kit of claim 19, wherein the consumable analytical processing device is a side-flow device.

21. The kit of claim 19, wherein the consumable analytical processing device is a liquid chromatography column.

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