Technical Field

This invention relates to a device for the detection of dextran. In particular, the invention relates to a test strip device for use in a competitive immunoassay for qualitative determination of dextran.

Background Art

Dextran, derived from bacteria, is a polymer of glucose. The presence of dextran in sugar industry causes several difficulties during the sugar production process, for example, increased viscosity, slower filtration rate, elongated sugar crystal shape, and increased sugar loss. In 2017/2018, statistical analysis showed that if 1000 ppm/Brix of dextran are found in cane juice in Thailand, the loss of sugar will be up to 125 million kilograms, costing around 1.5 billion baht. This will affect the mill’s revenue and country’s economy.

The current methods for dextran quantification in sugar industry are alcohol haze, Roberts’ copper and ASI II. These biochemical methods are time consuming and require instruments and large amount of sample for dextran measurement. Also, they are not specific for dextran detection as the results can be interfered by starch and other polysaccharides present in the reaction. In addition, a large variety of dextrans in terms of molecular weight and structure, resulted from bacteria strains and environmental conditions, leads to their different functional properties e.g. solubility. These factors could bring about dextran measurement error by biochemical methods.

The most accurate and precise analytical method for dextran determination is enzyme-HPLC analysis (Proc Comm Int Tech Suer (CITS), 1999, 21 : 259-265 and Proc S Afr Sug Technol Ass, 2000, 74: 317-327). First, dextran was ethanol precipitated. Then, dextranase enzyme was added to digest dextran, and the hydrolysis product is further quantified by HPLC. However, this method takes a lengthy time to process and the cost per unit is relatively high. Therefore, it was not designed as a routine analytical procedure but as a research tool.

The immunological assay for dextran has recently been introducted to the sugar industry (Proc Sug Ind Technol, 1999,58, 747). The method is based on the specific binding of monoclonal antibody to dextran. The reaction time is short because of rapid formation of antigen-antibody complex. The turbidity of this complex can be mesured by a nephelometer. Although this method is quick and easy to use, the cost is still high for routine test of large number of samples in the agricultural product processing. Therefore, there is a need for a simple, rapid, accurate and cost-effective assay for on-site analysis of dextran in all sugar streams, providing operations to be optimized accordingly to avoid slowdowns and loss of sucrose.

From the Science Citation Index Expanded database, there is no report related to the rapid dextran test kit based on immunochromatographic assay.

Thai patent application number TH 1301005362 involves semiquantitative detection of dextran based on competitive immunochromatographic assay. The device consists of a test band of immobilized dextran, a control band of goat anti-mouse IgG antibody, and an anti- dextramgold conjugate zone. It measures three dextran concentration levels in first juice dependent on the intensity of test band: <1000 ppm/Brix, >1000 ppm/Brix, 1000-2000 ppm/Brix with 85% confidence interval. However, the reading of band intensity for a large number of test samples is impractical and this device was designed for the test sample of first juice only. The technology needs to be further developed for other test samples in the sugar processing, which are different in term of chemical and physical characteristics, for example the purity and the viscosity.

The present invention involves rapid qualitative detection of dextran related to competitive immunoassay. The ratios of dextran immobilized on the test band to the antibody specific to dextran conjugated to gold nanoparticle (Au NP-anti-dextran) on the conjugate pad were optimized on the condition that the amount of immobilized dextran on the test band must be higher than signal saturation point leading to the decrease of false positive and false negative. The cut off value of this device is about 900 ppm/Brix, which is useful for determining dextran impurity in the sugar processing. The sensitivity, specificity and accuracy for dextran detection are 92.9%, 93.5%, and 93.3% with the reading time 5 minutes. Overall, the efficiency of this device is beter than the prior art.

Summary of Invention

The invention provides a device for qualitative detection of dextran in a test sample, including but not limited to sugarcane streams. The device has good performance characteristics (sensitivity 92.9%, specificity 93.5%, and accuracy 93.3%) and shows the result in a short time.

The present invention involves a test strip for qualitative detection of dextran. The device utilizes the principle of competitive immunoassay. The device comprising:

- backing (1) for supporting the device components;

- membrane (4) sitting on the backing for chromatography movement of samples; sample pad (2) for test sample loading area, at one end of membrane (4);

- conjugate pad (3) next to the sample pad (2), and immobilized by the antibody specific to dextran conjugated to gold nanoparticle;

- test band (5) positioned at a certain distance from the conjugate pad, and on the opposite side of the sample pad. The test band (5) has immobilized dextran for result judgement of the presence of dextran; and

- control band (6) next to the test band (5) on the other end of membrane (4) for confirming that the system is operating properly.

Brief Description of Drawings

Fig.l represents the dextran detection device of the present invention in exploded sideview, composed of backing (1), sample pad (2), conjugate pad (3), membrane (4), test band (5), control band (6), absorbent pad (7), and strip cassette (8).

Fig.2 illustrates the top-view of dextran detection device showing sample pad (2), membrane (4), test band (5), control band (6), and strip cassette (8).

Fig.3 illustrates schematic diagram of competitive immunochromatography for qualitative detection of dextran.

Fig.4 depicts the results for 45 test samples from cane comparing the test strip device of the present invention and the haze method.

Fig.5 illustrates the application of the test strip device of the present invention with palmyra palm juice.

Description of Embodiments

The present invention involves a device / test strip for qualitative detection of dextran. The device utilizes the principle of competitive immunoassay. The device comprising:

- backing (1) for supporting the device components;

- membrane (4) sitting on the backing for chromatography movement of samples; - sample pad (2) for test sample loading area, at one end of membrane (4);

- conjugate pad (3) next to the sample pad (2), and immobilized by the antibody specific to dextran conjugated to gold nanoparticle;

- test band (5) positioned at a certain distance from the conjugate pad (3), and on the opposite side of the sample pad (2). The test band (5) has immobilized dextran for result judgement of the presence of dextran; and

- control band (6) next to the test band (5) on the other end of membrane (4) for confirming that the system is operating properly.

The ratio of dextran immobilized on the test band (5) to the antibody specific to dextran conjugated to gold nanoparticle (Au NP-anti-dextran) on the conjugate pad (3) can be in the range from 1:1 to 1:10 (v/v) on the condition that the amount of immobilized dextran on the test band (5) must be 2-3 times of signal saturation point on the membrane (4).

In an embodiment of the present invention, the ratio of dextran immobilized on the test band (5) to Au NP-anti-dextran on the conjugate pad (3) can vary from 1 :4 to 1 :6 (v/v).

In an embodiment of the present invention, the size of gold nanoparticle can vary from 10 to 100 nm in diameter.

In a preferred embodiment of the present invention, the size of gold nanoparticle can vary from 20 to 50 nm in diameter.

In an embodiment of the present invention, the device has the cut off value ranging from 0.1 to 0.5 mg/mL dextran

In a preferred embodiment of the present invention, the device has the cut off value ranging from 0.15 to 0.3 mg/mL dextran

In an embodiment of the present invention, the control band (6) is immobilized with secondary antibody.

In a preferred embodiment of the present invention, the secondary antibody comprising at least one antibody selected from the group consisting of goat anti-mouse IgG, rabbit antimouse IgG, horse anti-mouse IgG, donkey anti-mouse IgG, or chicken anti-mouse IgG

In a preferred embodiment of the present invention, the secondary antibody is goat antimouse IgG. In an embodiment of the present invention, the device further comprising the strip cassete with at least 1 well and at least 1 window. The well is positioned at the sample pad (2) and the window is positioned at the test band (5) and control band (6).

In an embodiment of the present invention, the device further comprising absorbent pad (7), next to the control band (6), for collecting excess fluid and preventing backflow.

In an embodiment of the present invention, the membrane (4) comprising at least one membrane selected from the group consisting of nitrocellulose membrane, polyvinylidene fluoride, nylon, and polyestersulfone.

In an embodiment of the present invention, the conjugate pad (3) comprising at least one membrane selected from the group consisting of nitrocellulose membrane, cellulose filter, glass fiber, and polyester film.

In an embodiment of the present invention, the sample is selected from the group consisting of fresh cane juice, diluted cane juice, cane extracts, sugar crystals from cane, first juice, mixed juice, clarified juice, raw syrup, and raw sugar.

Conjugation of gold nanoparticle with antibody specific to dextran

The anti-dextran antibody in phosphate buffer or borate buffer, pH 8.0-9.5 is mixed with gold solution containing gold nanoparticle in the preferred range from 20 to 40 nm in diameter in phosphate buffer or borate buffer, pH 8.0-9.5, and incubated at 22-28 °C for 5- 15 minutes.

After that, 10% (w/v) BSA is added into the mixture and leaved at 22-28 °C for 10 minutes. Next, the mixture is centrifuged at 4 °C, 10,000 rpm for 15-30 minutes and the supernatant is discarded. The 20mM Tris-HCl, pH 8.0-9.5 buffer containing 1% (w/v) BSA, is used to resuspend the pellet, providing the solution of gold nanoparticle conjugated with antibody specific to dextran.

Principle and result analysis of the device

The principle of the device in the present invention is based on competitive theory as shown in Fig.3. The competition is between the migrating analyte and the immobilized analyte for the binding to the migrating Au NP-anti-dextran conjugate. The test sample, for example can juice, on the sample pad (2) of the device in the present invention for dextran determination, can be undiluted or diluted with a chase buffer in the ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (v/v)

In case of cane juice having high dextran, the Au NP-anti-dextran conjugate on the conjugate pad (3) binds to the dextran in the test sample, generating the Au NP-anti-dextran-dextran complex. The complex moves chromatographically on the membrane (4) to the test band (5) immobilized with dextran. However, there is no binding between the migrating conjugate and the dextran on the test band according to the fixed amount of Au NP-anti-dextran conjugate on the conjugate pad (3), already fully formed the complex with the dextran in the test sample. Therefore, if the test sample has a lot of dextran above the cut off value of the device, there is no color band on the test band (5).

The test sample, cane juice, travels by capillary action from the sample pad (2) to the absorbent pad. When the fluid is at the control band immobilized with the secondary antibody, which is goat anti-mouse IgG as an example in the present invention, the goat antimouse IgG binds to the anti-dextran antibody conjugated to gold nanoparticle in the case of test sample having no dextran and/or the Au NP-anti-dextran-dextran complex in case of test sample having dextran, providing a red color band in both cases. The control band is the quality indicator of the device. Dextran determination by the device in the present invention takes about 5 minutes.

Examples

Example 1: Dextran analysis of pure dextran with known concentrations in cane juice The device in the present invention is a strip based on the competitive immunochromatography. The strip comprises sample pad (2), which is glass fiber, membrane (4), which is nitrocellulose membrane, conjugate pad, absorbent ad, and backing made from plastic.

The control band (6) on the membrane (4), which is nitrocellulose membrane can be prepared by spraying goat anti-mouse IgG.

The test band (5) can be prepared by spraying dextran around 2 and 2.5 times of signal saturation point on membrane (4) for dextran detection. The conjugation pad (3) can be prepared by spraying the antibody specific to dextran conjugated to gold nanoparticle by using the ratio of dextran immobilized on the test band (5) to the antibody specific to dextran conjugated to gold nanoparticle (Au NP-anti-dextran) on the conjugate pad (3) of 1 :5 (v/v). Then, the strip is enclosed within a strip cassette (8).

The test samples are various concentrations of dextran in cane juice, which are dropped into the sample pad (2). After that, the result can be read at approximately 5 minutes. The analysis can be done by red-band visualization. The negative result is indicated by two red bands on both test and control bands (5 and 6) and the positive result is indicated by a red band on the control band (6).

The cut off value of the device can be determined by finding the lowest concentration of dextran giving the positive result on the device as shown in Table 1. The cut off value of the device in the present invention is approximately 0.2 mg/mL (between 0.2 and 0.3 mg/mL).

Table 1: The relationship of dextran concentrations in cane juice and color intensity on test band, providing the cut off value of dextran test strip.

Example 2: Dextran detection in cane juice

To evaluate the performance of the device in the present invention for dextran detection, the test samples from cane, i.e. first juice, mixed juice, clarified juice, raw syrup, and raw sugar are used and the results are compared to a gold standard haze method. The procedure for dextran analysis by the device in the present invention is as follows:

1. The test sample is applied to the sample pad. The test sample can be undiluted or diluted into a buffer at a dilution of sample to buffer of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (v/v). The preferred ratio for the defined cut off value and the most appropriate use of the device is in the range from 1:1 to 1:5 (v/v). When the test sample is in the solid form, the sample must be dissolved at the concentrations about 10%-40% (w/v) before applying to the device in the present invention.

2. The test result can be read at about 5 minutes

3. Result interpretation

3.1 Positive result: only the control band (6) appears visible color line, indicating that the sample test has higher dextran than the cut off value of the device

3.2 Negative result: both the test (5) and control (6) bands appear the color, indicating that the sample test has no dextran or lower dextran than the cut off value of the device.

3.3 Invalid result: both the test (5) and control (6) bands remain blank or only the test band (5) appears the color, indicating that the device does not operate properly.

Development of a device based on competitive immunoassay is initiated by choosing the right amount of the antigen, which is dextran, on the test band, which generally does not reach the signal saturation point. Other conditions are further optimized to gain the desired cut off value. However, the device in the present invention uses the dextran approximately 2 and 2.5 times of signal saturation point on the test band, giving higher efficiency than the prior art using the dextran below the signal saturation point for dextran detection. The number of false positives and false negatives decrease as can be seen in Table 2 and 3.

From Table 2, the amount of dextran at approximately 2 and 2.5 times of signal saturation point on the test band provides better results than those at approximately 1, 1.5 and 1.75 times of signal saturation point. These results can be explained by the complexity of various structure of dextran, leading to its dynamic binding to the antibody. The ratio of dextran immobilized on the test band to Au NP-anti-dextran on the conjugate pad in the range from about 1:4 to 1:10 (v/v) is also optimized and the one in combination with the optimization of dextran immobilized on the test band giving the best result is 1 :5 (v/v) as shown in Table 3-

The dextran detection of 45 agricultural samples by the device in the present invention (Fig.4) indicates the approximate cut off value of the device 900 ppm/Brix. The results are compared to a gold standard haze method to calculate the sensitivity, specificity, and accuracy of the device. From Table 3, the sensitivity, specificity, and accuracy of the device for dextran detection in the present invention are 92.9%, 93.5%, and 93.3%, respectively, whereas the efficiency of the prior art is about 15% lower than the device in the present invention, and its use is limited to the test sample first juice. The dextran test strip in the present invention is a very efficient device and easy to use for a non-technical person. Therefore, it is a suitable device for dextran detection in the industry, helping to improve the manufacturing process.

Table 2: The effect of dextran quantity (times of signal saturation point) immobilized on the test band on the efficiency of qualitative dextran test strip.

+: Dark red band. +/-: Faint red band. No red band. NA: Not available. FN: False negative.

FP: False positive. TN: True negative. TP: True positive. Table 3: The performance of the dextran test strip in the present invention by using dextran two times of the signal saturation point on the test band in combination with a range of the ratio of dextran immobilized on the test band to Au NP-anti-dextran on the conjugate pad.

Example 3: Dextran detection in palmyra palm juice

The dextran detection in palmyra palm juice is performed as described in Example 2. The results indicate the successful application of the device in the present invention with the test samples from palmyra palm juice. There is no band on the test band (positive result) for the sample with approximately >900 ppm/Brix of dextran, whereas there is a red band on the test band (negative result) for the sample with approximately <900 ppm/Brix of dextran as shown in Fig.5. These results suggest the high efficiency of the device in the present invention for dextran detection in various types of agricultural samples.

Best mode for carrying out the invention

As mentioned in the Description of Embodiments