WO2019175744A1 - System and methods for rapid analysis of biological samples - Google Patents

Abstract

A system, method and composition for rapid analysis of biological samples described. It discloses the test by manual or automated function achieved by centrifugation, gravitation, vacuum suction, pressure applications or any other method. Test sample obtained from bodily fluids (whole blood) are directly passed through a tube containing a plurality of reaction beads coated with capture antibody/antigen followed by performing sequence of steps of ELISA (enzyme-linked immunosorbent assay) by passing required volumes of wash buffer, HRP (horseradish peroxide) labeled antigen/antibody and Viscous-TMB (3,3',5,5' Tertramethyl benzidine) substrate. The substrate in the microtube is held in position till the appearance of color bands, which indicates the presence or absence of analyte. It significantly improves the sensitivity of rapid test and also allow simultaneous analysis of multiple analytes in a single test. Applicable in clinical diagnostics, molecular biology, agriculture, sericulture, food technology, environmental science, biomedical research and other related fields.

Description

System and methods for rapid analysis of biological samples

Background

The invention discloses a newer system and methods for rapid analysis of biological samples with sensitivities that have been equivalent to or more than that of an ELISA (enzyme-linked immunosorbent assay). In particular, it is applicable for rapid qualitative and quantitative analysis of proteins, nucleic acids, bacteria, virus and carbohydrates derived from bodily fluids (blood, serum, plasma, urine, milk, tears, mucus or saliva). The invention further discloses the use of the system and methods for simultaneous analysis of multiple analytes without compromising on sensitivity levels. The dominant model of laboratory testing throughout the world remains the centralized laboratory in which more and more of the analytical processes are automated to enable the analysis of large numbers of samples at relatively low cost. This trend is well established in biochemistry and hematology and is now extending to other disciplines including microbiology and anatomical pathology. However, healthcare is changing, partly as a result of economic pressures, and also because of the general recognition that care needs to be less fragmented and more patient-centered. The need to make healthcare more patient-centered is also a global trend and is based on the premise that healthcare should be organized more around the patient rather than the provider. Centralized testing does not represent a convenient process for many patients with the testing process often being disconnected from the consultation process such that more than one visit to the doctor is required to complete the assessment process. This problem applies particularly to those with a chronic disease such as diabetes who require regular monitoring including frequent blood tests. The growth in self- monitoring of blood glucose, which is by far the largest segment of Point-of-care testing (PoCT), is in part a testament to this need for more convenient and, in some cases, more effective care. Designers of PoCT devices begin with the needs of their users and these needs will to some extent depend on the clinical setting. However, some features are common to virtually all users in all settings. As documented by St John et al. these key requirements include: a. Simple to use b. Reagents and consumables are robust in storage and usage c. Results should be concordant with an established laboratory method d. Device together with associated reagents and consumables are safe to use With the growing potential for PoCT to improve healthcare in the developing world, particularly through timely detection of infectious diseases, developers of such devices have been guided by more specific design criteria. These are to ensure that the technology can address the needs of the user in a clinically and cost-effective manner and avoid the introduction of possibly expensive devices which fail to deliver the required outcomes. For PoCT devices that will be used in the developed world, some of the ASSURED criteria (as per WHO standards, ASSURED is Affordable, Sensitive, Specific, User-friendly, Rapid & Robust, Equipment-free, Delivered) will also remain relevant but others will be substantially different. Thus, instead of being equipment free, the need will be for relatively sophisticated equipment that at a minimum can provide a quantitative result, presentation of the results, decision support and, ideally, connectivity to other information systems such as the patient’s electronic health record. While the technology to provide all these features undoubtedly exists, they come at a cost which may be difficult to recover using the most common business model for PoCT that is used for the central laboratory, based on complexity and reagent costs, thus only charging for the test strips/cartridges. When one combines these equipment needs with what are seen as other competitive requirements such as a small sample volume, whole blood, production of a result within 10 minutes of applying the sample, ease of use and requisite analytical performance, it is possible to appreciate the technological challenges involved in building such devices. In recent times, a newer challenge has arisen, namely the ability to simultaneously measure multiple analytes on the same cartridge or multiplexing as already known. Multiplexing is a rather broad and undefined term but, with the exception of devices used in critical care such as blood gas analyzers, the number of multi-analyte PoCT devices is relatively few. Of the few that have appeared, those that have the ability for example to measure multiple cardiac enzymes or several different types of drugs are not universally popular with users because they will be charged for all such parameters, irrespective of whether they need the complete panel. However as more healthcare moves away from the hospital into the community, the demand grows for multi-analyte point of care platforms since these avoid the need for several devices, all with the attendant needs of multiple training, quality management and interfacing processes and the increased risk of errors. A typical classification of PoCT technology splits devices into small handheld ones including quantitative and qualitative strips, and those which are larger bench-top devices with more complex built-in fluidics, often variants of ones used in conventional laboratories. Majority of hand held once are based on Microfluidic Technology or Paper based technology. Two decades ago there was much discussion about a concept called Lab on a Chip (LOC) and the view was that this would become the dominant PoCT technology in the future. The development of LOC, also sometimes referred to as microchips, grew from the microelectronics industry through techniques of miniaturization and microfabrication. Such devices have been defined as ones that perform analysis at microscopic scales i.e. 1-500 pm and incorporate microfilters, microchannels, microarrays, micropumps, microvalves and bioelectronics chips. The microchip integrates into one reaction cell all the processes associated with analysis from the placement of the sample into the chip to the analysis itself. Microchips can be fabricated from silicon, glass or polymer. Due to slow commercialization of the LOC concept, lateral flow strip (LFS) technology has continued to dominate the PoCT market. But lateral flow strips have a number of limitations and described in relation to particular components of the strip by Wong et al.⁸ Collectively these limitations result in two major disadvantages which make it difficult for LFS to meet the needs of some PoCT applications. One such need is so-called multiplexing, namely the ability to measure multiple analytes on the same strip. Current technology is limiting LFS to analyze only two or three analytes. The second major problem for LFS technology is limited sensitivity and this has been brought into focus by the need for better technology for infectious disease testing, particularly in the developing world. While there have been incremental improvements in the performance of strip tests for various infectious diseases, a recent review shows that many do not meet the required sensitivity to be practically useful. Much has been published in the research literature on the development of paper- based PoCT devices including a comprehensive review by Yetisen et al (Point of care diagnostics for sexually transmitted infections). At this point it is important to distinguish nitrocellulose which is in lateral flow strips from paper-based devices. Nitrocellulose is a hydrophobic material which is good for binding to proteins but the hydrophobicity has other disadvantages which paper can potentially overcome including being able to be patterned to form microfluidic devices that are capable of multiplexing. Yetisen et al. identifies four capabilities of such paper devices: a. Sample can be dispensed into multiple spatially-segregated areas which enable simultaneous assays to be performed on the same device. b. Samples can move by capillary action so no pumps are needed. c. Only small sample volumes are required. d. No hazardous waste as the device can be incinerated. Many groups including organizations such as‘Diagnostics for All’ are developing such devices for the developing world primarily because paper is a significantly cheaper material than materials used in lateral flow strips. In addition, the potential applications are wider than the infectious disease area with one group developing a paper microfluidics device the size of a postage stamp for measurement of three liver functions tests - so-called‘Lab on a Stamp’. While the potential of this technology is high, Yetison et al. comment that no mature platform for this technology has yet appeared, let alone commercial applications. In spite of the advantages in LFS and paper based PoCT, there is still a need for the development of more sensitive, economical, robust and multiplex assays. Thus, there is an inherent need for the improved method for rapid analysis system. There is a further need for such methods and apparatus which are both economical and practical to use. None of the prior arts discussed herein, taken either singly or in combination, is seen to describe the instant invention as claimed.

Summary

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. The summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the disclosure or delineate the scope of the invention. Exemplary objective of the subject matter is to provide methods and system for rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide a tube packed with reaction beads for use in rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide a tube of different shapes and sizes for use in rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide a reaction bead of different shapes and sized for rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide the materials used for manufacture of tube and reaction beads. Another exemplary objective of the present disclosure is to provide different capture agents that can be used to coat the reaction beads. Another exemplary objective of the present disclosure is to provide different method for coating reaction beads. Another exemplary objective of the present disclosure is to provide different methods for packing of reaction beads in tube. Another exemplary objective of the present disclosure is to provide ways and means of quality check of reaction beads during coating, packing and performance. Another exemplary objective of the present disclosure is to provide ways and means of differentiation of reaction beads after completion of assay. Another exemplary objective of the present disclosure is to provide different labels for use in rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide different detection system for use in rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide different way of performing the assay for rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide different ELISA methods for analysis of biological samples. Another exemplary objective of the present disclosure is to provide the use of viscous enzyme substrate for localized color development for rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide a method for rapid qualitative and quantitative analysis of biological samples. Another exemplary objective of the present disclosure is to provide the use of manual and automated platform for rapid analysis of biological samples. Another exemplary objective of the present disclosure is to provide increased sensitivity of assay by passing same volume of analyte multiple time through the tube or by passing increased volume of analyte through the tube.

Drawings

The advantages and features of the present invention will become better understood with reference to the following more detailed description and claims taken in conjunction with the accompanying drawings. FIG 1 is the figure showing at tube, wherein the tube consists of a narrow hole at one end and a wide opened hole on the other end. The narrow hole can be of different diameters to control the flow of liquid. The wide opened hole is used to connect to a devise or for application of sample. The tube also consists of a capillary tube above the narrow hole for drawing a defined amount of analyte for analysis. The tube consists of a flat surface above the capillary tube to hold the reaction beads in position. FIG 2 is the figure showing a reaction bead. The reaction bead is designed to fit exactly in the tube. The reaction bead is designed to have pores or narrow gaps for smooth flow of liquid. The reaction bead with pores or narrow gaps was also designed to increase the contact between the liquid and the solid support. These reaction beads can be of different sizes, shapes to increase the surface area and visibility of color. FIG 3 is the figure showing reaction beads coated with different capture agents for immunoassay. The capture agent can be an antigen or an antibody or a nucleic acid. FIG 4 is the figure showing reaction beads with markings. The markings act as a quality control check for coating and packing. These marking can be of numbers (e.g 1 ,2,3); abbreviations (-Ve, +Ve, T1 , T2); symbols (-,+,*); alphabets (a,b,c,d); colors (red, blue, green, yellow) or any other means to distinguish beads. FIG 5 is the figure showing markings on the tube or marking on cover that fits the tube or marking on paper with the exact size and shape of tube. These markings can be of numbers (e.g 1 ,2,3); abbreviations (-Ve, +Ve, T1 , T2); symbol (-,+,*); alphabets (a,b,c,d); colors (red, blue, green, yellow) or any other means to distinguish between reaction beads during assay. FIG 6 is the figure showing the arrangement of reaction beads for single analyte and multi anlayte test. FIG 7 (a) is the figure showing a strategy of quantification using MicroTube Rapid ELISA. In this approach the color developed with analyte is compared with that of known standard (Reaction beads treated with different concentrations of analyte for the defined time). FIG 7 (b) is the figure showing a strategy of quantification using MicroTube Rapid ELISA. In this approach the analyte concentration is determined by using varying concentrations of capture antigen. Depending on the concentration of analyte in sample varying number of reaction beads will change color. The no. of beads that change color will help in determining the concentration of analyte. FIG 8(a) is the figure showing a dipstick approach for analysis of analyte using MicroTube Rapid ELISA method. FIG 8(b) is the figure showing a cassette method for analysis of analyte using MicroTube Rapid ELISA. FIG 9 is the figure showing the effect of concentration of capture antibody on sensitivity of MicroTube Rapid ELISA. FIG 10 is the figure showing the effect of concentration of Labeled antibody (secondary antibody) on the sensitivity of MicroTube Rapid ELISA. FIG 11 is the figure showing the sensitivity of MicroTube Rapid ELISA. FIG 12 is the figure show the effect of number of pipetting on sensitivity of MicroTube Rapid ELISA. FIG 13 is the figure showing the effect of sample volume on sensitivity of MicroTube Rapid ELISA. FIG 14 is the figure showing analysis of Human IgG in single analyte test format. FIG 15 is the figure showing analysis of Mouse IgG in single analyte test format. FIG 16 is the figure showing analysis of Human IgG and Mouse IgG in multi analyte (Two Analytes) test format. FIG 17 is the figure showing quantification of Mouse IgG using known standards. FIG 18 is the figure showing quantification of Mouse IgG after spiking in Human serum.

Detailed Description

It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”,“comprising” or“having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms“a” and“an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms“first”,“second”, and“third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

57. This invention relates to system and method for rapid analysis of biological samples. More particularly the invention relates to system and method herein can be applicable for rapid qualitative and quantitative analysis of proteins, nucleic acids, bacteria, virus and carbohydrates.

58. The invented system and method are rapid, simple, economical and sensitive compared to existing rapid methods which rely on lateral flow chromatography technique.

59. This invention is further explained with the help of the following examples and should not be construed to limit the scope of the invention

Materials:

60. Polystyrene tubes (FIG 1 ), Polystyrene reaction beads (FIG 2) were manufactured in-house. Goat anti-human IgG (GAH-lgG), Goat anti-mouse IgG (GAM-lgG), Goat anti-human IgG Horse Radish Peroxidase (GAH-lgG-HRP), Goat anti-mouse IgG-HRP (GAM-lgG-HRP), Human IgG, Carboxy methyl cellulose (CMC), glycerol, Poly Vinyl Pyrolidone (PVP), Hydrogen Peroxide (H2O2), 3,3’,5,5’Tetramethylbenzidine (TMB), Phosphate citrate buffer (pH 4.2), 100mM Carbonate buffer pH 9.6, Phosphate Buffer Saline (1xPBS), Phosphate Buffer Saline /0.1 %Tween 20 (1xPBS-T) and BSA.

Preparation of Reaction Beads for Coating:

61. The polystyrene reaction beads were washed with PBS-T for 5 minutes with mixing, followed by boiling Milli-Q water for 5 minutes. Reaction beads were then separated and air dried at room temperature. Coating of Reaction Beads:

62. Coating was carried out by placing the reaction beads in freshly prepared 100mM carbonate buffer (pH 9.6) containing required amount of capture antibody and incubated at room temperature for 4 hr. Reaction beads were then washed with 1xPBS and then blocked with 1 % BSA/PBS for 1 hr at room temperature. Following blocking reaction beads were washed with 1xPBS and air dried at room temperature. These coated reaction beads are ready for packing or can be stored in humidity free container at room temperature for several months. Reaction beads used as negative control were blocked in 1 % BSA/PBS for 1 hr, washed and dried.

Assembly of capture antibody coated reaction beads in tube:

63. Reaction beads were assembled by simply stacking them in the tube. Different analyte reaction beads and negative control were packed such that they are separated by 1 or more un coated reaction beads. After stacking reaction beads were held tightly in the tube by placing an O-ring on top.

MicroTube Rapid Test Procedure:

64. The test was performed the same way as that of sandwich ELISA described with minor modifications to volume and time of incubation. All the steps involved in conventional sandwich ELISA were performed in the tube with varying volume, number of pipettings, time and incubation temperature. In general the sequence of steps for MicroTube Rapid ELISA are as follows.

Step 1 : Equilibration- 200 ul of wash buffer was pipetted and held for 30 sec to wet the reaction beads.

Step 2: 10Oul of analyte was pipetted 10 times

Step 3: Reaction beads were washed with 200ul of PBS-T by pipetted 10 times

Step 4: 10Oul of labeled antibody (secondary antibody) was pipetted 10 times and

Step 5: Reaction beads were washed with 200ul of PBS-T by pipetting 10 times. Step 6: Finally, 10Oul of viscous enzyme substrate was pipetted and held in position till the appearance of blue color bands.

Step 7: Once the color was developed photo was taken to record the results.

The volume and number of pipetting of analyte and secondary in steps 2 and 4 varies depending on the number of inserts packed in the tube and the sensitivity required.

Example 1 : Optimization of concentration of capture antibody for MicroTube Rapid ELISA

65. Every immune assay involves a combination of antibody and antigen interactions, no one capture concentration is ideal and optimization is essential.

Optima! capture antibody concentration for coating of reaction bead was determined by keeping analyte (Human IgG) and labeled antibody (GAH-lgG- HRP) constant. Reaction beads coated with 0.061 , 0.125, 0.25, 0.5 and 1 mg/ml of GAH-lgG were packed in a tube by staking the reaction beads in ^'ascending order of capture antibody concentration. Uncoated reaction beads were used to separate reaction beads with capture antibody. MicroTube Rapid ELISA was performed as described in methods with the following modifications. 180ul of reagent was pipetted in step 2, 4 and 6 instead of 10Oul. Human-!gG was used at 10ug/ml, GAH-!gG-HRP was used at 1 :1000 dilution. Reaction bead with increasing concentration of capture antibody (GAH-lgG) showed increasing sensitivity as seen in FIG 9. The order of sensitivity was found to be 1 mg/mi > G.5mg/m! > Q.25mg/mi > 0.125mg/m! > 0.061 mg/m! respectively.

Example 2: Optimization of concentration of labeled antibody for MicroTube Rapid ELISA

66. The optimal labeled antibody concentration, which gives the best signal with minimum background, must be determined experimentally for each assay and is usually determined by using a series of dilutions in a titration experiment. A titration experiment is done by first selecting a fixed capture antibody and analyte concentration, and then a series of experimental dilutions of the labeled secondary antibody. In this experiment, we have chosen five tubes in which one reaction bead coated with 50 ug/ml of capture antibody (GAH-lgG) was assembled as described in method. Test was performed as described in method with 10 mg/ml analyte (Human IgG) and different dilutions of Labeled secondary antibody (GAH-lgG-HRP) dilutions (1 :100, 1 :200, 1 :400, 1 :800 and 1 : 1600). Labeled secondary antibody with a diluted 1 : 100 has given the best signal compared to other dilutions as seen in FIG 10.

Example 3: Sensitivity of MicroTube Rapid ELISA

67. Analytical Sensitivity or detection limit is the lowest level of analyte concentration that can be distinguished from background. Sensitivity highly depends on the affinity of antibody and needs to be determined for each and every paid of antibodies. Othe factos such as contact between analyte and capture antibody, volume of analyte and concentration of labeled antibody were also know to increase the sensitivity. In the current example sensitivity was tested by using all the possible combination such as no. of passes, volume of analyte, concentration of labeled secondary antibody were used to determine the sensitivity of MicroTube Rapid ELISA. In this experiment twelve tips were each packed with one 50 ug/ml capture antibody coated reaction bead as described in the methods. MicroTube Rapid ELISA was performed with varying conditions for different concentrations of analyte (Human IgG) as shown in Table - 2

The concentrations, no. of pipettings chosed for each set of experiments had resulted in increasing the sensitivity of MicroTube Rapid ELSA. From the FIG 11 it was clear that MicroTube Rapid ELISA can detect as low as 1 fg/m I of Human IgG.

Example 4: Effect of no. of passing of analyte on the sensitivity of MicroTube Rapid ELISA

68. As observed in Example-3, the analyte detection sensitivity can be increased by increasing number of passings. The effect of number of passings is tested in this experiment. Three tips were packed with one, 50 ug/ml GAH IgG coated reaction beads as described earlier. MicroTube Rapid ELISA was performed with varying number of pipetting. In step 2 and step 4 of the methods 100 ul each of 10 ug/ml of analyte (Human IgG) and 1 : 1000 diluted labeled secondary antibody (GAH- IgG-HRP) were passed once, 5 times and 10 times in different tubes respectively. The detection sensitivity increased with increase in the number of passing’s as shown in FIG 12.

Example 5: Effect of volume of analyte on the sensitivity of MicroTube Rapid ELISA

69. Since the number of pipetting had an effects the sensitivity, the effect of volume of analyte is tested in this experiment. Three tips were packed with one 50 ug/ml GAH IgG coated reaction beads as described earlier. MicroTube Rapid ELISA was performed on these tips with varying volumes of analyte. Since the volume of this micro tube design is limited to 200 ul, this experiment is performed by gravity flow method where the solutions are loaded at the top end of the tube and allowed the solutions to come in contact with reaction beads by gravity. Tubes were first equilibrated with 1000 ul of PBS-T and then 100, 500 and 1000 ul of 10 ug/ml analyte (Human IgG) is passed through different tube by gravity flow. Tubes were then washed each with 2000ul of PBS-T and 10Oul of 1 :1000 diluted labeled secondary antibody (GAH-lgG-HRP) was then passed through each of tube. Tubes were then washed each with 5000ul of PBS-T. Tubes were attached to multichannel micro pipettor and 10Oul of substrate was aspirated into each of the tube and held until colour development. From the FIG 13 it was clear that sensitivity of MicroTube Rapid ELISA increases with increase in the volume of analytes.

Example 6: Comparison of no. of passing to that of volume passed for sensitivity.

70. Since both number of passings and volume of analyte effect sensitivity of MicroTube Rapid ELISA, it is important to know if one is better that the other for sensitivity. In this experiment, number of times an analyte is passed through the reaction beads by repeated passings the same volume of analyte is compared to single passing by gravity flow. Since Analyte meets reaction beads twice in each passing by pipetting, 100 ul of analyte pipetted 5 times is compared to passing 1000ul of analyte. Two tubes were packed with 50 ug/ml GAH IgG coated reaction beads as described in methods. MicroTube Rapid ELISA was performed on both tubes as described earlier with the following changes. In step 2 of methods, Pipette was set at 180ul and 100 ul of 10ug/ml analyte (Human IgG) was passed 5 times in one tip while 1000ul of 10ug/ml of analytes is passed through the other tube by gravity flow. In step 4 of methods, 100 ul of 1 :1000 diluted labeled secondary antibody (GAH-lgG-HRP) is passed once in both the tubes. From the FIG 14 it was clear that sensitivity is same with both the methods.

Example 7: WlicroTube Rapid EUSA for Single Analyte 71. In this example two different analytes (Human IgG and Mouse IgG) were tested individually using single analyte test format as shown in FIG 6A. For both the tests the capture agent and the concentration of capture agent for different reaction beads (positive control, negative control, and test) are shown in TABLE 3. The reaction beads were then packed in 3 different tube for each test (HumanlgG and Mouse IgG). MicroTube Rapid ELISA for single analyte was performed as described in method with known concentration of analyte and labeled secondary antibody as shown in TABLE 4. Two different analytes were used in each assay to determine the specificity of the assay as well.

72. From the FIG 14 (Single Analyte Test For Human IgG) and FIG 15 (Single Analyte Test For Mouse IgG) it was clear that single analyte test was function with respect to sensitivity and specificity. Reaction beads coated with GAH-lgG- HRP only reacted with Human IgG. Similarly, Goat Anti Mouse IgG-HRP reacted with only Mouse IgG. Background and false positives were also ruled out the presence of no blue colored band development in negative control reaction bead.

Example 8: MicroTube Rapid ELISA for multiple analytes (2 analytes)

73. Multiplexing will reduce the number of assays that needs to be performed thereby making it simple and economical. In this example two different analytes (Human IgG and Mouse IgG) were tested in one single tube. The capture agent and concentration of capture agent for different reaction beads (positive control, negative control, and Test-1 (Human IgG) and Test-2 (Mouse IgG)) for multiplex assay were shown in TABLE 5. The reaction beads (positive control, negative control, Test-1 and Test-2) were then packed in 4 different tube for multiplex analysis. MicroTube Rapid ELISA for multiplex analysis of two different analytes was performed as described in method with known concentration of analyte and labeled secondary antibody as shown in TABLE 6.

TABLE 5:

74. From the FIG 16 it was clear that multiplex was functional with respect to sensitivity and specificity. Tube 1 which was treated with only Human IgG gave two bands. Tube 2 which was treated with only Mouse IgG gave two bands, Tube

3 which was treated with both Human IgG and Mouse IgG gave 3 band and Tube

4 which was treated with no analyte gave only one band. These results collectively indicate that the multiplex analysis of analytes using MicroTube Rapid ELISA was specific. The presence of background and false positives was also ruled out by the presence of no band with negative control beads in all the tubes and also lack of no bands in negative control bead and Goat Anti Human IgG and Goat Anti Mouse IgG in Tube 4.

Example 9: Quantitative Analysis of Analyte Using MicorTube Rapid ELISA.

75. Semi quantitative analysis of analytes using MicroTube Rapid ELISA can be performed in different approaches. Two such approaches are shown in FIG 7(A) and FIG 7 (B). In the current experiment we have optimized quantification of Mouse IgG using known standard (FIG 7 A). Standard were prepared by coating reaction bead with 100ug/ml GAM-lgG for 4hr. The reaction beads each were then treated with known concentration of Mouse IgG (5, 10, 15ug/ml) for 2 minutes and washed with 1xPBS. The beads were then packed in a tube as shown in FIG 7(a) in descending order from top to bottom. Quantification was initially tested with 5, 10 and 15ug of analyte (Mouse IgG) using three tubes packed with one reaction bead each coated with 100ug/ml GAM-gG. MicroTube Rapid ELISA was performed as described in method by pipetting the analyte (Mouse IgG) and holding for 2 minutes and standard tube for 2 min with 1xPBS. All the standards and quantification tubes were treated with 1 : 1000 diluted labeled GAM-lgG-HRP for 2min. Images of color development were captured and were shown in FIG 17. From the figure it was clear that all the analytes 5, 10 and 15 ug/ml Mouse-lgG had given similar color development to that of known standard.

76. Quantification was further tested using serum spiked sample. In this study 11 ug of Mouse-lgG was added to Human serum and repeated quantification procedure as above. Serum spiked Mouse-lgG resulted in similar colored band to that of 10ug/ml standard in standard tube in FIG 18. This further confirms the use of MicroTube Rapid ELISA for quantification.

Claims

Claims im:

1. A MicroTube Rapid ELISA comprising: a tube packed with a plurality of reaction beads, the said tube contains a narrow hole on one end to reduce the flow rate of liquid being drawn and released and wide opening on the other end for connecting it to a device or for applying a sample; a reaction bead coated with capture agent; an O-ring to hold the plurality of reaction beads in position;

2. The tube of claim 1 wherein, the said tube is selected from a group consisting of but not limited to tube, capillary, tip, housing, column, and tapered column; or a combination thereof.

3. The tube of claim 1 wherein, the said tube is of any shape or size.

4. The tube of claim 1 wherein, the said tube is of a volume between 0.0001 and 100 milliliters, and length of the said tube is between 0.1 -1000 mm, and internal diameter of the said tube is between 0.0001 -100 mm.

5. The tube of claim 1 wherein, multiple units of the said tube are joined together in any type of configuration including but not limited to 2-unit, 8-unit, 48-unit, 96- unit, 384-unit or 1536-unit formats.

6. The tube of claim 1 wherein, the said tube is made of one or more materials selected from a group consisting of but not limited to polytetrafluoroethylene, polysulfone, polyethersulfone, cellulose acetate, polystyrene, polystyrene/ acrylonitrile copolymer, PVDF and glass and combination thereof.

7. The tube of claim 1 wherein, the said tube is made of transparent material for direct visualization or detection.

8. The tube of claim 1 wherein, the said tube has white color on one side for clear visibility of colored bands formed on the reaction beads.

9. The tube of claim 1 wherein, the said tube contains markings on the tube or on a separate cover that fits the tube to distinguish between reaction beads during assay.

10. The tube of claim 1 wherein, the said tube contains a capillary tube of 0.001 -3mm diameter and 0.001 to 20mm length on one end of the tube to draw a defined amount of sample for analysis.

11. The tube of claim 1 wherein, the narrow hole of the tube is below or above the capillary tube for collecting defined amount of sample for analysis.

12. The tube of claim 1 wherein, the said tube contains a flat surface above the capillary tube or narrow hole to hold the reaction beads in position.

13. The tube of claim 1 wherein, the said O-ring is made of one or more materials se l ecte d fro m g ro u p co n si sti n g of b ut n ot l i m i ted to si l i co n , polytetrafluoroethylene, polysulfone, polyethersulfone, cellulose acetate, polystyrene, polystyrene/acrylonitrile copolymer, PVDF and glass and combination thereof.

14. The tube of claim 1 wherein, the said reaction beads is of any shape and size.

15. The tube of claim 1 wherein, the said reaction beads is made of one or more materials selected from the group consisting of but not limited to polytetrafluoroethylene, polysulfone, polyethersulfone, cellulose acetate, polystyrene, polystyrene/acrylonitrile copolymer, PVDF and glass and combination thereof.

16. The tube of claim 1 wherein, the said reaction beads is made of transparent or colored material.

17. The tube of claim 1 wherein, the said reaction beads is coated with antibodies, antigens, receptors, ligands, oligo-nucleotides, haptenes, aptamers, and prokaryote and eukaryote cells.

18. The tube of claim 1 wherein, the said reaction bead is coated by non-covalent or covalent boding or a combination of both.

19. The tube of claim 1 wherein, the said reaction bead is coated in a buffer selected from buffers having a pH, in the range of from 6 to 12, with molarity ranging from 0.005 to 0.2M.

20. The tube of claim 1 wherein, the said reaction beads is coating by spraying or soaking in coating buffer or equivalent method for a duration of 1 min to 24 hours at 0-37 degrees Celsius.

21. The tube of claim 1 wherein, said reaction bead is marked with number, symbol, alphabets or a combination of the above for quality cheque during manufacturing.

22. The tube of claim 1 wherein, the reaction beads packed in the tube consists of Positive Control reaction bead, Negative Control reaction bead and a Test reaction bead.

23. The tube of claim 22 wherein, the reaction bead for Positive Control reaction bead always results in colored band, Negative Control reaction bead results in no colored band and the Test reaction bead gives blue colored band in the presence of analyte and no colored band in the absence of analyte.

24. A method in a MicroTube Rapid ELISA comprising: a plurality of volumes of analyte, wash buffer, labeled antige/antibody being passed in sequence for the assay; a viscous enzyme substrate is passed at the end of the assay and is held in the tube till the development of colored bands due to enzyme reaction;

25. The method according to claim 24, where in methodology is selected from the group consisting of but not limited to Direct ELISA, Indirect ELISA, Sandwich ELISA, Competitive/Inhibition ELISA.

26. The method according to claim 24 wherein, the said analyte is a protein, nucleic acids, bacteria, virus and carbohydrates.

27. The method according to claim 24 wherein, the said label is fluorescent, chemilluminescent, radioactive or a colorimetric label.

28. The method according to claim 24, wherein in the method involves mixing of analyte and labeled antibody/antigen in a tube before applying it to tube.

29. The method according to claim 24, wherein the said viscous enzyme substrate is of varying viscosities and chromogenic agents.

30. The method according to claim 24, wherein the method is used for both qualitative and quantitative analysis of biological sample.

31. The method according to claim 24, wherein the method shows increasing sensitivity with the increase in the volume, number of passes and reducing the flow of analyte and increasing concentrations of labeled antibody/antigen.

32. The method according to claim 24, wherein the method shows increased sensitivity with the use of signal amplification strategies which include but not limited to poly-HRP, Biotin-Streptavidin signal amplification.

33. The method according to claim 24, wherein the method is used to analyze one or more analytes (multiplex analysis) in a single test.

34. The method according to claim 24, wherein the method is performed manually, with the use of devises which function semi-automatically and automatically.

35. The method according to claim 24, wherein the result is either directly visible to naked eye or can be detected with the use of instrument.

36. The method according to claim 24, wherein, the said method is applicable in clinical diagnostics, molecular biology, agriculture, sericulture, food technology, environmental science, biomedical research and other related fields.

37. The method according to claim 24, wherein the method is used for development of rapid kits.

38. The method of claim 37 wherein, the said rapid kit is used at bed side, medical emergency sites, in medical labs and home based medical care.

39. The method according to claim 24, wherein the method is adaptable across technologies used for rapid analysis of biological samples using immunological methods.