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Testing device for identifying antigens and antibodies in biofluids

a biofluid and antigen technology, applied in the field of identification of antigens and antibodies within biofluids, can solve the problem of no convenient low cost disposable tests available for “on the spo

Inactive Publication Date: 2012-12-20
MONASH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]It has been found that red blood cell agglutination, triggered by specific antigen interaction, drastically decreases blood wicking and dispersion on paper or chromatographic media. The agglutination process also considerably enhances the chromatographic separation (elution) of the individual blood components, especially the red blood cells from the serum. The testing device according to the present invention can allow direct analysis of blood cells because of this visual indication. This can be performed instantaneously, either with a detection / reporting system built-in to the device or in conjunction with other off-line analytical equipment. The testing device may also allow for the identification and quantification of specific biomolecules (eg antigens and antibodies) based on induced coagulation, followed by the wicking and elution (separation) of the soluble protein fraction from the blood sample onto the porous substrate. The blood colloids, whose coagulation directly affects their wicking / separation, can either be present in the fluid of interest, such as the red blood cells of blood, or introduced as nanoparticles (gold, silver, micro-silica, zeolite, titanium dioxide and the like). In the latter case, the nanoparticle is typically covered with the specific counter-biomolecule or molecule of interest used as sensitive reporter component. The colloid particles may be of a size ranging from 1 nm to 100 μm and may be introduced into the biofluid being analysed.
[0018]In the application of the present invention for determining the type of blood group, the present invention may determine the antigens present within a blood sample, the antigens determining whether the blood type is type A, B, O, AB and Rhesus+ / −. Antibodies A, B and D (Rhesus) are deposited into separate detection zones. It may also be preferable to include an untreated control zone in one of the detection zones. A drop of blood is then deposited on the central collection zone, the blood sample being transferred by capillary action to each of the detection zones. When the blood sample contacts an appropriate antibody, the reaction of the red blood cells antigen with its corresponding antibody results in agglutination or coagulation of the red cells. This agglutination results in a drop in velocity of the movement of the blood sample along the microfluidic channel providing the detection zone and separation of the red blood cells from the serum. The velocity of the blood samples travelling along other detection zones with non-specific antibodies is unaffected. This visual contrast facilitates easy and rapid identification of the blood type of the blood sample.
[0019]The applicant has developed a low cost paper based microfluidic system which is described in International patent application no. PCT / AU2009 / 000889 details of which are incorporated herein by reference. The microfluidic systems described in this application utilise a paper based substrate, with the described fabrication methods producing hydrophilic microfluidic channels on the paper based substrates. It should be noted that the term paper is used in this application to refer to all cellulosic materials including woven fabrics and non-woven cellulosic material as well as paper. The microfluidic systems described in these applications can be readily adapted for the purpose of the present invention.

Problems solved by technology

Surprisingly, and in spite of its vital importance, there are no convenient low cost disposable tests available for “on the spot” analysis of blood type.

Method used

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  • Testing device for identifying antigens and antibodies in biofluids
  • Testing device for identifying antigens and antibodies in biofluids
  • Testing device for identifying antigens and antibodies in biofluids

Examples

Experimental program
Comparison scheme
Effect test

example 1

Sequential Agglutination / Coagulation of Blood Followed by Wicking on Paper: B+ (Two Step Process) (See FIG. 1)

[0044]Antibody A and B (Epiclone™ Anti-A, Anti-B, and Anti-D; CSL, Australia) solutions were used. Anti-A and Anti-B come as blue and yellow colour reagents, respectively. ‘B+’ blood was used in this study. The blood sample was supplied into plastic vials with anti-coagulant. ‘B+’ blood was separately mixed with pure Anti-A and Anti-B (as received) to prepare 100 μL solution. Paper strips (70 mm×2 mm) were made from Whatman#4 filter paper on which 2 mm unit marks were printed. The paper strips were soaked into phosphate buffer saline (PBS). Excess PBS was removed from the paper strips using standard blotting papers (Drink Coster Blotting, 280 GSM). The paper strips were then placed on Reflex Paper (80 GSM). 20 μL of every mixed solution was dispensed at the centre of paper strip using a calibrated micro-pipette. Pictures were taken after 4 minutes wicking.

[0045]It can be see...

example 2

Sequential Agglutination / Coagulation of Blood Followed by Wicking on Paper: O+ (Two Step Process) (See FIG. 2)

[0052]Antibody A and B (Epiclone™ Anti-A, Anti-B and Anti-D; CSL, Australia) solutions were used. Anti-A and Anti-B come as blue and yellow colour reagents, respectively. ‘O+’ blood was used in this study. The blood sample was supplied into plastic vials with anti-coagulant. ‘O+’ blood was separately mixed with Anti-A and Anti-B to prepare 100 μL solution. Paper strips (70 mm×2 mm) were made from Whatman#4 filter paper on which 2 mm unit marks were printed. The paper strips were soaked into phosphate buffer saline (PBS). Excess PBS was removed from the paper strips using standard blotting papers (Drink Coster Blotting, 280 GSM). The paper strips were then placed on Reflex Paper (80 GSM). 20 μL of every mixed solution was dispensed at the centre of paper strip using a calibrated micro-pipette. Pictures were taken after 4 minutes wicking.

[0053]It can be seen that:

[0054]O+ bloo...

example 3

Simultaneous Agglutination / Coagulation of Blood Followed by Wicking on Paper: Effect of Antigen Concentration (One Step Process) (See FIG. 3)

[0060]In another embodiment of the invention, the paper is first treated with specific antibodies, dried or conditioned before been exposed to a sample of pure blood. This example provides a single step treatment in which the only requirement is to deposit a drop of blood on the paper. This example also illustrates the effect of diluting the antibody solution on the wicking and separation performance of blood on paper. Antibody dilution affects the ratio blood (with its antigen) antibody.

[0061]Antibody A and B (Epiclone™ Anti-A and Anti-B; CSL, Australia) solutions were used. Anti-A and Anti-B come as blue and yellow colour reagents, respectively. “AB+” and ‘B+’ blood were used in this study. The blood sample was supplied into plastic vials with anti-coagulant. Paper strips (70 mm×2 mm) were made from Whatman#4 filter paper on which 2 mm unit m...

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Abstract

A testing device for identifying an antigen or antibody within a biofluid sample including: a substrate having a hydrophilic surface thereon; the surface including a collection zone, and at least one detection zone extending therefrom; wherein the biofluid sample can be mixed with a specific antigen or antibody, and deposited on the collection zone and transferred by capillary action to the detection zone; the antigen or antibody in the biofluid sample reacting with an appropriate said antibody or antigen thereby resulting in a visual indication within the detection zone.

Description

FIELD OF THE INVENTION[0001]The present invention is directed to the identification of antigens and antibodies within a biofluid. While the invention will be described with specific reference to its use in determining a person's blood type, it is to be appreciated that other applications of the invention are also envisaged.BACKGROUND TO THE INVENTION[0002]Blood is essential for sustaining living tissue, with the most important roles of supplying oxygen and other soluble nutrients, immune protection and metabolic turnover. While it is a tissue in its own right, blood in a chemical sense can be considered as a stable, highly packed colloid suspension made of red blood cells (erythrocytes, 4-6 million / mL, 6-8 μm), white cells (leukocytes, 4000-6000 / mL, 10-21 μm), platelets (150,000-400,000 / mL, 2-5 μm) carried within a fluid solution (serum) containing a host of biomolecules (eg albumins, fatty acids, hormones), metabolites and electrolytes. A subset of these biomolecules, such as the b...

Claims

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Application Information

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IPC IPC(8): C12M1/34G01N21/82
CPCG01N30/90G01N33/558G01N21/82G01N33/80G01N2021/752G01N2021/757G01N2021/825G01N33/54389
Inventor GARNIER, GILSHEN, WEIKHAN, MOHIDUS SAMADLI, XUTHOUAS, GEORGE
Owner MONASH UNIV
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