The present invention relates to a rapid immunoassay assay device for simultaneous determining human ABO/RH blood type with inbuilt controls and method thereof. Present invention provides an alternative to the conventional agglutination tests and methods, lateral flow tests and methods, tests and methods using enzyme substrate and uses control sera. These tests require skilled manpower, very complicated in structure, method and test procedure is not user friendly, time consuming, costly and subjective and does not provide in built controls for accuracy of the test.

Background of the Invention:

[003] The determination human blood type is essential for blood transfusion or are planning to donate blood or appropriate medical treatment. Receiving blood that’s incompatible with the blood type could trigger a dangerous immune response.

[004] Most blood contains antigens, which are substances that make the body produce antibodies. Usually antibodies are for things like viruses and bacteria, but in a wrong transfusion the immune system sees the new blood as an intruder that must be destroyed. Which can prove fatal.

[005] Two blood group systems are important for transfusions:

[006] In the ABO Grouping System, there are four types of blood: Type A, Type B, Type AB, and Type O, which are determined by the presence or absence of certain antigens on the surface of red blood cells.
Group A: has only the A antigen on red cells (and B antibody in the plasma). Group B: has only the B antigen on red cells (and A antibody in the plasma). Group AB: has both A and B antigens on red cells (but neither A nor B antibody in the plasma).
Group O: has neither A nor B antigens on red cells (but both A and B antibody are in the plasma).

[007] Immune systems are adapted to individual blood types. If someone from Group B donated blood to someone from Group A, the B antibodies would recognize the B antigen as a threat and blood clumping would occur. But if someone from Group B donated to someone in Group AB there are
no B antibodies, so the immune system doesn’t recognize the intruder.

[008] additionally, there’s the Rh factor blood grouping system. The Rh antigen is either present (+) or absent (-) in the blood. Typically, Rh negative blood goes to patients without the antigen, and Rh positive blood goes to patients who have the antigen but an Rh positive patient can receive Rh negative blood without any problems.

[009] So there are eight blood groups i.e. A Rh+, A Rh-, B Rh+, B Rh-, AB Rh+, AB Rh-, O Rh+, and O Rh -. To figure out a person’s blood type, doctors can use two methods: ABO Typing or back typing.

[010] In ABO typing, doctors take blood and mix it with serums containing the antibodies in Type A and B blood. If your blood cells stick together when mixed with Anti-A serum, one has type A blood; Anti-B serum, one have type B blood; Both anti-A and anti-B serums, one have type AB blood; If blood cells do not stick together when anti-A and anti-B are added, one have type O blood.

[011] People with O Rh- blood are universal donors, but there are also universal receivers, they have the AB Rh+ blood type. The opposite is true for plasma donors. O plasma contains antibodies for both A and B, so it would cause an immune reaction in any other blood type. AB plasma doesn’t have either.

[012] Blood typing is done prior to a blood transfusion or when classifying a person’s blood for donation. Blood typing should be fast and easy to ensure that the patient receive the right kind of blood during surgery or after an injury. If patient has received incompatible blood, it can lead to blood clumping, or agglutination, which can be fatal.

[013] Blood typing is especially important for pregnant women. If the mother is Rh-negative and the father is Rh-positive, the child will likely be Rh-positive. In these cases, the mother needs to receive an appropriate drug which will keep her body from forming antibodies that may attack the baby’s blood cells if their blood becomes mixed, which often happens during pregnancy.

[014] The US Application No. 12/995,288 teaches about the method of determining human blood type and test kit, however, structure of the device, test procedure and interpretation of the test is very complicated, not user friendly and not have inbuilt controls for accuracy of the test.
Objectives:

[015] The object of the present invention is to provide a novel rapid immunoassay assay device for simultaneous in-vitro determination of human ABO/Rh blood type.

[016] It is another object of the present invention to provide a novel rapid immunoassay device for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control.

[017] Yet another object of the present invention to provide a novel rapid flow- through immunoassay device for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control.

[018] It is another object of the present invention is to provide a method for spotting the membranes with anti A monoclonal antibody, anti B monoclonal antibody, anti D monoclonal antibody, anti-goat IgG antibody and anti-human RBC antibody for rapid immunoassay method for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt negative and positive control.

[019] It is another object of the present invention is to provide a novel rapid immunoassay device and method for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control which is highly sensitive and high specific.

[020] Yet another object of the present invention to store a novel rapid flow through immunoassay device for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control at room temperature which is easy for handling and will reduce transportation cost.

[021] The object of the present invention is to reduce time for simultaneous determination of human ABO/Rh blood type with positive and negative control to determine the test results visually within two minutes.

[022] It is another object of the present invention is to reduce cost for simultaneous determination of human ABO/Rh blood type with positive and negative control which does not require any instruments or skilled person to perform the test.

[023] Yet another object of the present invention is to provide an improved and simple rapid immunoassay device for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control to the man of ordinary prudence which will take short time to perform the method.

Summary of the Invention:

[024] To achieve the objectives, this invention provides a novel rapid flow through immunoassay device (TA) for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control having close assembly comprising:

a. upper casing (UC) having at least five inlet holes/windows (H1 to H5) for dropping sample, wash buffer and for visibility of the test result; and

b. Spacer/separator pad followed by spotted membrane mounted side by side three times over large backing card (B1) for testing and two times over small backing card (B2) for controls. More particularly the testing backing card (B1) mounted with spacer/separator pad (P1) followed by membrane (M1) spotted with anti A monoclonal antibody, thereafter spacer/separator pad (P2) followed by membrane (M2) spotted with anti B monoclonal antibody, thereafter spacer/separator pad (P3) followed by membrane (M3) spotted with anti D monoclonal antibody and the spacer/separator pad (P4) is mounted after the third spotted membrane (M3). The controls backing card (B2) mounted with spacer/separator pad (P5) followed by membrane (M4) spotted with anti-goat IgG antibody, thereafter spacer/separator pad (P6) followed by membrane (M5) spotted with anti-human RBC antibody and the spacer/separator pad (P7) is mounted after the spotted membrane (M5). The said spacer/separator pads (P1 to P7) and spotted membranes (M1 to M5) are mounted on backing cards (B1 & B2) with adhesive material and the spotted membranes are visualized through each inlet holes/windows (H1 to H5) of the upper casing (UC); and

c. lower casing (LC) having five square ribs (R1 to R5) below the inlet holes/windows (H1 to H5) of the upper casing (UC) to support backing cards (B1 & B2) with spacer/separator pads (P1 to P7) and spotted membrane (M1 to M5) and control flow of sample and wash buffer; seven medium thin horizontal ribs (R6 to R12) parallel to square ribs R1 to R5, two small vertical tin ribs (R13 & R14) on left and right side to each square ribs (R1 to R5) to support backing cards (B1&B2); one large thin rib (R15 & R16) at top of each raw and one bracket thin rib (R17 & R18) at the bottom of each raw to hold backing cards (B1 & B2) mounted with absorbent pads (P1 to P7) and spotted membranes (M1 to M5).

[025] The present invention also provides a method for spotting the membranes with anti A monoclonal antibody, anti B monoclonal antibody, anti D monoclonal antibody, anti-goat IgG antibody and anti- human RBC antibody with dilution buffer as herein described for rapid flow through immunoassay method for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt negative and positive control.

[026] The present invention also provides a rapid flow through immunoassay method for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control with application of blood sample followed by the wash buffer as herein described through inlet holes/windows (H1 to H5) of the assay device (TA) and visually determine human blood type with negative and positive control as herein described.
[027] The subject invention may best be understood with reference to the accompanying drawings which are for example purpose and non-limiting.

Brief Description of the Accompanying Drawings:

FIGURE - 1 represents the rapid immunoassay device (TA) for simultaneous in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control
FIGURE - 2(a) & 2(b) represent upper casing (UC) and lower casing (LC) respectively
FIGURE - 3(a) & 3(b) represent mounted absorbent pads (P1 to P4) with spotted membranes (M1 to M3) over testing backing card (B1) and mounted absorbent pads (P5 to P7) with spotted membranes (M5 to M5) over control backing card (B2) respectively
FIGURE – 4 Backing cards (B1 & B2) with mounted pads spacer/separator pads (P1 to P7) and spotted membrane (M1 to M5) locked on the lower casing (LC)
FIGURE - 5 represents principle of rapid immunoassay device and method
FIGURE - 6 represents the interpretation of the rapid immunoassay test result

Detail description of the Invention:

[028] The present invention describes a novel rapid flow-through immunoassay device (TA) for simultaneous determination of human ABO/Rh blood type with inbuilt positive and negative controls and method thereof.

[029] The flow-through immunoassay device (TA) comprises upper casing (UC), lower casing (LC), support cards (B1 & B2), spacer /separator pads (P1 to P7) and spotted membranes (M1 to M5).

[030] According to the preferred embodiment immunoassay device (TA) [fig.1] for carrying out the method can be of any shape preferable in rectangle shape comprise closed assembly having 40 x 80 mm with thickness of 3.5 to 5 mm having upper casing (UC) [fig.2(a)] and lower casing (LC) [fig.2(b)] made preferably from polypropylene although one may use wide variety of organic or inorganic material, natural or artificial or combination of any polymers. The upper casing (UC) [fig.2(a)] contains 5 inlet holes/windows H1 to H5 of any shape, preferably three holes/windows H1 to H3 having in one row and two holes/windows H4 & H5 in another raw, for dropping of blood sample and wash buffer. According to the preferred embodiment the three holes/windows are round from upper side (US) having 10.42 mm diameter and round at lower side (LS) having 4.80 mm diameter in one raw for testing and two holes/windows are round from upper side (US) having 10.42 mm diameter and square at lower side (LS) having 4.8mm x 4.8mm in another row for controls to free flow the sample and buffer. The lower casing (LC) [fig.2(b)] having five square ribs R1 to R5, having height on 1 mm below the inlet holes/windows H1 to H5 of the upper casing (UC) to support backing cards (B1 & B2) and for proper pressure to control flow of sample and wash buffer; seven medium thin horizontal ribs R6 to R12 having height of 1 mm parallel to square ribs R1 to R5 to support backing cards (B1 & B2), two small thin vertical ribs R13 & R14 on left and right side of each square ribs R1 to R5, one large thin rib (R15 & R16) at top of each raw and one bracket thin rib (R17 & R18) at the bottom of each raw all having to hold backing cards (B1 & B2) mounted with spacer/separator pad (P1 to P7) and spotted membranes. The height of each vertical ribs (R13 & R14), large thin ribs (R15 & R16) and bracket thin ribs (R17 & R18) are higher than the support ribs (R1 to R12).

[031] According to the preferred embodiment the support card (B1 & B2) made preferably from plastic material such as Polyvinyl Material or PVC having thickness of 1 to 1.5 mm. The testing support card B1 is preferably 80 mm x 300 mm and controls support card B2 is preferably 50 mm x 300 mm as described in fig. 3(a) & 3(b).

[032] According to the preferred embodiment the spacer/separator pad (P1 to P7) is a medium of thick weight, cellulosic material lies cotton linter material. Each spacer/separator pad (P1 to P7) should have the sufficient absorbent capacity of 99.2 mg/cm2, so as to retain the absorbed liquid material i.e. wash buffer and sample that filter through or flow through the porous membrane (M1 to M5). The thickness of the said spacer/separator pad (P1 to P7) is preferably 1.5 to 2.0 mm with capillary flow rate 63.3 s/4cm. The spacer/separator pad (P1 to P7) can be cut into variety of shapes of limited size, so as to mount on the support card (B1 & B2) and also fit inside the immunoassay device (TA). According to the present invention the spacer/separator pad at P1, P2, P3, P5 & P6 having size of 15 mm x 5 mm, P4 having size of 8 mm x 5 mm and P7 having size of 8 x 5 mm as described in fig. 3(a) and 3(b).

[033] According to the preferred embodiment membranes M1 to M5 having pore size of 12 micron made up of cellulosic material preferably of glass fiber and the membranes should be unbacked with plastic sheet, allowing either belt or air side of the membranes to be used. Also the pore size of the membranes should not be too large so that a good volume of surface ratio can be obtain. Additionally, the membranes material must allow agglutination of the red blood cells with the antibody spotted on the membrane. The membrane can be cut in to variety of shapes suitable to the dimensions of the inlet holes/windows H1 to H5 of upper casing (UC) of the test apparatus (TA) so as to allow sample and wash buffer to pass through porous membranes (M1 to M5) only, preferably in square or rectangle shape having size of 6 mm x 5 mm and with 1 to 1.25mm thickness.

[034] According to the preferred embodiment the absorbent pad (P1 to P7) and spotted membrane (M1 to M5) mounted side by side three times over the backing card (B1) for testing and two times over control backing card (B2) for controls. More particularly the testing backing card mounted with absorbent pad P1 followed by membrane M1 spotted with anti A monoclonal antibody, thereafter absorbent pad P2 followed by membrane M2 spotted with anti B monoclonal antibody, thereafter absorbent pad P3 followed by membrane M3 spotted with anti D monoclonal antibody and absorbent pad P4 is mounted after the spotted membrane M3 over the testing backing card (B1). The controls backing card (B2) mounted with absorbent pad P5 followed by membrane M4 spotted with anti-goat IgG antibody, thereafter absorbent pad P6 followed by membrane M5 spotted with anti-human RBC antibody and the absorbent pad P7 is mounted after the membrane M5. The absorbent pads (P1 to P7) and membranes (M1 to M5) are mounted on backing cards (B1 & B2) with adhesive material and the membranes (M1 to M7) are visualized through each inlet holes/windows (H1 to H5) of the upper casing (UC).

[035] According to the preferred embodiment the membrane of the test device (TA) contains three test regions for A, B and D, one negative control (NC) and one positive control (PC) on the upper casing (UC). According to the preferred embodiment blood type test dot(s) includes distinct spotting of monoclonal antibody i.e. anti A monoclonal antibody, anti B monoclonal antibody and anti D monoclonal antibody is immobilized at region A, B and D respectively on the membrane M1 to M3 respectively and negative control (NC) and positive control (PC) includes distinct spotting of anti-human antibody i.e. anti-goat IgG antibody and anti-human RBC antibody is immobilized near region at NC and PC respectively on the membrane M4 & M5 as shown in the figure 1 & 3(b). According to the preferred embodiment the antibodies to be diluted separately with diluent material preferably TE buffer having 7.5 pH. The dilution ratio per each test A, B and D regions will be 1:1 and for each controls (NC & PC) regions will be 1:5. The spotting volume will be 1 uL/mm and the size of each dots is from 4.5 mm to 5.5 mm preferably in round shape. Thereafter, put the spotted membranes (M1 to M5) in to DH chamber at 25 °C for 4 hours for drying and after drying seal the membranes (M1 to M5) until process of mounting on the backing cards (B1 & B2).

[036] According to the preferred embodiment fix the testing backing card (B1) mounted with absorbent pads (P1 to P4) and spotted membrane (M1 toM3) with anti A, B and D monoclonal antibody respectively on support ribs and lock with the locking ribs R15 & R17 and control backing card (B2) mounted with absorbent pads (P5 to P7) and spotted membrane (M4 toM5) with anti-goat IgG antibody and anti-human RBC antibody respectively on support ribs and lock with the locking ribs R16 & R18 on lower casing (LC), [fig.4] put upper casing (UP) and ensure that the spotted membranes (M1 to M5) are visualized through each inlet holes/windows (H1 to H5) of the upper casing (UC) and thereafter seal the housing by pressing with the lower casing (LC) to active immunoassay device (TA) for simultaneous determination of human ABO/Rh blood type with inbuilt negative and positive control.

[037] According to the preferred embodiment the wash buffer of PBS, 2% SDS and the pH of the solution is 7.5 to 8.0 in volume 50µ1.

[038] According to the preferred embodiment the testing method will be performed with testing device (TA) and wash buffer. For this purpose, take the testing device (TA) and label it with the appropriate identification and put it on flat clean surface. Thereafter drop 5-10 µ1 of blood sample in to center of the each holes/windows H1 to H5 of the testing device (TA) and allow it to soak completely. Thereafter drop 50µ1 (2 drops) of wash buffer in to center of the each holes/windows H1 to H5 of the testing device (TA) and allow it to soak completely and interpret the test results within two minutes.

[039] According to the preferred embodiment the presence or absence of the A, B and D antigens on human red blood cells can be determined by testing the red blood cells with the respective antisera, specifically Anti-A, Anti-B and Anti-D. The procedure is based on the principle of agglutination (fig. 5) and interpretation of test results shown in fig. 6 of the accompanying drawings.
a. If coloured spot appeared in test region A and D with control region PC, then blood type/group A Rh+;
b. If coloured spot appeared in test region B and D with control region PC, then blood type/group B Rh+;
c. If coloured spot appeared in test region A, B & D and control region PC, then blood type/group AB Rh+;
d. If coloured spot appeared in test region D and control region PC, then blood type/group O Rh+;
e. If coloured spot appeared in test region A with control region PC, then blood type/group A Rh-;
f. If coloured spot appeared in test region B with control region PC, then blood type/group B Rh-;
g. If coloured spot appeared in test region A and B with control region PC, then blood type/group AB Rh-;
h. If coloured spot appeared in control region PC only, then blood type/group O Rh-;
i. If coloured spot appeared in control region NC, irrespective of the other dots, then the test is invalid;
j. If none of the coloured spot appeared, then the test is invalid.

[040] According to the present invention the inbuilt control dot at region NC will work as quality control dot which confirms the proper functioning of the device and correct procedural application.

[041] According to the present invention the kit containing test device to provide a novel rapid immunoassay device and method for simultaneously in-vitro determination of human ABO/Rh blood type with inbuilt positive and negative control having 100% sensitivity and 100% specificity.

[042] According to the present invention the kit containing the test device and wash buffer can be stored at +4 to +40 ? and the self-life of the kit is 12 months from the date of manufacturing.

[043] The invention can be described more clearly with reference to following examples, but these are for illustrative purpose only and same should not be construed to restrict the scope of the invention.

Example – 1: Assembly of Test Device (TA)

a. Test Backing Card (B1):
Take 80 mm x 300 mm support card having thickness of 1 mm. Mount absorbent pad P1 followed by membrane M1 spotted with Anti-A monoclonal antibody, then mount absorbent pad P2 followed by membrane M2 spotted with Anti-B monoclonal antibody then mount absorbent pad P3 followed by membrane M3 spotted with Anti-D monoclonal antibody, thereafter mount absorbent pad P4. Mount absorbent pads followed by spotted membranes side by side with adhesive material on backing card B1. Thereafter mount the said backing card on support ribs at tests regions and lock with the locking ribs on the lower casing (LC)
b. Control Backing Card (B2) :
Take 50 mm x 300 mm support card having thickness of 1 mm. Mount absorbent pad P5 followed membrane M4 spotted with anti-goat IgG antibody, again stick absorbent pad P6 followed membrane M5 spotted with anti-human RBC antibody, thereafter stick absorbent pad P7. Mount absorbent pads followed by spotted membranes side by side with adhesive material. Thereafter mount the said backing card on support ribs at controls regions and lock with the locking ribs on lower casing (LC).

c. After mounting both the backing cards (B1 & B2) mounted with absorbent pads (P1 to P7) and spotted membrane (M1 to M5) on the lower casing (LC) put the upper casing (UC) and ensure that the spotted membranes (M1 to M5) are visualized through each inlet holes/windows (H1 to H5) of the upper casing (UC) and thereafter seal the housing with pressing.

Example – 2 : Spotting of membranes (M1 to M5):

Take a 6 mm size glass fiber membrane and arrange the membranes separately in the tray for spotting anti-A monoclonal antibody, anti-B monoclonal antibody anti-D monoclonal antibody, anti-goat IgG antibody and anti-human RBC antibody with spotting identification of the tray. Arrange the membranes (M1 to M5) on separate tray in such a way that membrane should not be touched and maintain the record of exposure to Thermo Lab Stability Chamber for each tray.

Prepare the test spots solution by taking anti-A monoclonal or polyclonal antibody, anti-B monoclonal or polyclonal antibody anti-D monoclonal or polyclonal antibody separately and dilute in normal saline and the dilution ration is 1:1 and spotted separately on membrane M1 to M3 respectively. Similarly, prepare the negative control (NC) spot solution by taking anti-goat IgG antibody and positive control (PC) spot solution by taking anti-human RBC antibody from the same species from ABO antibodies raised from, separately and dilute in normal saline and the dilution ration is 1:5 and spotted separately on membrane M4 & M5 respectively.

The spotting volume will be 1 uL/mm and the size of each dot is from 4.5 mm to 5.5 mm in round.

Thereafter, put the spotted membranes in to DH chamber at 25 °C for 4 hours for drying and after drying seal the membranes until process of mounting on the plastic support cards.

[044] It should be noted that the description merely illustrates the principles of the present subject matter. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described herein, embody the principles of the present subject matter and are included within its spirit and scope. Furthermore, all examples recited herein are principally intended expressly to be for explanatory purposes to aid the reader in understanding the principles of the present subject matter and the concepts contributed by the inventor(s) to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and implementations of the invention, as well as specific examples thereof, are intended to encompass equivalents thereof.

[045] Although implementations of present subject matter have been described in language specific to structural features and/or methods, it is to be understood that the present subject matter is not necessarily limited to the specific features or methods described. Rather, the specific features and methods are disclosed and explained in the context of a few example implementations of the above-mentioned aspects of the present subject matter.

We Claim:

1. A rapid immunoassay device (TA) for simultaneous in-vitro determination of human ABO/Rh blood type comprising:

- upper casing (UC) having at least five inlet holes/windows (H1-H5), preferably three holes/windows for test regions (H1-H3) and two holes/windows for controls regions (H4-H5)
- at least two support cards (B1 & B2)
- at least seven absorbent pads (P1-P7)
- at least five membranes (M1-M5), separately coated/spotted with Anti A monoclonal antibody, Anti B monoclonal antibody and Anti D monoclonal antibody at test regions, anti-goat IgG antibody at negative control (NC) region and anti-human RBC antibody at positive control (PC) region
- lower casing (LC) having ribs (R1-R5) below each inlet holes/windows (H1-H5)

2. A rapid immunoassay device (TA) as claimed in claim 1 wherein each coated/spotted antibody are diluted separately with TE buffer having 7.5 pH

3. A rapid immunoassay device (TA) as claimed in claim 1 wherein coated/spotted antibody dilution ration is 1:1 for each test regions and 1:5 for each control regions

4. A rapid immunoassay device (TA) as claimed in claim 1 wherein coated/spotted volume is 1 uL/mm for each regions

5. A rapid immunoassay device (TA) as claimed in claim 1 wherein coated/spotted preferably in round shape with coated/spotted size 4.5 to 5.5 mm

6. A rapid immunoassay device (TA) as claimed in claim 1 wherein each rib (R1-R5) on lower casing (LC) having height of 1mm
7. A rapid immunoassay device (TA) as claimed in claim 1 wherein each support card having thickness preferably of 1 to 1.5mm

8. A rapid immunoassay device (TA) as claimed in claim 1 wherein each absorbent pad having capacity of 99.2 mg/cm2

9. A rapid immunoassay device (TA) as claimed in claim 1 wherein each absorbent pad having thickness preferably 1.5 to 2mm with capillary flow rate 63.3 s/4cm

10. A rapid immunoassay device (TA) as claimed in claim 1 wherein each membrane having pore size of 12 micron

11. A rapid immunoassay device (TA) as claimed in claim 1 wherein each membrane preferably made-up of glass fiber.

12. A rapid immunoassay device (TA) as claimed in claim 1 substantially as herein described with reference to and as illustrated in the accompanying drawing.