WO2008104081A1

WO2008104081A1 - Parallel immunoassay device

Info

Publication number: WO2008104081A1
Application number: PCT/CA2008/000391
Authority: WO
Inventors: Richard A. Galli; John Huang
Original assignee: Biolytical Laboratories Inc.
Priority date: 2007-03-01
Filing date: 2008-02-29
Publication date: 2008-09-04
Also published as: CA2578313A1

Abstract

The present invention relates to test devices comprising a porous membrane, a control and distinctly applied multiple recombinant antigens, of different combinations of epitopes for the same disease marker, that are capable of accurate, parallel detection of the presence of disease specific antibodies in a given specimen.

Description

TITLE OF THE INVENTION

Highly Accurate Rapid Parallel Immunoassay Device

BACKGROUND OF THE INVENTION

The rapid tests' ability to produce accurate and reliable results depends not only on the intrinsic quality of the tests themselves but also on the extrinsic factors such as the quality/type of biological specimens, the ability of the user to correctly perform the tests, and the prevalence rate of the target disease in a given population. The probability that a single rapid test will accurately determine the true infection status of a person varies with the prevalence of the infectious disease in that population.

Generally, when there is a higher prevalence of an infectious disease in a population, there's also a greater probability that a person from that population testing positive is truly infected (i.e. the greater the positive predictive value). This means that the proportion of biological specimens testing false-positive for a given disease decreases in low prevalence settings. Similarly, specimens showing negative test results in high prevalence settings may have a higher likelihood of being falsely negative (i.e. negative predictive value).

According to World Health Organization and UNAIDS' recommendations, published on 21 March 1997, in the context of rapid antibody testing for HIV diagnosis, while taking into consideration of different prevalence of infection settings, three disease testing strategies have been presented to statistically improve the accuracy of the test results while minimizing the cost. These strategies are again published by WHO/UNAIDS in 2001 in "Guidelines for Using HIV Testing Technologies in Surveillance". The strategies are described below.

Strategy I: Requires one test; for use in diagnostic testing in populations with an HIV prevalence >30% among persons with clinical signs or symptoms of HIV infection; for use in blood screening, for all prevalence rates; and for use in surveillance testing in populations with an HIV prevalence >10%.

Strategy II: Requires up to two tests; for use in diagnostic testing in populations with an HIV prevalence <30% among persons with clinical signs or symptoms of HIV infection or >10% among asymptomatic persons; and for use in surveillance testing in populations with an HIV prevalence <10%.

Strategy III: Requires up to three tests; and for use in diagnostic testing in populations with an HIV prevalence <10% among asymptomatic persons.

Strategies II and III, as shown in Fig. 9, can be performed in serial or in parallel. WHO/UNAIDS adopts the serial testing algorithms with the concern that parallel testing algorithms require higher cost as two or more tests are always used simultaneously. Serial testing may be lengthy and may require more than one specimen collection. Advantages for performing parallel testing include: Reduction in the risk of false negative results in high prevalence populations; reduction in the risk of false positive results in low prevalence populations; require only a single specimen, avoiding the collection of additional specimens; favorable perception that two or more tests are better than one; and reduction in the stigma of the patient being called back for the second test. It is also noted in WHOAJN AIDS' recommendations that the tests used for the above testing algorithms should contain different antigens. Fig. 8 shows a parallel testing algorithm for HIV (source: Global AIDS Program, Centers for Disease Control and Prevention, USA).

Flow-through tests employ solid-phase capture technology, which involves the immobilization of antigens on a porous membrane. The specimen flows through the membrane and is absorbed into an absorbent material on the opposite side. A dot or a line visibly forms on the membrane when developed with a signal reagent (usually a colloidal gold or selenium conjugate). Some tests allow the detection of multiple diseases or disease subtypes by immobilizing antigens from disease specific markers to different locations on the membrane. One such example is Bio-Rad Laboratories' Multispot™ HIV-l/HiV-2 Rapid Test. The flow-through tests usually require a few steps for the addition of specimen, wash buffer, and signal reagent. Several flow-through devices include a procedural control on the membrane; the appearance of a colored dot or line at this location confirms the test has been performed correctly. US Patent Publication No. 20030165970 and International Patent Publication No. WO 03098215 describe examples of flow-through devices.

Lateral-flow strips incorporate both antigen and signal reagent into a strip of porous membrane and absorbent materials. The specimen, sometimes followed by a buffer, is applied to the absorbent/sampling area of the device. Alternatively, the specimen is diluted in a vial of buffer, into which the test device is inserted or from which a quantity is drawn out and applied to the test device. The specimen combines with the signal reagent and laterally migrates through the porous membrane. A positive reaction results in a visual line on the membrane where antigens have been applied. A procedural control line is usually applied to the strip at a location beyond the antigen line. A visual line at both the test and control locations indicates a positive test result, a line only at the control location indicates a negative test result, and the absence of a line at the control location means the test is invalid. Some tests apply different antigens in different locations and allow differentiation of antibodies to two or more disease specific antibodies. In most lateral-flow devices, the test strip is encased in a plastic cartridge. EP 0306772, GB 2204398, EP 38619, EP 0225054, EP 0183442, and EP 0186799 describe examples of lateral-flow devices.

SUMMARY OF THE INVENTION

In light of the above strategies published by WHO/UNAIDS and CDCs parallel testing algorithm, the present invention relates to diagnostic test kit designs comprising multiple recombinant antigens, of different combinations of epitopes for the same disease marker, that are capable of accurate, simultaneous, parallel detection of the presence of disease specific antibodies in a single specimen applied to a single test device, thus avoiding the need to perform multiple rapid tests or to repeatedly collect specimens. The present invention therefore greatly improves the accuracy of current immunoconcentration (flow-through) and immunochromatographic (lateral-flow) rapid tests.

In one aspect, the invention provides a rapid test device comprising a porous membrane, a procedural control applied to the porous membrane, and two or more recombinant antigens applied to the porous membrane. The recombinant antigens comprise differing combinations of epitopes for a disease marker and are applied at visually distinct locations on the porous membrane to simultaneously capture antibodies specific to the disease marker in a single collected specimen.

In another aspect, the rapid test device is a flow-through rapid test device. In yet another aspect, the rapid test device is a lateral-flow test device. In one aspect, the differing combinations of epitopes are partially identical to one another in terms of their amino acid sequences and/or surface structures.

In a further aspect, the specimen is of either human or animal origin and comprises one of the following: serum, plasma, whole blood, saliva, mucous, skin cells, and urine.

In another aspect, the invention provides a rapid test device comprising one or more porous membranes, a procedural control applied to the one or more porous membranes, and two or more recombinant antigens applied to the one or more porous membranes. At least one of the recombinant antigens are applied to each of the porous membranes. The recombinant antigens comprise differing combinations of epitopes for a disease marker and are applied at visually distinct locations on the porous membranes to simultaneously capture antibodies to the disease marker in a single collected specimen.

The foregoing was intended as a broad summary only and of only some of the aspects of the invention. It was not intended to define the limits or requirements of the invention. Other aspects of the invention will be appreciated by reference to the detailed description of the preferred embodiment and to the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by reference to the detailed description of the preferred embodiment and to the drawings thereof in which:

Fig. 1 illustrates an example of a flow-through rapid test device according to an embodiment of the present invention;

Fig. 2A and Fig. 2B illustrate examples of immobilizing a procedural control and three antigens of different combinations of epitopes of the same disease marker on four different locations on the same membrane in a single flow-through rapid test device according to an embodiment of the present invention;

Fig. 3A and Fig. 3B illustrate examples of immobilizing a procedural control and two antigens of different combinations of epitopes of the same disease marker on three different locations on the same membrane in a single flow-through rapid test device according to an embodiment of the present invention;

Fig. 4 illustrates an example of a lateral-flow rapid test device according to an embodiment of the present invention;

Fig. 5A and Fig. 5B illustrate examples of immobilizing two antigens of different combinations of epitopes of the same disease marker on two different locations on the same membrane or two separate membranes in a single lateral-flow rapid test device according to an embodiment of the present invention;

Fig. 6 illustrates an example of immobilizing antigens of different combinations of epitopes of the same disease marker on 2 separate membranes in a single lateral-flow rapid test device according to an embodiment of the present invention;

Fig. 7A and Fig. 7B illustrate examples of test strips that are housed within the lateral-flow rapid test device in Fig. 6;

Fig. 8 is a diagram of a parallel testing algorithm sourced from Global AIDS Program, Centers for Disease Control and Prevention, USA; and

Fig. 9 is a diagram of testing strategies adopted by of WHO/UNAIDS.

DETAILED DESCRIPTION OF THE INVENTION

Referring to Fig. 1, an example of a flow-through rapid test device with a solid casing 4 composed of a well-shaped receptacle 2 into which specimen fluid and complementary buffers and reagents can be poured, a porous membrane 1 immediately at the bottom of the receptacle that comprises an immobilized procedural control and antigens of different combinations of epitopes of the same disease marker, and a reservoir 3 within which holds an absorbent material meant to collect and retain fluids that are flown through the membrane 1 and to support the membrane. According to one embodiment of the present invention, the flow-through rapid test device's porous membrane 1 comprises three immobilized antigens and one procedural control. Fig. 2A and Fig. 2B show two examples of possible arrangements of the immobilized antigens and procedural control at four locations on the porous membrane 1. One of the four locations at 6, 7, 8, or 9 shown in Fig. 2A comprises a quantity of immobilized procedural control. Each of the three remaining locations, other than the location comprising the immobilized procedural control, comprises a quantity of immobilized antigens. Each of the three immobilized antigen locations comprises a recombinant or a multiplex antigen of a different combination of epitopes of the same disease marker. Instead of recombinant antigens, the present invention may also use antigens comprising synthetic or naturally-occurring peptides. The procedural control and/or each of the three different antigens can be applied at one of the four locations as a dot, line, or any visually distinguishable shapes. The different combinations of epitopes may be partially identical to one another in terms of their amino acid sequences and/or surface structures.

According to another embodiment of the present invention, the flow-through rapid test device's porous membrane 1 comprises two immobilized antigens and one procedural control. Fig. 3A and Fig. 3B show two examples of possible arrangement of the immobilized antigens and procedural control at three locations on the porous membrane 1. One of the three locations at 10, 11, or 12 shown in Fig. 3 A comprises a quantity of immobilized procedural control. Each of the two remaining locations, other than the location comprising the immobilized procedural control, comprises a quantity of immobilized antigens. Each of the two immobilized antigen locations comprise a recombinant or a multiplex antigen of a different combination of epitopes for the same disease marker. The procedural control and/or each of the two different antigens can be applied at one of the three locations as a dot, line, or any visually distinguishable shapes.

The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention. For example, although gρ41 from Human Immunodeficiency Virus Type 1 and gp36 from Human Immunodeficiency Virus Type 2 are exclusively exemplified as disease markers, a variety of other disease makers are also applicable. Furthermore, serum, plasma, and whole blood are exclusively exemplified as specimen types, but a variety of other bodily fluids and tissues are also applicable. These may include saliva, mucous, skin cells, and urine. These specimen types may be of either human or animal origin.

Example 1 According to this example, the flow-through test device contains two different recombinant antigens; each of the two different antigens has a different combination of epitopes of the disease marker gp41 from Human Immunodeficiency Virus Type 1. Both combinations of epitopes are capable of capturing antibodies specific to gp41 in an HIV-I infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity. The arrangement of the locations on the porous membrane of the two immobilized antigens and the procedural control is similar to Fig. 3 A, where the procedural control is immobilized at 10, one of the recombinant antigens for HIV-I is immobilized at 11, and the other is immobilized at 12. Fig. 3B shows another possible arrangement of the three locations.

Example 2

According to this example, the same flow-through test device in Example 1 also contains two immobilized recombinant antigens of different combinations of epitopes of the disease marker gp36 from Human Immunodeficiency Virus Type 2, that are separately immobilized at 11 and 12, that are capable of capturing antibodies specific to gp36 in an infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity.

Example 3

According to this example, the flow-through test device contains three different recombinant antigens; each of the three different antigens has a different combination of epitopes of the disease marker gp41 from Human Immunodeficiency Virus Type 1. All three combinations of epitopes are capable of capturing antibodies specific to gp41 in an HIV-I infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity. The arrangement of the locations on the porous membrane of the three immobilized antigens and the procedural control is similar to Fig. 2A, where the procedural control is immobilized at 6 and the three antigens each occupies 7, 8, and 9. Fig. 2B shows another possible arrangement of the four locations. Example 4

According to this example, the same flow-through test device in Example 3 also contains three immobilized recombinant antigens of different combinations of epitopes of the disease marker gp36 from Human Immuno deficiency Virus Type 2 that are separately immobilized at 7,8, and 9 on the porous membrane. All three combinations of epitopes are capable of capturing antibodies, which can bind to the disease marker gp36 from Human Immunodeficiency Virus Type 2, in an infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity.

Now referring to Fig. 4, an example of lateral-flow rapid test device with a solid casing 14 that houses a test strip composed of absorbent materials and a porous membrane 13 that contains an immobilized procedural control and antigens of different combinations of epitopes of the same disease marker. The casing also has a receptacle 16 immediately above the absorbent materials 15 of the test strip into which specimen fluid and complementary buffers and reagents can be added, and a viewing window 17 immediately above the porous membrane 13 to allow visual reading of the testing result.

According to one embodiment of the present invention, the lateral-flow rapid test device's porous membrane 13 contains two immobilized antigens and one procedural control. Fig. 5 A shows an example of possible arrangement of the immobilized antigens and procedural control at three locations 18, 19, and 20 on the porous membrane. One of the three locations shown in Fig. 5A contains a quantity of immobilized procedural control. Each of the two remaining locations, other than the location containing the immobilized procedural control, contains a quantity of immobilized antigens. Each of the two immobilized antigens locations contains a recombinant or multiplex antigen of a different combination of epitopes of the same disease marker. Instead of recombinant antigens, the present invention may also use antigens comprising synthetic or naturally-occurring peptides. The procedural control and/or each of the two different antigens can be applied at one of the three locations as a dot, line, or any visually distinguishable shapes. The different combinations of epitopes may be partially identical to one another in terms of their amino acid sequences and/or surface structures.

According to another embodiment of the present invention, as illustrated in Fig. 6, the modified lateral-flow rapid test has an additional porous membrane 21 containing one or more immobilized antigens and one optional procedural control. The additional membrane 21 can be supported on the same test strip Fig. 7A or on a separate test strip Fig. 7B housed within a single casing 14. The casing has a single receptacle 16 immediately above the absorbent materials 15 of the test strip(s) into which specimen fluid and complementary buffers and reagents can be added, two viewing windows 17 and 22 immediately above the porous membrane 13 and 21, which contain immobilized procedural control and antigens of different combinations of epitopes for the same disease marker. Further additional porous membranes may also be used.

Example 5 According to this example, the lateral-flow test device contains two different recombinant antigens on the same porous membrane 13; each of the two different antigens has a different combination of epitopes of the disease marker gp41 from Human Immunodeficiency Virus Type 1. Both combinations of epitopes are capable of capturing antibodies specific to gp41 in an HIV- 1 infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity. The arrangement of the locations on the porous membrane of the two immobilized antigens and the procedural control is similar to Fig. 5A, where the procedural control is immobilized at 18, one of the two recombinant antigens for HIV-I is immobilized at 19, and the other is immobilized at 20.

Example 6

According to this example, the lateral-flow test device contains two different recombinant antigens on two separate porous membranes 13 and 21; each of the two different antigens has a different combination of epitopes of the disease marker gp41 from Human Immunodeficiency Virus Type 1. Both combinations of epitopes are capable of capturing antibodies specific to gp41 in an HIV-I infected patient's serum, plasma, or whole blood, but differ in sensitivity and specificity. The arrangement of the locations on the porous membrane of the two immobilized antigens and the procedural control is similar to Fig. 5A and 5B, where the procedural control is immobilized at 18 and 23, while one antigen is immobilized at 19, and the other is immobilized at 22.

Numerous modifications and variations in practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing descriptions of preferred embodiments thereof. It is well within the skill in the art to practice the present invention accordingly to a wide variety of methods and formats. Consequently, only such limitations should be placed on the invention as appear in the following claims.

Claims

1. A rapid test device comprising: a porous membrane; a procedural control applied to said porous membrane; and two or more recombinant antigens applied to said porous membrane, wherein said recombinant antigens comprise differing combinations of epitopes for a disease marker and wherein said recombinant antigens are applied at visually distinct locations on said porous membrane to simultaneously capture antibodies specific to said disease marker in a single collected specimen.

2. The rapid test device according to claim 1, wherein said rapid test device is a flow- through rapid test device.

3. The rapid test device according to claim 1, wherein said rapid test device is a lateral- flow rapid test device.

4. The rapid test device according to any one of claims 1 to 3, wherein said differing combinations of epitopes are partially identical to one another in terms of their amino acid sequences and surface structures.

5. The rapid test device according to any one of claims 1 to 3, wherein said differing combinations of epitopes are partially identical to one another in terms of their amino acid sequences.

6. The rapid test device according to any one of claims 1 to 3, wherein said differing combinations of epitopes are partially identical to one another in terms of their surface structures.

7. The rapid test device according to claim 1, wherein said disease marker is a protein molecule introduced and generated by a disease-causing vector in the body.

8. The rapid test device according to claim 1, wherein said disease marker is a protein molecule introduced by a disease-causing vector in the body.

9. The rapid test device according to claim 1, wherein said disease marker is a protein molecule generated by a disease-causing vector in the body.

10. The rapid test device according to any one of claims 7 to 9, wherein said disease- causing vector comprises viruses.

11. The rapid test device according to any one of claims 7 to 9, wherein said disease- causing vector comprises bacteria.

12. The rapid test device according to any one of claims 1 to 3, wherein said specimen is of human origin and comprises one of the following: serum, plasma, whole blood, saliva, mucous, skin cells, and urine.

13. The rapid test device according to any one of claims 1 to 3, wherein said specimen is of animal origin and comprises one of the following: serum, plasma, whole blood, saliva, mucous, skin cells, and urine.

14. A rapid test device comprising: a porous membrane; a procedural control applied to said porous membrane; and two or more peptides applied to said porous membrane, wherein said peptides comprise differing combinations of epitopes for a disease marker and wherein said peptides are applied at visually distinct locations on said porous membrane to simultaneously capture antibodies specific to said disease marker in a single collected specimen.

15. The rapid test device of claim 14, wherein said peptides are synthetic peptides.

16. The rapid test device of claim 14, wherein said peptides are naturally-occurring peptides.

17. The rapid test device of any one of claims 14 to 16, wherein said rapid test device is a flow-through rapid test device.

18. The rapid test device of any one of claims 14 to 16, wherein said rapid test device is a lateral-flow rapid test device.

19. A rapid test device comprising: two or more porous membranes; a procedural control applied to at least one of said two or more porous membranes; and two or more recombinant antigens applied to said two or more porous membranes, wherein: at least one of said two or more recombinant antigens are applied to each of said two or more porous membranes; said recombinant antigens comprise differing combinations of epitopes for a disease marker; and said recombinant antigens are applied at visually distinct locations on said two or more porous membranes to simultaneously capture antibodies specific to said disease marker in a single collected specimen.