WO2024117982A1

WO2024117982A1 - Strip test kit for tuberculosis diagnosis

Info

Publication number: WO2024117982A1
Application number: PCT/TH2022/000048
Authority: WO
Inventors: Watchara Kasinrerk; Chatchai Tayapiwatana; Ponrut PHUNPAE; Supansa PATA; Umpa YASAMUT; Weeraya THONGKUM; Witida LAOPAJON; Nuchjira TAKHEAW
Original assignee: Chiang Mai University; Office Of National Higher Education Science Research And Innovation Policy Council
Filing date: 2022-11-30
Publication date: 2024-06-06

Abstract

An immunochromatographic strip test kit for tuberculosis diagnosis is based on immunological principles by using 3 clones of monoclonal antibodies specific to the antigen-85 (Ag85) protein; i.e., anti-Ag85 clone AM85B-5, anti-Ag85 clone AM85B-8, and anti-Ag85 clone AM85B-9. The strip test kit for tuberculosis diagnosis according to this invention can identify both Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacterial (NTM) infections in the liquid medium of Mycobacterium culture. This strip test kit can provide rapid test results than detection with the currently used standard methods, thereby allowing patients to quickly access treatments, and reducing transmission to the community.

Description

STRIP TEST KIT FOR TUBERCULOSIS DIAGNOSIS

Technical Field

Biotechnology in relation to the strip test kit for tuberculosis diagnosis

Background of the Invention

Tuberculosis (TB) is a chronic communicable disease that represents a global public health problem. It is estimated that one third of the world's population is infected with TB, which is caused by infection with bacteria in the Mycobacterium tuberculosis (M. tuberculosis') complex (MTBC), with M. tuberculosis being the most common cause. This disease is transmitted by inhalation of contaminated droplets. It is a severe and chronic communicable disease that can affect every organ in the body. While pulmonary tuberculosis is most common (over 80%), extrapulmonary tuberculosis can also be found, such as in pleura, lymph nodes, spine, joints, abdomen, urinary system, reproductive system, and nervous system, etc. TB can affect general population, but it is often found in the elderly or those who have been physically weakened by other diseases, such as cold, measles, pertussis, drug addicts, AIDS, as well as those in close contact with someone having the disease, like sleeping in the same room or staying in the same house. Nevertheless, only one-tenth of infected individuals develop TB, while most others usually remain in the latent stage. Furthermore, there have been reports of increasing TB cases from non- tuberculous mycobacterial (NTM) infection, which shares similar symptoms but requires different treatments, as well as infection with multidrug-resistant M. tuberculosis (MDR-TB), which are virulent strains that require a longer duration of treatments than a normal tuberculosis infection. In 2021, the World Health Organization (WHO) reported TB infection in as many as 9.9 million people (new and recurrent cases), among which 1.1 million were children with 0-14 years of age, and also 1.3 million deaths in TB patients. In Thailand, the incidence of new and recurrent TB cases is approximately 105,000 or 150 per 100,000 population.

At present, the diagnosis of TB is of great importance. If TB patients get diagnosed correctly and quickly, they can be efficiently treated, and thus, the spread of the disease can be controlled. To diagnose patients with TB, several factors must be taken into consideration simultaneously, such as risks of infection, symptom manifestation, chest X-ray, tubulin skin test, microscopic examination of sputum for acid-fast bacilli (AFB) staining, and in vitro culture (Mycobacterium growth indicator tube or MGIT). However, chest X-ray examination, tubulin skin test, and acid-fast bacilli (AFB) staining of sputum offer low sensitivity and specificity, leading to misdiagnoses. In contrast, Mycobacterium culture can only be performed in the hospitals where a Mycobacterium laboratory is available. Moreover, since it is a slow-growing bacterium, culturebased diagnosis takes as long as about 6-8 weeks to generate test results. Currently, several molecular biological methods have been developed for TB diagnosis. While these methods provide high specificity, the major drawbacks are not only their complicated procedures, which need to be carried out by someone with expertise only in those laboratories equipped with molecular biology techniques, but also the high costs of examination. Thus, they are not suitable for underdeveloped and developing countries, where the rates of TB outbreaks are usually high. As a result, we can see that the methods currently used for TB diagnosis are not quite appropriate. As rapid TB diagnosis and early recognition of infection allow for both effective treatments and better control of disease outbreaks, studies and development of novel approaches for TB diagnosis are therefore essential and in demand by the public health industry.

A review of patents related to tuberculosis diagnosis revealed existing works, for example:

European Patent Publication No. EP3726219A1 is related to a method for diagnosing tuberculosis consisting of an acidic substance for the isolation of tuberculosis antigen complexes from patient specimens, a neutralizing reagent (basic buffer solution) and antigen specific antibodies. This kit can test antigens, namely MPT6 (64-kDa early secreted antigenic target; ESAT6), 10-kDa culture filtrate protein (CFP10), antigen 85 complex (Ag85), Ag85A, Ag85B, Ag85C, HspX, LAM, and 38-kDa protein, and this test kit can be applied to the technique of immunochromatography kit or enzyme-linked immunosorbent assay (ELISA) kit

Chinese Patent Publication No. CN108693345A is related to a test kit for testing and diagnosing tuberculosis. It contains a reagent that can test indicators of tuberculosis in serum samples, including tuberculosis Ag85B antibody, interleukin-6, interleukin-8 and interleukin- 18. This test kit can be used with a variety of assays such as ELISA (enzyme-linked immuno-sorbent assay), Luminex assay, single-molecule array, flow immunoassay and nucleic acid aptamers.

Chinese Patent Publication No. CN103954754A is related to a test kit for testing and diagnosing tuberculosis that can detect cytokines 1-309, MIG and IL-8, and can also detect antigens of Mycobacterium tuberculosis with an IgG antibody specific to 38-kDa, 32-kDa, 14-16-kDa and AG85B antigens.

Thai Petty Patent Application No. 1903001324 is related to a human tuberculosis antibody test kit (indirect ELISA) consisting of coating individual proteins to the wells of the microplates: ESAT6, CFP10, MPT64, PPE18 (Rvll96), RpfD (Rv2389c), RelA (Rv2583c) and DosR (Rv3133c).

Thai Petty Patent Application No. 2003001666 is related to a test kit for antibodies against tuberculosis in macaques using specific protein strips based on immunohistochemistry principles specific to seven types of recombinant proteins as follows: ESAT6, CFP10, A60 (Rv0440), Ag85B (Rvl886c), PPE18 (Rvll96), DosR (Rv3133c) and RpfD (Rv2389c), PPE18 (Rvl96), DosR (Rv3133c) and RpfD (Rv2389c). This test kit can identify tuberculosis infection and differentiate latent-TB or active-TB, thus shortening diagnosis time when compared to other methods used in the diagnosis of tuberculosis, and it helps to isolate tuberculosis infection caused by Mycobacterium tuberculosis complex and non-tuberculous mycobacteria.

Although the above existing works are related to tuberculosis test kits based on the immunohistochemistry technique, there is no such invention related to a test kit for tuberculosis diagnosis in the form of a test strip capable of isolating Mycobacterium tuberculosis complex (MTB) and non-tuberculous mycobacteria (NTM). It can also shorten the time of tuberculosis diagnosis compared to the standard tuberculosis culture test.

Summary of the Invention

Development of a strip test kit for tuberculosis diagnosis is based on immunological principles by using 3 clones of monoclonal antibodies specific to the antigen-85 (Ag85) protein, i.e., anti-Ag85B clone 5B, anti-Ag85B clone 8B, and anti-Ag85B clone 9B. The strip test kit for tuberculosis diagnosis according to this invention can identify both Mycobacterium tuberculosis complex (MTB) and non-tuberculous mycobacterial (NTM) infections in the liquid medium of Mycobacterium culture (Mycobacterium growth indicator tube or MGET).

The aim of this invention is the development of a strip test kit for tuberculosis diagnosis for the diagnosis and differentiation of Mycobacterium tuberculosis complex (MTB) and non- tuberculous mycobacterial (NTM) infections by using antigen- 85-specific antibodies. This strip test kit can provide rapid test results and takes less time than detection with the currently used standard test methods, thereby allowing patients to access treatment quickly and reducing transmission to the community.

Brief Description of the Drawings

Figure 1. Principles of the strip tests and key elements in IC strip tests 8B and IC strip test 98. Figure 2. Principles of the strip tests and interpretation of the test results for people infected with Mycobacterium tuberculosis complex (MTB).

Figure 3. Principles of the strip tests and interpretation of the test results for people infected with non-tuberculous mycobacteria (NTM).

Detailed Description of the Invention

The development of a strip test kit for tuberculosis diagnosis wherein the strip test kit according to this invention consists of specific protein strips, which are nitrocellulose pads that have been sprayed and immobilized with the antigen-85-specific monoclonal antibodies.

It is characterized in that the antigen- 85-specific monoclonal antibodies consist of 3 clones of monoclonal antibodies, including anti-Ag85 clone AM85B-5, anti-Ag85 clone AM85B-8, and anti-Ag85 clone AM85B-9.

One control strip was sprayed and immobilized with an immunoglobulin- specific monoclonal antibody.

And two strip test kits in which a monoclonal antibody specific to the antigen-8 protein was sprayed and immobilized with unique characteristics as follows:

Strip test kit I was sprayed and immobilized with the monoclonal antibody clone of anti- Ag85 clone AM85B-8 or anti-Ag85 clone AM85B-9.

Strip test kit II was sprayed and immobilized with the monoclonal antibody clone of anti- Ag85 clone AM85B-5 bonded with gold particles on absorbent pads.

There are two sets of strip test kits for tuberculosis diagnosis as follows:

The strip test kit I of tuberculosis diagnosis is for those infected with mycobacterium tuberculosis complex (MTB) where nitrocellulose sheets with two clones of anti-Ag85 clone AM85B-8 and anti-Ag85 clone AM85B-5 are prepared with a kit called the “IC strip test 8B.”

The strip test kit II of tuberculosis diagnosis is for those infected with non-tuberculous mycobacteria (NTM) where nitrocellulose sheets with two clones of anti-Ag85, anti-Ag85 clone AM85B-9 and anti-Ag85 clone AM85B-5 are prepared with a kit called the “IC strip test. 9B.”

Example 1 Preparation of recombinant protein antigen-85 The AG85 B gene are increased by PCR using the M. tuberculosis H37 Rv DNA model. The hybrid protein is then produced using plasmid pAK400CB and host Escherichia coli cells. The product is recombinant biotinylated-Ag85B protein.

Example 2 Preparation of monoclonal antibody specific to antigen-85 protein

The obtained recombinant protein AG85B is attached to streptavidin-coated magnetic particles for the beads in monoclonal antibody production. Then, it is injected into mice (strain A BALB/c) intraperitoneally. The beads are injected 3 times over a 2 weeks period before immunogenicity testing. When the measured titers are higher, monoclonal antibodies will be collected and proliferated by hybridoma technique, then purified by affinity chromatography.

Example 3 Specificity test of monoclonal antibodies specific to antigen-85 protein

Monoclonal antibodies specific to the antigen-85 protein (anti-Ag85 clone AM85B-5, anti- Ag85 clone AM85B-8, and anti-Ag85 clone AM85B-9) are coated onto ELISA plates and block plates with 2% skimmed milk (dissolved in PBS solution). Then, TB culture medium is added and incubated at 37°C for 1 hour. Next, HRP-conjugated anti-rabbit immunoglobulin antibody is then added and incubated at 37°C for 1 hour. After the end of the washing cycle to remove excess antibody, TMB substrate is added and inactivated with HC1. The absorbance is measured at 450 nm.

Example 4 Tuberculosis rapid diagnostic test kit (IC strip test)

Tuberculosis Rapid Diagnostic Test Kit consists of 4 parts as follows:

(1) Sample pad is used for absorbing samples. The fiberglass is used as material.

(2) Conjugate releasing pad is used for spraying and immobilizing the monoclonal antibody clone of anti-Ag85 clone AM85B-5.

(3) Nitrocellulose membrane is used for spraying and immobilizing the monoclonal antibody clone of anti-Ag85 clone AM85B-8 or anti-Ag85 clone AM85B-8 (strip test) and goat anti-mouse immunoglobulins (strip control).

(4) Absorbent pad is used for absorbing liquid caused by the diffusion of liquid.

Example 5 Testing of tuberculosis test kit with culture filtrate

It is to develop the tuberculosis diagnosis by using a tuberculosis assay kit to detect culture water obtained from sputum culture in liquid medium. Several details are as follows: The sputum is cultured in liquid medium and BACTEC MGET Mycobacteria culture system to determine bacterial growth for at least 42 days. If the fertilized specimens develop and signal on any days, the filtered water will be examined with both 8B and 9B tuberculosis testing kits. The samples cultured for 42 days without the alarm signal are reported as no TB growth and examined with the test kit as well. The results are compared with those of the commercially available MPT-64 protein assay. Then, the results of infectious test kit are compared with the findings of mycobacterium by standard culture system.

M. tuberculosis testing of 129 samples showed 99 positive samples and 30 negative samples.

The experimental results showed that of the 99 samples detected with M. tuberculosis infection (Table 1 ), 93 of 8B and 85 of 9B of tuberculosis assays are positive for 94% and 86%, respectively. This indicates that the 8 B tuberculosis assays for antigen-85 protein are valid and quite consistent with the diagnosis of M. tuberculosis infection. Interestingly, the results from MPT-64 protein assays are fairly consistent with only 87% of M. tuberculosis infection diagnosis.

Thirty samples of filtered water without the growth of M. tuberculosis are examined with 8B, 9B, and MPT-64 assay kits. The results are shown in Table 2. The 8B and 9B of tuberculosis assays are each negative in 28 samples (93%), while MPT-64 was negative in all cases (100%). Thus, the TB test kits of 8B and 9B yield very few false positives.

Table 1 Comparison of samples detected with M. tuberculosis by standard culture methods combined with 8B, 9B, and MPT-64 of filtered water tests

IC strip test Culture IC strip test Culture

No. MPT method No. MPT method

8B 9B 8B 9B

64 identification 64 identification

1 + + + MTB 51 + + + MTB

2 + + + MTB 52 + + + MTB

3 + + + MTB 53 + + + MTB

4 + + + MTB 54 + + + MTB

5 + + + MTB 55 + + + MTB

6 + + + MTB 56 + + + MTB

7 + + + MTB 57 + + + MTB

8 + + + MTB 58 + + + MTB MTB 59 + + + MTB

MTB 60 + + + MTB

MTB 61 + + + MTB

MTB 62 + + + MTB

MTB 63 + + + MTB

MTB 64 + + + MTB

MTB 65 + + + MTB

MTB 66 + + + MTB

MTB 67 + + + MTB

MTB 68 + + + MTB

MTB 69 + + + MTB

MTB 70 + + + MTB

MTB 71 + + + MTB

MTB 72 + + + MTB

MTB 73 + + + MTB

MTB 74 + + + MTB

MTB 75 + + + MTB

MTB 76 + + + MTB

MTB 77 + + + MTB

MTB 78 + + + MDR

MTB 79 + + + MTB

MTB 80 + + + MTB

MTB 81 + + + MTB

MTB 82 + + + MTB

MTB 83 + + MTB

MTB 84 + + MTB

MTB 85 + + MTB

MTB 86 + + MTB

MTB 87 + + MTB

MTB 88 + + MTB

MTB 89 + + MTB

MTB 90 + MTB 41 + + + MTB 91 - + - MTB

42 + + + MTB 92 - + - MTB

43 + + + MTB 93 - + - MTB

44 + + + MTB 94 - - - MTB

45 + + + MTB 95 - - - MTB

46 + + + MTB 96 - - - MTB

47 + + + MTB 97 - - - MTB

48 + + + MTB 98 - - - MTB

49 + + + MTB 99 - - - MTB

50 + + + MTB

Table 2 Comparison of samples without M. tuberculosis detected by standard culture methods combined with 8B, 9B and MPT-64 of filtered water test kits

No. IC strip test Culture method

MPT-64 8B 9B Identification

1 - - - No Growth

2 - - - No Growth

3 - - - No Growth

4 - - - No Growth

5 - - - No Growth

6 - + - No Growth

7 - - - No Growth

8 - - - No Growth

9 - - - No Growth

10 - - - No Growth

11 - - - No Growth

12 - - - No Growth

13 - - - No Growth

14 - - - No Growth

15 - - - No Growth

16 - - - No Growth No. IC strip test Culture method

MPT-64 8B 9B Identification

17 - - - No Growth

18 - - - No Growth

19 - - - No Growth

20 - - - No Growth

21 - - - No Growth

22 - - - No Growth

23 - - - No Growth

24 - - - No Growth

25 - + + No Growth

26 - - - No Growth

27 - - - No Growth

28 - - - No Growth

29 - - + No Growth

30 - - - No Growth

Example 6 Test results of Mycobacterium tuberculosis complex (MTBC) and Non-tuberculous mycobacterium (NTM) isolation by 8B and 9B tuberculosis assays

Because the 8B tuberculosis test kit is positive for the antigen-85 protein produced by Mycobacterium spp., while the 9B Tuberculosis test kit is positive only for the antigen-85 protein produced from Mycobacterium tuberculosis complex (MTBC), the simultaneous use of 8B and 9B assays can isolate MTB and NTM from each other. NTM is detected in 1 9 samples as shown in Table 3 . The combination of 8B and 9B assays is able to identify NTM infection in 12 samples, i.e. the samples with culture results as NTM are positive for 8B tuberculosis assays, but negative for 9B tuberculosis assays. When being considered in conjunction with Table 1 which is an example where the MTB culture results are positive for both 8B and 9 B tuberculosis test kits. Thus, both tuberculosis test kits can be used to isolate Mycobacterium tuberculosis complex (MTBC) and Non-tuberculous mycobacterium (NTM).

Table 3 Comparison of samples with Non-tuberculous mycobacterium by standard culture method combined to the filtered-water culture by 8B and 9B tuberculosis test kits No. IC strip test Culture method

8B 9B Identification

1 + - NTM

2 + - NTM

3 + - NTM

4 + - NTM

5 + - NTM

6 + - NTM

7 + - NTM

8 + - NTM

9 + - NTM

10 + - NTM

11 + - NTM

12 - - NTM

13 + - NTM

14 + + NTM

15 - - NTM

16 - - NTM

17 - - NTM

18 - - NTM

19 - - NTM

Example 7 Tuberculosis diagnosis results by using the developed strip test kits for tuberculosis diagnosis

Based on the above results, the developed 8 B and 9 B strip test kits for tuberculosis diagnosis can be applied to detect the filtered water culture from the tuberculosis culture system in liquid media. Because the standard culture method takes 6-8 weeks for tuberculosis diagnosis, the developer has questioned whether this strip test can shorten the time for tuberculosis diagnosis by analyzing the dates of interpreting results from 8B test kits for M. tuberculosis in 99 samples as shown in Table 4 . Results are found to be interpreted from No. 1 -28 and this strip test for TB diagnosis is able to identify approximately 60% of M. tuberculosis (59 out of 99 samples) within 5 days of culture, while indicating 80% (81 out of 99 samples) of such M. tuberculosis within 19 days after culture. Therefore, this developed strip test can perform TB diagnosis faster than the standard culture method.

Table 4 The time (days) for interpreting results from 8 B tuberculosis test kit in cases with M. tuberculosis culture and frequency of 99 samples

Date of detection Frequency of sample

7 14

8 1

9 6

6 2

10 17

11 10

12 17

13 14

14 3

15 3

16 5

17 3

18 4

19 4

21 5

23 1

24 1

26 2

27 1

28 1 Example 8 Effect of isoniazid and rifampicin resistance for Mycobacterium tuberculosis in the developed TB strip test kit

M. tuberculosis resistance to isoniazid and rifampicin affects the detection of antigen-85 protein. It was found that M. tuberculosis resistant to isoniazid and rifampicin was ineffective against the developed 8B tuberculosis strip test kit. Thus, this developed TB strip test kit can be applied to any samples without considerations whether or not those samples carry drug-resistant M. tuberculosis.

Table 5 Results of 8B TB test kit for drug-resistant M. tuberculosis and non-drug resistant M. tuberculosis to isoniazid (H) and rifampicin (R) in filtered water of 20 samples

Drug susceptibility IC strip test Culture method

Sample test

8B Identification

H R

1 + MTB S S

2 + MTB S S

3 + MTB R S

4 + MTB S S

5 + MTB S S

6 + MTB S R

7 + MTB S S

8 + MTB S S

9 + MTB S S

10 + MTB S S

11 + MTB S S

12 + MTB S S

13 + MTB S S

14 + MTB S R

15 + MTB S S

16 + MTB S S

17 + MTB R S

18 + MTB S S

19 + MTB R R

20 + MTB R R S = sensitive; R = resistance

Best Mode of the Invention

As disclosed under the Detailed Description of the Invention

Claims

1. A strip test kit for tuberculosis diagnosis consists of specific protein strips, which are nitrocellulose pads that have been sprayed and immobilized with monoclonal antibodies specific to the antigen- 85 protein.

Wherein it is characterized in that the antigen- 85-specific monoclonal antibodies consist of 3 clones of monoclonal antibodies, including anti-Ag85 clone AM85B-5, anti-Ag85 clone AM85B-8, and anti-Ag85 clone AM85B-9.

2. The strip test kit for tuberculosis diagnosis according to Claim 1, wherein the specific protein strips contain control lines and test lines.

One control strip was sprayed and immobilized with an immunoglobulin- specific monoclonal antibody. and 2 strip test kits in which a monoclonal antibody specific to the antigen- 8 protein was sprayed and immobilized with unique characteristics as follows:

3. The strip test kit for tuberculosis diagnosis according to Claim 1, wherein the first strip test kit for tuberculosis diagnosis in people infected with Mycobacterium tuberculosis complex (MTB) consists of 2 clones of the antigen- 85-specific monoclonal antibodies, namely anti-Ag85 clone AM85B-8 and anti-Ag85 clone AM85B-5.

4. The strip test kit for tuberculosis diagnosis according to Claim 1, wherein the second strip test kit for tuberculosis diagnosis in people infected with non-tuberculous mycobacteria (NTM) consists of 2 clones of the antigen- 85-specific monoclonal antibodies, namely anti-Ag85 clone AM85B-9 and anti-Ag85 clone AM85B-5.

5. The strip test kit for tuberculosis diagnosis according to Claim 1, wherein the immunoglobulinspecific monoclonal antibodies are goat anti-mouse immunoglobulins.

6. The strip test kit for tuberculosis diagnosis according to Claim 1, wherein the test specimens are the liquid medium of Mycobacterium culture (Mycobacterium growth indicator tube or MGIT).