WO2012008821A1

WO2012008821A1 - Rapid serodiagnosis of melioidosis

Info

Publication number: WO2012008821A1
Application number: PCT/MY2010/000167
Authority: WO
Inventors: Zainoodin Sheik Abdul Kader; Asma Ismail; Subramania Aiyar
Original assignee: Universiti Sains Malaysia
Priority date: 2010-07-12
Filing date: 2010-09-09
Publication date: 2012-01-19

Abstract

The present invention relates to a method in rapid serodiagnosis of the disease melioidosis by using a modified method for an acidified glycine extraction of the cell surface proteins (CSP) antigen of Burkholderia pseudomallei and the use of the extracted cell surface proteins (CSP) to develop a rapid and simple modified dot enzyme immunoassay (dotEIA) test. Accordingly, a method for serodiagnosis of melioidosis in a patient, the method comprises steps of (a) preparing a medium for growing Burkholderia pseudomallei; (b) growing the Burkholderia pseudomallei in broth; (c) harvesting the Burkholderia pseudomallei from step (ii) by centrifugation; (d) extracting Burkholderia pseudomallei cell surface proteins (CSP) from step (iii) by acidified glycine extraction method; (e) standardizing and optimizing the CSP antigens from step (iv) and enzyme conjugates reagents to develop a dot enzyme immunoassay; (f) designing assay protocol for dot enzyme immunoassay (dot EIA) based on the concentrations obtained from standardization assay in step (v); (g) preparing serum / plasma from blood specimen obtained from a patient; and (h) detecting antibody isotypes in serum/plasma from patient against the Burkholderia pseudomallei CSP antigens using the dot enzyme immunoassay (dot EIA) from step (vi)and serum/plasma from patient from step (vii). Results from the test can be determined by visible observation of the reaction. This method of diagnosis allows simultaneous detection of serum immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) antibody isotypes for serodiagnosis of melioidosis. Also provided is a kit for serodiagnosis of melioidosis wherein the kit includes (a) test strips dotted with optimized concentration of cell surface protein (CSP); (b) anti-human immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA); (c) nitroblue tetrazolium (solution A); (d) C 1 -5 bromo-4-chloro-3-indolylphosphate p-toluidine salt (solution B); (e) phosphate buffered saline buffer at pH 9.6 (substrate buffer); (f) 20 mM tris buffered saline at pH 7.4; (g) controls for reaction determination; and (f) reaction tray.

Description

Rapid Serodiagnosis of Melioidosis

Field of Invention

The invention relates to a method for serodiagnosis of melioidosis by dot enzyme immunoassay (dot EIA).

Background of Invention

Melioidosis is an environmental related and potentially fatal bacterial infectious disease involving multi-organ systems of man and a wide range of domestic and wild animals. It is caused by a soil saprophyte, Burkholderia pseudomallei. Most endemic cases occur in South East Asian countries and also in the northern part of Australia. This bacterium is ubiquitous in soil and water, therefore the risk of infection or contracting this disease is significant in rice farming population, plantation workers or farmers and gardeners and any individual participated in leisure activity or sport related activity that involved direct contact with soil and surface water. The more severe forms of melioidosis are often seen among people with underlying conditions that compromise the immune system. These conditions include diabetes, alcoholism, cancer, advanced age and chronic liver, kidney or lung disease and patient undergoing surgical procedures.

The clinical presentation of melioidosis is variable, ranging from subclinical infections to fatal septicaemia associated with broad-spectrum of clinical diseases involving multi-organ systems. Laboratory diagnosis of the disease has been limited to isolation of the bacteria by culture technique and detection of antibody by serological method. Most of the time, many cases go undiagnosed or underdiagnosed due to lack of awareness and laboratory facilities. Besides that, the use of culture isolation method can take up to days for confirmation of diagnosis. In addition, several attempts were made to develop molecular tests, however, these tests are still in research phase and need further improvement to make these tests more reliable before can used in routine laboratory diagnosis. The delay in the identification of . bacteria in laboratory and also lack of rapid diagnostic methods will delay the administration of treatment and increases the mortality rate of melioidosis patients.

In addition, current laboratory diagnostic methods have always been hampered by lack of sensitivity and specificity. Prior art as mentioned in the US Patent (20080108104), the invention utilizes extractable free lipids present in the bacteria for identification and to distinguish the clinical isolate of Burkholderia pseudomallei from other species by using thin layer chromatographic techniques as an alternative to conventional biochemical technique.

Although this can be a defining step in identifying the bacteria, the procedure is time consuming, not cost effective and requires specialized training.

In another prior art as in the patent WO 1997010351 , the invention relates to methods and kits in diagnosing of an infection by Burkholderia pseudomallei. The invention provides an in vitro diagnosis of Burkholderia pseudomallei infection by detection of exopolysaccharides antigen using monoclonal antibodies and detection of antibodies against exopolysaccharides antigen in sampled of body fluids and tissue. However, the steps may be complicated and requires a huge amount of time, which again does not address the issue of rapidity and easy application. Hence, there is a need for a rapid and simplified diagnostic method which is specific and sensitive in the laboratory diagnosis of melioidosis.

Summary of Invention

The present invention relates to a method in rapid serodiagnosis of the disease melioidosis by using a modified method for an acidified glycine extraction of the cell surface proteins (CSP) antigen of Burkholderia pseudomallei and the use of the extracted cell, surface proteins (CSP) to develop a rapid and simple modified dot enzyme immunoassay (dot EIA) test. Accordingly, a method for serodiagnosis of melioidosis in a patient, the method comprises steps of (a) preparing a medium for growing Burkholderia pseudomallei; (b) growing the Burkholderia pseudomallei in broth; (c) harvesting the Burkholderia pseudomallei from step (ii) by centrifugation; (d) extracting Burkholderia pseudomallei cell surface proteins (CSP) from step (iii) by acidified glycine extraction method; (e) standardizing and optimizing the CSP antigens from step (iv) and enzyme conjugates reagents to develop a dot enzyme immunoassay; (f) designing assay protocol for dot enzyme immunoassay (dot EIA) based on the concentrations obtained from standardization assay in step (v); (g) preparing serum / plasma from blood specimen obtained from a patient; and (h) detecting antibody isotypes in serum/plasma from patient against the Burkholderia pseudomallei CSP antigens using the dot enzyme immunoassay (dot EIA) from step (vi) and serum/plasma from patient from step (vii). Results from the test can be determined by visible observation of the reaction. This method of diagnosis allows simultaneous detection of serum immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) antibody isotypes for serodiagnosis of melioidosis. Also provided is a kit for serodiagnosis of melioidosis wherein the kit includes (a) test strips dotted with optimized concentration of cell surface protein (CSP); (b) alkaline phosphatase (AP) conjugated anti-human immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA); (c) nitroblue tetrazolium (solution A); (d) C 1 -5 bromo-4-chloro-3-indolylphosphate p- toluidine salt (solution B); (e) phosphate buffered saline buffer at pH 9:6 (substrate buffer); (f) . 20 mM tris buffered saline at pH 7.4; (g) controls for reaction determination; and (f) reaction tray.

Description of the Preferred Embodiments

The present invention, provides a rapid serodiagnosis of melioidosis by using patient serum or plasma. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, it is to be understood that, limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications and equivalents without departing from the scope of the appended claims. The term "patient" and "patients" used in the description refers to a patient who is suspected of having meliodosis and also, a patient who is diagnosed with meliodosis. The invention includes the use of cell surface proteins (CSP) as antigens in the dot enzyme immunoassay (dot EIA) assay for detection of IgG, IgM and IgA antibody isotypes. The first stage in the extraction requires the preparation of broth culture of the bacteria Burkholderia pseudomallei. Four 500 ml_ volume conical flasks containing 250 ml. of nutrient broth were prepared and autoclaved at 121 °C for 15 minutes prior to inoculation. Actively growing overnight cultures in 10 mL of nutrient broth after three consecutive subculturing was used as inoculums. Inoculation was performed at 1 :100 ratio in four conical flasks with 250 mL nutrient broth. All cultures were incubated overnight at 37 °C in incubator shaker (Innova 4080 incubator shaker, New Brunswick Scientific Co., Inc., USA) set at 200 revolutions per minute (rpm). The cultures were harvested by centrifugation (Eppendorf Centrifuge 581 OR, Hamburg, Germany) at 8000 xg for 10 minutes and washed twice with sterile saline. The final pellets of bacterial cells were then resuspended in 15 mL 0.2 M glycine at pH 2.2 and placed in a laboratory shaker at room temperature for 15 minutes. The suspensions were then subjected to centrifugation at 8000 xg for 10 minutes to remove the whole cell. Supernatants containing CSP proteins were saved. The pH of the supernatants were adjusted to pH 7.4 using 1 N sodium hydroxide (NaOH) and two volumes of ice cold absolute ethanol were added to the supernatants and stored at temperatures between -20°C to -80 °C, preferably at -20 °C overnight to allow the proteins to precipitate. The preparations were then centrifuged at 8000 xg for 10 minutes at 4 °C to 10 °C to pellet the proteins. The protein pellet was dissolved in 10 mM Tris pH 7.4, containing the protease inhibitors cocktail mixtures (Boehringer Mannheim Ltd., UK) and stored at -20 °C to -80 °C.

The subsequent stage would be the optimization of all the essential reagents for the development of dot EIA test and development of dot EIA test protocol for diagnostic application. Two minimum concentrations of the CSP antigens that were required to carry out the dot EIA test were determined. The standardization of_. the assay is for optimization of the minimum concentration of antigen, the dilution of the patient's serum, the dilution of conjugates and also the volume of colour development reagent that is needed. This also prevents wastage of resources.

The optimization of the assay was carried out by dividing a piece of nitrocellulose membrane into smaller strips. A nitrocellulose membrane is used as the solid phase carrier for the CSP antigens wherein the membrane with a pore size of 0.45 μιη (Microfiltration System, Ca., USA) is divided into 3 cm long and 0.4 cm width small rectangles. These strips were then further divided into smaller squares with the size of 0.4 cm by 0.4 cm. These membrane strips were used to perform optimization assay with panel of sera from melioidosis cases and healthy subjects.

Panels of sera from culture proven cases of melioidosis and sera obtained from healthy subjects were used to standardize the assay. Serial dilution of the cell surface proteins in .tris- buffer saline (TBS) containing protease inhibitor was prepared at the concentration ranging from 0.15625 Mg/mL to 0.50 μg/mL. Each concentration of the diluted antigen was spotted onto the respective square in the nitrocellulose membranes. Several numbers of similar strips, containing six different concentrations of cell surface protein antigens, were prepared to perform antigen optimization test. The nitrocellulose membrane was then treated with the blocking reagent of tris-buffered saline (TBS, pH 7.5) containing 5% of non-fat skim milk to block unbound sites for 30 minutes. This is to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay. After the blocking, membranes were washed in distilled water and air dried. The prepared strips were cut according to the divided size of 3 cm x 0.4 cm and placed into separate wells of the western blot incubation tray. These plates containing the strips are stored in a temperature of 4°C before using. The two minimum concentrations of the CSP antigens which have been determined were then spotted onto the middle of each rectangle and were air-dried. There are two spots of the optimized concentrations of antigens on each of the divided rectangle.

The panels of the five positive sera and three sera from healthy subjects were diluted 1 :100 in TBS and 1 mL of each diluted sera was reacted with each of the dotted strips for 1 hour at room temperature. The serum samples were then aspirated and the strips washed for 5 minutes with 1 ml_ of TBS. The washing procedure was repeated 3 times. After the final washing, 1 mL of diluted alkaline phosphatase (AP) conjugated goat anti-human immunoglobulin M, G and A (Zymed, USA) were added into the respective wells in the incubation tray and followed by incubation for 1 hour at room temperature. Anti-human IgM and IgG were diluted at 1 :4000 while anti-human IgA was diluted at 1 :2000. Upon completion of incubation, the conjugate solutions were aspirated off and the membranes were washed three times in TBS, pH 7.5, as described above. The test strips were then ready for colour development. The bound antibody was visualized using a chromogenic substrate detection system (NBT-BCIP, Bio-Rad alkaline phosphatase substrate kit, USA) comprising of three solutions which include nitroblue tetrazolium (solution A), C 1 -5 bromo-4-chloro-3- indolylphosphate p-toluidine salt (solution B) and phosphate buffered saline buffer at pH 9.6 (substrate buffer). Each of the chromogenic substrate solutions was mixed together in substrate buffer according to the recommended ratio before use and 1 mL of the prepared substrate solution was added into each well for colour development. The strips were incubated with the substrate solution for 15 minutes at room temperature for optimum colour reaction. The reaction was stopped by aspirating off the substrate and followed by thorough washing with distilled water for 5 minutes. A positive reaction was indicated by the development of insoluble purple colour. Optimum concentrations of antigen were determined by comparing the colour intensity observed in the panel of positive and negative sera. The colour intensity of each dot present in the test strips was then visually inspected while the strips still kept wet. The two lowest dilutions of the antigen that produced visible colour intensity to clearly differentiate between positive and negative reaction were selected to develop the dot EIA for the serodiagnosis of melioidosis.

The following results shows the distribution of the specific IgM, IgG and IgA antibody isotypes against the CSP of B. pseudomallei in the sera obtained from 65 culture positive melioidosis cases detected by dot EIA at 1 :100 dilution. Table 1 : The number of sera positive for one or a combination of any antibody isotypes and the sensitivity for each antibody calculated against culture isolation as the gold standard.

Table 2: The dot EIA results positive for total single antibody isotypes and their sensitivity values. The abbreviation of CP is the culture positive for B. pseudomallei.

Evaluation of the specificity of the dot EIA assay performed against control groups comprising of healthy subjects and diseased subjects confirmed for variety of infections by culture or serological methods. Table 3: The summary of the overall performance of the dot EIA and overall specificity of the dot EIA. Abbreviation used in the table: NM, non-melioidosis; ED, endemic; HIV, human immunodeficiency virus.

Table 4: The total number of positivity in dot EIA for each antibody isotypes observed in non- melioidosis diseased and healthy subjects and specificity values for each antibody isotypes. Abbreviation used in the table: NM, non-melioidosis; ED, endemic; HIV, human immunodeficiency virus.

Preliminary analysis of the distribution of the specific IgM, IgG and IgA antibody isotypes against the CSP of B. pseudomallei in the 60 representative clinical cases of melioidosis tested positive by the dot EIA. Table 5: The number of sera positive for one or a combination of any antibody isotypes.

Table 6: The result for total number of sera positive for single antibody isotypes by the dot EIA test.

This prompt dot EIA assay based on antibody detection provides a faster result compared to microbiological methods which requires isolation and identification that will take up to 4 or 5 days compared to the 3 hours using this assay. The results of the assay can be visibly observed with the appearance of two insoluble bluish black dots which indicates a positive reaction. This assay protocol can be completed in three hours which is useful in obtaining results of diagnosis rapidly. The detection of various antibody isotypes simultaneously increases the sensitivity and specificity of the test which is essential in diagnosing melioidosis. A kit for the serodiagnosis of meliodosis is useful in rapid and onsite serodiagnosis. The kit will include test strips dotted with two optimized concentration of cell surface protein antigens (CSP), anti-human immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA), commercially available chromogenic substrate reagents, tris buffered saline, positive. and negative controls and a 48 well reaction tray. The chromogenic substrate reagents are comprised of nitroblue tetrazolium (solution A), G 1 -5 bromo-4-chloro-3-indolylphosphate p- toluidine salt (solution B) and phosphate buffered saline buffer at pH 9.6 (substrate buffer). The buffered saline is a 20 mM tris buffered saline pH 7.4 which is used as a diluent and washing solution.

Claims

1.. A method for serodiagnosis of melioidosis in a patient, the method comprises steps of: i. preparing a medium for growing Burkholderia pseudomallei;

ii. growing the Burkholderia pseudomallei in broth;

iii. harvesting the Burkholderia pseudomallei irom step (ii) by centrifugation;

iv. extracting Burkholderia pseudomallei cell surface proteins (CSP) from step (iii) by acidified glycine extraction method;

v. standardizing and optimizing the CSP antigens from step (iv) and enzyme conjugates reagents to develop a dot enzyme immunoassays

vi. designing assay protocol for dot enzyme immunoassay (dot EIA) based on the concentrations obtained from standardization assay in step (v); vii. preparing serum / plasma from blood specimen obtained from a patient; and viii. detecting antibody isotypes in serum/plasma from patient against the Burkholderia pseudomallei CSP antigens using the dot enzyme immunoassay (dot EIA) from step (vi) and serum/plasma from patient from step (vii).

2. The method as claimed in claim 1 wherein the extracting of Burkholderia pseudomallei cell surface proteins (CSP) comprises steps of:

i. resuspending harvested Burkholderia pseudomallei in glycine

ii. placing the suspension in a laboratory shaker at room temperature for 15 minutes;

iii. obtaining supernatant containing the CSP by centrifuging the suspension;

iv. neutralizing pH of the supernatant from step (iii);

v. precipitating the CSP in the supernatant;

vi. centrifuging the supernatant to obtain the CSP in a pellet;

vii. dissolving the CSP with buffer and protease inhibitors; and

viii. storing of the CSP from step (vi)

The method as claimed in claim 2 wherein the glycine used is 0.2 M and of pH 2.2.

The method as claimed in claim 2 wherein the supernatant was neutralized to pH 7.4 using 1 N sodium hydroxide (NaoH).

The method as claimed in claim 2 wherein the precipitation of proteins were carried out using ice cold ethanol at a temperature range of -20 °C to -80 °C.

The method as claimed in claim 2 wherein the centrifugation was carried out at 8000 xg for 10 minutes at a temperature range of 4 °C to 10 °C.

The method as claimed in claim 2 wherein the CSP obtained is stored in 10 mM Tris pH 7.4 containing protease inhibitors cocktail mixtures and stored -20 °C to -80 °C.

The method as claimed in claim 1 wherein the standardizing an assay using cell surface proteins (CSP) comprises steps of:

preparing a nitrocellulose membrane;

i. diluting serially the CSP in buffer to different concentrations;

iii. spotting each concentration of the CSP from step (iii) onto the nitrocellulose membrane;

iv. blocking unbound sites of CSP on the spotted nitrocellulose membrane from step (iii);

v. washing and drying of the spotted nitrocellulose membrane; and

vi. storing of the spotted nitrocellulose membrane in incubation tray.

9. The method as claimed in claim 8 wherein the nitrocellulose membrane has a 0.45 μπν pore size.

10. The method as claimed in claim 8 wherein the serial dilution was prepared between the range of 0.15 Mg/mL to 0.50 g/mL.

1 1 . The method as claimed in claim 8 wherein the nitrocellulose membrane was blocked using a blocking reagent preferably tris-buffered saline of pH 7.5 containing 5% of nonfat skim milk for 30 minutes.

12. The method as claimed in claim 1 wherein the designing assay protocol in dot enzyme immunoassay (dotEIA) based on the concentrations obtained from standardization of assay comprises steps of:

i. reacting small strips of nitrocellulose membranes spotted with CSP antigens with buffer and sera/plasma obtained from infected patients or suspected patients;

ii. incubating the reacting nitrocellulose strips in an incubation tray;

iii. removing the sera and washing the nitrocellulose strips;

iv. adding conjugate solution into the incubation tray and incubating it;

v. removing the conjugate solution and washing the nitrocellulose strips with a buffer;

vi. adding substrate solution or detection reagents into the incubation tray and incubating the mixture;

vii. removing the substrate solution or detection reagents and washing the incubation tray; and

viii. visualizing results of reaction.

13. A kit for serodiagnosis of melioidosis wherein the kit includes

i. test strips dotted with optimized concentration of cell surface proteins (CSP); ii. alkaline phosphatase conjugated anti-human immunoglobulin G (IgG), immunoglobulin M (lgM). and immunoglobulin A (IgA);

iii. nitroblue tetrazolium (solution A);

iv. C1 -5 bromo-4-chloro-3-indolylphosphate p-toluidine salt (solution B);

v. phosphate buffered saline buffer at pH 9.6;

vi. 20 mM tris buffered saline at pH 7.4;

vii. controls for reaction determination; and

viii. reaction tray.

14. A kit for serodiagnosis of melioidosis as claimed in claim 13 wherein the test strips is dotted with at least optimized concentration of cell surface protein (CSP).

15. A kit for serodiagnosis of melioidosis as claimed in claim 13 wherein the kit is provided with at least one control for determining positive or negative reaction.