Description:FIELD OF INVENTION:
The present disclosure relates to immunochromatographic device. More particularly, to an immunochromatographic device for detecting of antigens on S.typhi and S.paratyphi causing Typhoid and Paratyphoid respectively. The present disclosure also relates to an immunochromatographic kit for detecting of antigens of S.typhi and S.paratyphi and a method for detection thereof.
BACKGROUND:
Typhoid is one of the most contagious infections which spread in a body and retain therein for several weeks affecting various parts of the body. It is also known as enteric fever caused by a Gram-negative bacterium. One of the bacterium pathogens is Salmonella enterica serovar Typhi (S.typhi). Paratyphoid has also similar symptoms to that of Typhoid but less severe. The common mode of infection is by ingestion of an infecting dose of the organism, usually through contaminated water or food. Some patients have also been found to be infected with mixed antigens of both the bacteria.
Detection of infection due to Typhoid or Paratyphoid requires combination of various procedures such as clinical evaluation, laboratory tests, culture/microbiological test, PCR test and sometimes imaging studies, and so on. Conventional techniques used in the laboratories for diagnosis of infection with S.typhi or S.paratyphi include such as serological tests WIDAL, ELISA and antibody detection based rapid tests. However, there are many drawbacks associated with each of such conventional techniques as enlisted in following table:
S. No. Test / Technique Descriptions Specimen Limitations
1 Clinical Evaluation The doctor takes a detailed medical history and conducting a physical examination. He inquiries about any recent travel to areas where typhoid fever is prevalent and ask about symptoms such as high fever, headache, abdominal pain, weakness, and other signs associated with the disease Not applicable Lack of detection accuracy due similar clinical symptoms in to the patients.
2.0 Serological Tests Serological tests detect antibodies produced by the body in response to the infection by S.typhi/ S.paratphi. These tests may be helpful if blood or stool cultures are negative or inconclusive.
However, they are more useful during the later stages of the disease when antibodies have had time to develop. Serum / plasma / whole blood Antibodies are developed in the patient at the later stage of infection and takes longer time.

Can not be used for early detection of Typhoid.
2.1 WIDAL Test Agglutination based slide / tube method test. Qualitatively / semi-quantitatively detects antibodies against ‘O’ and ‘H’ antigen of S.typhi and S.paratyphi.

Most popular, widely used and cost-effective technique.
Serum / plasma Detects antibodies

Poor sensitivity and specificity. False-positive or false-negative results.

Storage need refrigerated conditions and shelf life is less than 24 months from date of manufacturing.

Can not used for early detection of Typhoid.
2.2 Rapid test kits Test based upon immuno-chromatographic principle for qualitative detection of antibodies against ‘O’ and ‘H’ antigen of S.typhi and S. paratyphi.

Simple and quick procedure with visual interpretation.

Shelf life 24 months if stored at 2-30°C. Serum / plasma Detects antibodies

Poor sensitivity and specificity.

Can not used for early detection of Typhoid.
3.0 ELISA ELISA-based antigen detection tests use specific antibodies to capture and detect Salmonella antigens in clinical samples. The sample is added to a plate with wells coated with anti-Salmonella antibodies. If the antigen is present in the sample, it binds to the antibody, and the test produces a color change or signal that can be measured and interpreted as positive Blood culture / Isolated bacteria Longer and multistep procedure

Needs trained technologies and equipment.

Storage needs refrigeration.
Not cost effective

Can not be used with single specimen
4.0 Rapid Test
Kits Test based upon immuno-chromatographic principle for qualitative detection of antigens of S.typhi and S. paratyphi.

Shelf life 24 months if stored at 2-30°C. Stool / Feces Collection & handling of stool / feces is not a preferred choice of specimen.
Discharge / Shredding of bacteria in the stool at later stage of infection.

Poor Sensitivity

Can not be used for the early detection.
5.0 Imaging Studies In some cases, imaging studies like X-rays or ultrasound may be performed to assess the extent of complications or to check for features like an enlarged spleen (splenomegaly) or liver (hepatomegaly) that can be associated with typhoid fever Not applicable Needs the imaging equipment and expert radiologist

No diagnostic accuracy

Can not used for early diagnosis
6.0 PCR / DNA Test Most accurate diagnostic technique.

Detects DNA of the bacteria in the clinical specimen using PCR technique.

This method offers high sensitivity and specificity in identifying the bacterium Whole blood / Blood culture
7.0 Biochemicals Tests Suspected Salmonella typhi colonies are subjected to a series of biochemical tests to confirm their identity. These tests include the triple sugar iron (TSI) agar test, citrate utilization test, urease test, and others, as mentioned in previous responses.
Identified bacteria from blood / stool culture Provides only relevant and supporting information.
Can not be used for early diagnosis

Equipped lab setup and trained technologist required

Lengthy procedure
8.0 Blood culture Test Blood Culture is considered as gold standard for diagnosis of typhoid and is best when performed within a week of onset of symptoms.

Can be used for the early and accurate diagnosis with help of microbiological / biochemical, serological and PCR procedures.

Blood Identifying S.typhi / S,paratyphi from a blood culture requires a careful assessment of the culture results and additional laboratory tests

Needs multiple steps in the identification and confirmation of bacterial using microbiological / biochemical and serological procedures

Only microbiological and biochemical tests are performed but antigen detection

Can not be used for the early diagnosis.

In a nutshell, majority of the conventional techniques for detecting typhoid/paratyphoid infections involve detection of antibodies for detection of the infections in a sample. However, infections to immune system, lead to presence of antigens of the infection causative agents first in the blood, followed by production of antibodies in response to the antigens which requires a time. Thus, antigen detection would ensure early detection of infection as compared to that of antibodies, which is a known fact.
There are some conventional techniques such as Rapid tests as tabulated above. Such techniques detect antigens to determine the Typhoid infection. However, such techniques involve collection of a sample as stool/feces which delays the detection of infection as typhoid infection in stomach develops during later stage (third week) of typhoid infection. Such techniques additionally lack sensitivity, specificity and delayed timeline of diagnosis. Collection of stool specimen is actually a very tedious and unhygienic. Also, the S.typhi bacteria can be shed intermittently in the stool. This means that a single stool sample may not always contain the bacteria, leading to false negatives. Multiple samples collected over several days may be needed to increase the chances of detection. The sensitivity of stool culture methods, which involve growing the bacteria in a laboratory, can be relatively low. This is because the number of bacteria shed in the stool can be low, especially in the early stages of the disease. This can result in false-negative results. Other bacterial infections, such as non-typhoidal Salmonella infections, can cause similar symptoms to typhoid fever. These bacteria can be present in the stool and may lead to confusion in diagnosis.
Other serological tests compromise with the sensitivity and specificity and not suitable for early diagnosis of infection with S.typhi or S.paratyphi.
Although tests based upon PCR technique are more accurate and reliable but not being used in the general lab settings due to high cost, need of sophisticated lab equipment with costly instruments. Such a technique is not suitable for early and quick diagnosis of infection with S.typhi and S.paratyphi. None of the conventional tests is able to detect infections caused with either one or both of S.typhi and S.paratyphi with high sensitivity and specificity.
Therefore, there exists a need to overcome the existing prior arts.
OBJECTS OF THE INVENTION:
The principal object of the present invention here is to overcome the drawbacks in the prior art and provide an immunochromatographic device (100) for detection of antigens on the surface of either one or both of typhoid or paratyphoid causing S.paratyphi or S.typhi in a sample.
Another object of the present invention is to provide the immunochromatographic device which has 100% sensitivity and 100% specificity.
Another object of the present invention is to provide the immunochromatographic device which is stable at room temperature and stable at extreme temperatures of up to 40 deg C.
Another object of the present invention is to provide the immunochromatographic device which does not require power supply and can be used in remote settings where good laboratory set up is not available
Another object of the present invention is to provide the immunochromatographic device which is simple to use.
Another object of the present invention is to provide the immunochromatographic device which has shelf life of 24 months from the date of manufacturing stored at temperature in the range of about 2 to 40 deg C.
Another object of the present invention is to provide the immunochromatographic device which can detect the antigens within a time duration in the range of 5 to 20 minutes.
quick, early, visual, qualitative, differential and accurate detection of the aforementioned antigens
These and other objects and advantages of the present subject matter will be apparent to a person skilled in the art after consideration of the following detailed description taken into consideration with accompanying drawings in which preferred embodiments of the present subject matter are illustrated.
SUMMARY OF THE INVENTION:
In one aspect of the present disclosure, an immunochromatographic device (100) for detection of antigens in a whole blood cultured sample is disclosed. The device (100) includes a sample pad (102) to receive the sample; a conjugate pad (104) containing colloid gold particles conjugated with a particular pair of monoclonal antibodies against S.typhi and S.paratyphi, and a control antibody including either rabbit IgG or mouse IgG. A nitrocellulose membrane (106) is immobilized with monoclonal antibodies of S.typhi and S.paratyphi and a control line protein consisting of either goat anti-rabbit IgG or goat anti-mouse IgG.
In another aspect of the present disclosure, an immunochromatographic kit for detection of antigens in a whole blood cultured sample is disclosed. The kit includes the immunochromatographic device (100) and venipuncture equipment to collect the sample. The kit may also include an optional bottle containing culture media in which the sample is inoculated immediately after collection through the venipuncture equipment.
In another aspect of the present disclosure, a method (200) for detection of antigens in a whole blood cultured sample is disclosed. The method (200) includes collecting 5-10 ml of the sample through a venipuncture equipment, followed by inoculating the sample immediately into a bottle containing culture media and shaking the bottle gently. The method (200) further involves incubating the bottle in an incubator for time duration in the range of about 12 to about 24 hours, followed by placing the immunochromatographic device (100) on a flat surface. Then, the method (200) includes transferring an amount of the sample from the bottle and visualizing appearance of color lines in a window of the device (100) and interpreting if presence of antigens on the surface of either of one or both of S.paratyphi or S.typhi in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the figures to reference like features and components. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
Figures 1A-1C illustrate schematic views of immunochromatographic device (100) where Figure 1A shows a schematic top view of the device (100), Figure 1B shows a schematic view of housing of the device (100), and Figure 1C shows a schematic view of internal components of the device (100), in accordance with an illustrative embodiment of a present disclosure;
Figure 2 shows a flowchart depicting different steps of a method (200) for early and visual detection of antigens on one or both of S.typhi and S.paratyphi, in accordance with another illustrative embodiment of the present disclosure;
Tables
Table 01 shows results of sensitivity test conducted on the device (100) while detection of each of S.typhi and S.paratyphi bacteria, in accordance with the illustrative embodiment of the present disclosure; and
Table 02 shows results of specificity test conducted on the device (100) while detection of each of S.typhi and S.paratyphi bacteria, in accordance with the illustrative embodiment of the present disclosure.
DETAILED DESCRIPTION
Various modifications and alternative forms, specific embodiment thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
The terms “comprises”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
The present disclosure discloses an immunochromatographic device (100) for detection of antigens on the surface of either one or both of S.paratyphi or S.typhi in a sample. The device (100) provides quick, early, visual, qualitative, differential and accurate detection of the aforementioned antigens.
Figure 1A shows a schematic top view of the device (100). The device (100) has a cover which when removed, exposes internal components of the device (100). Figure 1B shows a schematic internal view of the device (100) having a housing (120) to accommodate different internal components of the device (100). The different internal components are clearly shown in a schematic view of the device (100) in Figure 1C.
As shown in Figure 1C, the device (100) includes a sample pad (102) which is configured to receive a sample in a well thereof. The sample here is a whole blood cultured sample collected from a subject through venipuncture technique. The collected sample is cultured, steps thereof in detail are discussed hereinafter. The device (100) includes a conjugate pad (104) next to the sample pad (102). The conjugate pad (104) contains colloid gold particles conjugated with a particular pair of monoclonal antibodies against two particular bacterial antigens causative of different diseases. One of the two particular bacterial antigens is S.typhi causative of Typhoid disease. While another of the two particular bacterial antigens is S.paratyphi causative of Paratyphoid disease. The conjugate pad (104) also includes a control antibody selected from either rabbit IgG or mouse IgG.
The nitrocellulose membrane (106) is immobilized with each of the particular pair of monoclonal antibodies i.e., immobilized with monoclonal antibodies against S.typhi, anti S.paratyphi. Outer layer of the nitrocellulose membrane (106) contains two test lines (T1, T2) and control line (C). Either of the test line regions T1 or T2 or vice versa may be coated with monoclonal antibodies of S.paratyphi and S.typhi separately. A control line protein selected from either goat anti-rabbit IgG or goat anti-mouse IgG corresponding to rabbit IgG or mouse IgG of the conjugate pad respectively.
When an adequate volume of the sample containing S.typhi and S.paratyphi is applied in a well of the sample pad (102), the sample migrates immuno-chromatographically by capillary action across the nitrocellulose membrane (106) along with complex of colloidal gold conjugate containing with S.typhi and S.paratyphi antigens present in the sample.
Visual appearance of exemplary pink or purple lines at test line regions (T1) or (T2) in addition to exemplary pink / purple line at the control line region (C), indicates that there are antigens of S.typhi or S.paratyphi in the sample and may be considered as positive. In some embodiments, there is a chance that the sample has antigens of both the bacteria, causing mixed typhoid to the patient. In such an embodiment, there may be appearance of color on both the test line regions (T1) and (T2) in addition to the control line region (C). No colored line at (T1) or (T2) regions indicate there are no antigens of the aforementioned bacteria and may be considered as negative.
In case, there is no appearance of colored line on the control line region ‘C’, it means result of detection is invalid and new device (100) may be tried.
The present invention also relates to a kit for detection of one or both the antigens of aforementioned bacteria in the sample. The kit includes the device (100) along with venipuncture equipment to collect the sample. The kit may also include a bottle containing culture media in which the sample is inoculated immediately after collection through the venipuncture equipment.
In another embodiment, a method (200) for detecting of antigens of one or both of the bacteria including S.paratyphi or S.typhi is disclosed, as shown in a flowchart in Figure 2. The method (200) involves collecting 5-10 ml of the sample through a venipuncture equipment, followed by inoculating the sample immediately into a bottle containing culture media. Shaking the bottle gently, followed by incubating the bottle in an incubator for time duration in the range of about 12 to about 24 hours. Thereafter, the method (200) involves placing the device (100) on a flat surface away from direct exposure to high blow of air. Then, an adequate amount of the sample from the bottle is drawn using a micropipette from the bottle and then transferred into the sample pad (102) of the device (100). Visual appearance of color lines in a result window of the device (100) indicates presence of antigens on the surface of either of one or both of S.paratyphi or S.typhi in the sample.
Thus, the device (100) and other embodiments thereof are based upon immunochromatography principle having pair of highly specific monoclonal antibodies for the accurate detection of bacterial antigen in the sample. No segregation and confirmation of bacteria using additional techniques is required for the concluding detection, thereby reducing turnaround time and saving the costs. Thus, the device (100) provides quick, early, visual, qualitative, differential and accurate detection of the aforementioned antigens. The device can be stored at room temperature and up to 40°C. Hence, the device (100) can be used in remote settings where good laboratory set up is not available and does not require to be stored under refrigerated conditions for which continuous power supply is required.
The device (100) has 100% accuracy of detection of the aforementioned bacterial antigens, defined in terms of sensitivity and specificity for which experimental values for different bacteria have been shown in Tables 01 and 02 respectively. The device (100) has shelf life of 24 months from date of manufacturing if stored at temperatures in the range of about 2 deg C to about 40 deg C. The device (100) provides a single step procedure of early detection of the antigens and visual interpretations within 5-20 minutes of the sample provide quick detection and interpretation of the results. In causative infection of typhoid and paratyphoid, antigens get firstly develop in the blood, followed by infection in stomach in third week. Hence, detection of the antigens in the blood provides early detection thereof via the device (100). Only a single procedure on the device (100) is enough for the detection of antigens of S.paratyphi or S.typhi in the sample.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).
Although embodiments for the present subject matter have been described in language specific to structural features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/component of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.

, Claims:We claim:
1. An immunochromatographic device (100) for detection of antigens in a whole blood cultured sample, the device (100) comprising:
a sample pad (102) to receive the sample;
a conjugate pad (104) comprising colloid gold particles conjugated with a particular pair of monoclonal antibodies against S.typhi and S.paratyphi, and a control antibody comprising either rabbit IgG or mouse IgG;
a nitrocellulose membrane (106) immobilized with monoclonal antibodies of S.typhi and S.paratyphi and a control line protein consisting of either goat anti-rabbit IgG or goat anti-mouse IgG; and
an absorbent pad (108).
2. The device (100) as claimed in claim 1, wherein the monoclonal antibodies of S.paratyphi and S.typhi are coated on test lines (T) of the nitrocellulose membrane (106) separately while the control line protein is coated on the control line (C).
3. The device (100) as claimed in claim 1, wherein the device (100) is stable at temperature in the range of about 2 deg C to about 40 deg C.
4. The device (100) as claimed in claim 1, wherein the device (100) has shelf life of at least 24 months.
5. The device (100) as claimed in claim 1, wherein the device (100) works without requiring power supply.
6. The device (100) as claimed in claim 1, wherein visual appearance of colour on any one or both the test lines, and control line (C) is indicative of either Typhoid or Paratyphoid or mixed infection thereof.
7. The device (100) as claimed in claim 1, wherein the device (100) detects antigens present on the surface of either of one or both of the S.paratyphi or S.typhi in the sample within a time duration in the range of 5 to 20 minutes.
8. An immunochromatographic kit for detection of antigens in a whole blood cultured sample, the kit comprising:
an immunochromatographic device (100) comprising:
a sample pad (102) to receive the sample;
a conjugate pad (104) comprising colloid gold particles conjugated with a particular pair of monoclonal antibodies against S.typhi and S.paratyphi, and a control antibody comprising either rabbit IgG or mouse IgG;
a nitrocellulose membrane (106) immobilized with monoclonal antibodies of S.typhi and S.paratyphi and a control line protein consisting of either goat anti-rabbit IgG or goat anti-mouse IgG; and
an absorbent pad (108); and
venipuncture equipment to collect the sample.
9. The kit as claimed in claim 8, wherein the kit comprising an optional bottle containing culture media in which the sample is inoculated immediately after collection through the venipuncture equipment.
10. A method (200) for detection of antigens in a whole blood cultured sample, the method (200) comprising:
(i) collecting 5-10 ml of the sample through a venipuncture equipment;
(ii) inoculating the sample immediately into a bottle containing culture media;
(iii) shaking the bottle gently;
(iv) incubating the bottle in an incubator for time duration in the range of about 12 to about 24 hours;
(v) placing an immunochromatographic device (100) on a flat surface, the device (100) comprising a sample pad (102) to receive the sample; a conjugate pad (104) comprising colloid gold particles conjugated with a particular pair of monoclonal antibodies against S.typhi and S.paratyphi, and a control antibody comprising either rabbit IgG or mouse IgG; a nitrocellulose membrane (106) immobilized with monoclonal antibodies of S.typhi and S.paratyphi and a control line protein consisting of either goat anti-rabbit IgG or goat anti-mouse IgG; and an absorbent pad (108);
(vi) transferring an amount of the sample from the bottle at step (iv) into the sample pad; and
(vii) visualizing appearance of color lines in a window of the device (100) and interpreting if presence of antigens on the surface of either of one or both of S.paratyphi or S.typhi in the sample.