RAHMAH NOORDIN (MY)
LAMMIE P.J. ET AL.: "Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis-a multicenter trial", FILARIA J., vol. 3, no. 9, 2004, XP021007587
LALITHA P. ET AL.: "Development of antigen detection ELISA for the diagnosis of Brugian and Bancrofian Filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and WB-SXP-1", MICROBIOL. IMMUNOL., vol. 46, no. 5, 2002, pages 327 - 332, XP008147847, DOI: doi:10.1111/j.1348-0421.2002.tb02703.x
BASKAR L.K.V. ET AL.: "Development and evaluation of a rapid flow through immuno filtration test using recombinant filarial antigen for diagnosis of brugian and bancrofian filariasis", MICROBIOL. IMMUNOL., vol. 48, no. 7, 2004, pages 519 - 525, XP008147870, DOI: doi:10.1111/j.1348-0421.2004.tb03547.x
What is claimed is:
1. A method for rapid detection of lymphatic filariasis in a biological sample, comprising the steps of: - a) Adding a buffer to reconstitute drLed mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent prepared separately in a mαcrowell; b) Adding a biological sample to tϊie sample receiving end of a chromatographic element; c) Allowing the biological sample to flow from the sample receiving end to the reaction zoλe of the chromatographic element wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction scne,- d) Placing the chromatographic element in (c) into the microwell containing the reconstituted mouse monoclonal anti-human IgG4 ancibody conjugated to a detection reagent in (a) ; e) Allowing the mouse monoclonal anti-i-iuman igG4 antibody conjugated to a detection reagent to flow from the microwell to the reaction zone of the chromatographic element; and f) Detecting the complex formed in (e) .
2. The method of claim 1 wherein the method further comprises the steps of : - g) Allowing the unbound mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent in (e) to further flow from the reaction zone to the control zone of the chromatographic element wherein anti-mouse IgG antibody is immobilized within the control zone,- and h) Detecting the complex formed in (g) .
3. A method for rapid detection of lymphatic filariasis in a biological sample, comprising the steps of : - a) Adding a biological sample to the sample receiving end of a chromatographic element; b} Allowing, the biological sample to flow from the sample receiving end to the reaction zone of the chromatographic element ' wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction zone; c) Adding a buffer to the reagent releasing end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein; d) Allowing -the mouse monoclonal aήti-buman IgG4 antibody conjugated to a detection reagent to flow from the. reagent releasing end to the reaction zone of the chromatographic element; and e) Detecting the complex formed in (d) .
4. The method of claim 3 wherein the method further comprises the steps of:- f) Allowing the unbound mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent in (d) to further flow from the reaction zone to the control zone of the chromatographic -element wherein anti-mouse IgG antibody is immobilized within the control zone; and g) Detecting the complex formed in (f) .
5. A method for rapid detection of lymphatic filariasis in a biological sample, comprising the steps of : - a) Adding a biological sample to the sample S receiving end of a chromatographic element; b) Allowing the .biological sample to flow 1 from the sample receiving end to the reaction zone of the chromatographic element wherein SXP/SXP-1 ^recombinant antigen is immobilized within the reaction zone;
10 c) Adding a buffer to the reagent feleast.ng end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein; d) Allowing the mouse monoclonal anti-human IgG4 5 antibody conjugated to a detection reagent: to flow from the reagent releasing end to the control zone of the chromatographic element wherein anti- mouse IgG antibody is immobilized within the control zone; e) Allowing the mouse monoclonal anti-human IgG4 0 antibody conjugated to a detection reagent to further flow from the control zone to the reaction Zone of the chromatographic element; and f) Detecting the complex formed in (d) and (e) .
6. The method according to any one of claims 1 to 5 5 wherein the method is for detection of Wuchereria bancrofti and/or Brugia malayi and/or ξrugia t±mori infections .
7. The method according to any one of claims 1 to β wherein the chromatographic element is a membrane or 0 any other suitable chromatographic element. • 8.- The method of claim ■ 7 wherein the membrane is an absorbent nitrocellulose membrane.
9. The method of claim 7 wherein the other suitable chromatographic elements include nylon and/or mixed esters .
10. The method according to any ' one of claims 1 to 9 wherein the recombinant antigen is ' expressed by SXP/SXP-I gene in Brugia malayi and/or IVuchereria bancrofti and/or Brugia timori .
11. The method according to any one of claims 1 ' to 10 wherein the detection reagent is gold particles, latex particles or any other suitable particles.
■ 12. ' The method of claim 11 'wherein ' the other suitable
■ particles ...include any one of , silver or non-metal colloidal particles such as selenium, tell-uriura and sulfur.
13. A diagnostic kit for rapid detection of lymphatic filariasis in a biological sample according to- the method of any one of claims 1 to 12 comprising a detection device wherein the detection. device comprises a chromatographic element ■ wherein • the chromatographic element comprises a sample receiving end, a reaction zone and a control zone characterized in that SXP/SXP-l recombinant antigen is 5.πw«obilized within the reaction zone and dried mouse ϊήonoclonal anti-human IgG4 antibody conjugated to a detection reagent is in a separate rhicrowell.
14. A diagnostic kit for rapid detection of lymphatic filariasis in a biological sample according to the method of any one of claims 1 to 12 con prising a detection device wherein the detection device comprises a chromatographic element wherein the chromatographic element comprises a sample receiving end, a reagent releasing end, a reaction zone and a control zone characterized in that SXP/SXP-1 recombinant antigen is immobilized within the reaction zone.
15. The diagnostic kit of claim 13 or 14 wherein the kit further comprises a buffer wherein the buffer is in a separate container from the detection device.
16. The diagnostic kit of claim 14 wherein mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is immobilized within the reagent releasing end of the chromatographic element
17. The diagnostic kit according to any one of claims 13 to 16 wherein the detection reagent is gold particles, latex particles or any other suitable particles .
18. The diagnostic kit of claim 17 wherein the other suitable particles include any one of silver or non- metal colloidal particles such as selenium, tellurium and sulfur.
19. The diagnostic kit according to any one of claims 13 to 18 wherein anti-mouse IgG antibody is immobilized within the control zone.
20. The diagnostic kit according to any one of claims 13 to 19 wherein the chromatographic element is a membrane or any other suitable chrowatographic element .
2 * 1. The diagnostic kit of claim 20 wherein the membrane is an absorbent nitrocellulose membrane .
22. The diagnostic kit of claim 20 wherein the other suitable chromatographic elements include nylon and/or mixed esters.
23. The diagnostic kit according to any one of claims 13 to 22 wherein the kit is for detection of Wuchereria bancrofti and/or Bxugia ma.la.yi and/or Brugia timori infections.
24. The diagnostic kit according to any one of claims 13 to 23 wherein the recombinant antigen is expressed by SXP/SXP-1 gene in Brugia ma.la.yl and/or Wuchereria bancrofti and/or Brugia timori .
25. The diagnostic kit according to any one of claims 13 to 24 wherein the detection device is a dipstick.
26. The diagnostic kit according to any one of claims 13 to 24 wherein the detection device is a cassette.
27. The use of a diagnostic kit according to any one of claims 13 to 26 for the detection of lymphatic filariasis in a biological sample. |
Method for Rapid Detection of Lymphatic Filariasis
1. Technical Field of the Invention
The present invention relates to a method for rapid detection of lymphatic filariasis, particularly a method that detects anti-filarial IgG4 antibodies in a biological sample using a SXP/SXP-1 recombinant antigen and the technique of immunochromatόgraphy.
2. Background of the Invention,
Lymphatic filariasis is a- parasitic and infectious tropical disease caused by a number of slender and thread-like parasitic filarial worms which invade blood circulation, lymphatics, lymph nodes and • other parts of the human body.
One hundred and ' twenty million people in 83 countries of the world are infected with lymphatic filarial parasites, and it is estimated that more than
1 billion (20% of the world's population) are at risk of acquiring the infection. Ninety percent of ' these infections are caused by Wuchereria bancrofti and the remainder by Brυgia malayi and Brugia timori. The disease has been identified by the World Health
Organization (WHO) as the second leading cause of permanent and long-term disability in the world.
Lymphatic filariasis is transmitted to man by mosquitoes, -which introduce a large number of infective larvae into human. The female worijis produce
microfilariae, which make their way i_o blood circulation and are taken up by a suitable mosquito species. On reaching the mosquito's body, the microfilariae (first stage larvae, Li larvae) undergo several moultings to form infective larvae (L 3 larvae)', which reach the blood circulation of the definite host through wound made by the bites of the mosquitoes . Soon the infective larvae enter into the lymphatic system where they slowly mature into adult male and female worms. The male and 'female adult mate- to produce microfilariae which find their way to peripheral blood ' circulation.
The clinical manifestations of lymphatic filariasis can be divided into three types : 1. Asymptomatic: these individuals are outwardly non-symptomatic, but will demonstrate lymphatics and/or renal damage if appropriately tested.-
2. Acute manifestations: these include "filarial fever" or acute attacks associated with inflammation of the lymphatic flodes and channels .
3. Chronic manifestations: these arise from adult worm's damage .to the lymphatic system, and include hydrocoele, lymphoedema, chγluria and elephantiasis. ■
The traditional or routine method to diagnose filarial infection depended on the direct demonstration of the microfilariae in blood using relatively cumbersome techniques and having to take
into account the periodicity of microfi iariae in blood. This traditional method severe iy lacks sensitivity (25% - 40% sensitive) , thus missing many positive cases. This is due to the inability of the method to detect cryptic infections (before microfilariae are produced and after microfilariae ceased to be produced) , single sex infections, occult infections and low levels of microfilariae. Blood concentration techniques such as the Knoti ' s method and membrane filtration increases the sensitivity of detection but are usually not performed because they require ' venous blood taking. Polymerase chaia reaction
(PCR) -based detection methods are very sensitive to
• detect low levels of microfilariae,- however it is not suitable for detection of cryptic, occult or single sex infections .
In year 1998, WHO initiated a Global Program for Elimination of Lymphatic Filariasis (GPELF) The main aim of this program is to eliminate lymphatic filariasis as a public health problem by reducing the level of the infection in endemic populat Ions to a .point, whereby active transmission no longer occurs. The main approach that is being taken is -to provide mass drug treatment once yearly to the entire λ at risk' population for a period of 4-6 years. Availability of diagnostic tools is one of the important factors for success of this program. The tools are needed for accurate ■ mapping σfi endemic areas, for monitoring activities, certification of elimination and surveillance activities post- elimination. For bancroftian filariasis, a rapid
antigen, test is commercially available for mapping and monitoring activities, but a rapid test based on antibody" detection is also needed, particularly for the certification and post -elimination surveillance phases of the program.
SXP or SXP-I gene (Genbank accession No. M98813) was previously identified by imtmmoscreening of Brugia ma.la.yl cDNA library with immune sera from microfilariae positive patients with brugian and brancroftian filariasis (Dissanayake S., Xu M., Piessens WF. A cloned antigen 5 for serological diagnostic of Wuchereria hancrofti microfilariae with daytime blood samples. MoI B±ochem pa.ra.sito! 1992: 256-26) . The Brugia malayi-derived recombinant protein (JBm-SXP-I) was reported to be successful in identifying 83% (64/72) of bancroftian filariasis patients when tested with IgG4 -ELISA (Chandrashekar R., et al Molecular cloning of • Brugia malayi antigens for diagnosis of lymphatic filariasis. MoI Biochem Parasitol 1994:64 (2) : 262-271). With this method
(IgG4 -ELISA) , Wuchereria hancrofti-derived protein homologue of SXP-I (Wb-SXP-I) was reported to detect
100% (72/72) of the Wuchereria bancroftl infected patients. (Rao KV., efc al The Wuchereria Bancroft! orthologue of Brugia malayi SXP-I and the diagnosis of bancroftian filariasis. MoI Biochem Pa.rasx.tol 2000; 107 (1) : 71-80) .
Although the expression products of SXP/SXP-1 gene has been reported over the years and proven . to successfully detect lymphatic filariasis in. infected
patients, the SXP/SXP-1 gene was applied for the development of tests using ELISA format, . which requires several hours to perform. There is no report of the application of the expression products of
5 SXP/SXP-1 gene using the immunochrόrnatography technique for rapid detection ' of filariasis . Thus, there remains a need in the art for a rapid detection of lymphatic filariasis that employs the SXP/SXP-1 gene and the technology of immunochromatography for
10 the reason discussed above.
3. Summary of the Invention
It is a general object of the present invention to provide a diagnostic method, which is capable of detecting lymphatic filariasis in infected patients by
"L 5 detecting anti-filarial IgG4 antibodies in a biological sample.
It is also an object of the present invention to provide a specific and sensitive diagnostic method, which is capable of detecting anti-filarial IgG4 antibodies in a biological sample by using SXP/SXP-1 recombinant antigen and the technique of immunochromatography.
Another object of the present invention is ' to provide a simple and ' rapid diagnostic kit employing the method outlined- above that can be performed by untrained personnel in a minimum amount of time.
These and other objects -of the present invention
are achieved by,
A method for rapid detection of lymphatic filariasis in a biological sample, comprising the steps of: - a) Adding a buffer to reconstitute dified mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent prepared separately in a mierowell; b) Adding a biological sample to the sample receiving end of a chromatographic element; c) Allowing the biological sample to flow from the sample receiving end to the reaction zoλe of the chromatographic element wherein SXP/SXP-1 ^recombinant antigen is immobilized within the reaction zone; d) Placing the chromatographic element in (c) into the microwell containing the reconstituted mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent in (a) ; e) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent to flow from the microwell to the reaction zone of the chromatographic element; and f) Detecting the complex formed in (e) .
A method for rapid detection of lymphatic filariasis in a biological sample, comprising the steps of: - a) Adding a biological sample to the sample receiving end of a chromatographic element; b) Allowing the biological sample to flow from the sample receiving end to the reaction zone of the
chromatographic element wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction zone; c) Adding a buffer to the reagent releasing end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein,- d) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent to flow from the reagent releasing end to the reaction zone of the chromatographic element; and e) Detecting the complex formed in (d) .
A method for rapid detection of lymphatic filariasis in a biological sample, comp.fising the steps of : - a) Adding a biological sample to the sample receiving end of a chromatographic element; b) Allowing the biological sample to floW fr.om the sample receiving end to the reaction zol\e of the chromatographic element wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction zαie; c) Adding a buffer to the reagent releasi ng end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein; d) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagen-i to flow from the reagent releasing end to the control zone of the chromatographic element wherein anti- mouse IgG antibody is immobilized within the control zone,- e) Allowing the mouse monoclonal anti-human IgG4
antibody conjugated to a detection reagent -fco further flow from the control zone to the reaction zone of the chromatographic element; and f) Detecting the complex formed in (d) • & (e) .
A diagnostic kit for rapid detection of lymphatic filariasis in a biological sample comprising a detection device wherein the detectioa device comprises a chromatographic ' element wherein the chromatographic, element comprises a sample receiving end, a reaction zone and a control zone characterized in that SXP/SXP-1 recombinant antig'en is immobilized within the reaction zone and dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is in a separate miσrowell .
A diagnostic kit for rapid detection of lymphatic filariasis in a biological sample corrprising a detection device wherein the detection device comprises a chromatographic element wherein the chromatographic element comprises a sample receiving end, a reagent releasing end, a reaction zone ' and a control zone characterized in that SXP/SXP-L recombinant antigen is immobilized within the reaction zone .
4. Brief Description of the Accompanying Drawings
Other aspects of the present invention and their advantages will be discerned after studying the detailed description' in ■ conjunction with the accompanying drawings in which:
Figure 1 shows the plasmid map of SXP/SXP-I recombinant gene .
Figure 2 shows the SDS-PAGE profile of SXP/SXP-I recombinant protein.
S Figure 3 shows the appearance of tests in a. dipstick and cassette format employing the method for rapid detection of lymphatic filariasis .
5. Detailed. Description of the Invention
The method for rapid detection of lymphatic
10 filariasis in accordance with ' the present invention detects anti-filarial IgGA antibodies in a biological sample based on a specific antibody-antigi :α binding reaction ' , which comprises a recombinant antigen, expressed by the SXP/SXP-1 . gene binding to the anti- 5 filarial Ig<34 antibodies in the biological ' sample. The
SXP/SXP-1 gene is a recombinant gene derived from filarial parasites Brugia malayi, Wuαhereria bancrofti or Brugia timor±.
In the present ' invention, SXP/SXP-1 gene was ' 0 cloned from Brugia malayi cDMA library by established
PCR cloning methodology using the following primers:
Forward; 5' GTC ACT TCA TCA CTC AAT 3' and Reverse: 5'
CTA TTT ATT ACT TTT TGT CG 3 ' . The recomb Lnant gene was recloned into a bacterial expression vector 5 (pPROξXHT, Life Technologies) and the His-tagged recombinant gene as shown in DNA sequence ID No . : 1
and Figure 1 was transformed into E. coli TOP 10 host (Invitrogen) . Any other expression vector such as prokaryote, insect or mammalian expression vector may be used in the present invention.
Expression of the SXP/SXP-1 recombinani gene was then induced with isopropyl-b-D-thiogalactosϊde (IPTG) to produce recombinant protein and followed by purifying the recombinant protein by affinity
'chromatography. In the purification step, AKTA Prime Purification System (Pharmacia) and chromatography column packed with Ni-WTA resin (Qiagen, USA) were employed. The protein containing fractions v f &re pooled and passed through using spin columns (MWCO 1OkDa) for buffer exchange and concentration of the recombinant protein.
SDS-PAGE analysis of the SXP/SXP-1 recombinant protein in Figure 2 shows that it has an apparent molecular weight of approximately 30 kDa . Western blot analysis of the purified recombinant protein showed that the SXP/SXP-1 recombinant antigen produced was sensitive and specific for detection of lymphatic filariasis.
IgG4 assay in an ELISA format was then developed using SXP/SXP-1 recombinant antigen and the assay was evaluated using serum samples from individuals infected with various infections and. normal individuals. The results showed that the igG4 assay developed was highly sensitive and specific for detection of Nuchereria bancrofti infection. The
π
recombinant antigen also reacted with serum samples from Brugia malayi and Brugia timori patients, however the sensitivity was found to be lower.
The assay was further developed into a rapid assay that employs the SXP/SXP-1. recombinant antigen and the technique of immunoσhromatograpihy . This • ■ technology of lateral flow or immunochrømatography refers to capillary flow of immunological components through an absorbent membrane to mix and s-ybsequentIy separate the various components.
The rapid immunochromatography assay for detecting filariasis of the present invention includes a chromatographic element comprising three generally contiguous sections : sample receiving end, reaction zone and control zone. Chromatographic eletn&αt refers to a solid matrix upon which the sample can foe applied and allowed to migrate during the assay procedure . The chromatographic element particularly preferred in this invention is an absorbent nitrocellulose membrane. Other chromatographic elements that can be used include nylon and/ or mixed esters. SXP/SXP-1 recombinant antigen is immobilized within the reaction zone and anti-mouse IgG antibody is immobilized within the control zone.
In one embodiment of the present invention, dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is prepared separately in a microwell . In another embodiment of the present invention, the dried mouse monoclonal anti-human IgG4
antibody conjugated to a detection reagent is incorporated within the reagent releasing end of the chromatographic element. Any substance that is capable of producing a detectable signal can be used as the detection reagent conjugated to the mouse monoclonal anti-human Ig.G4 antibody including colloidal metallic particles such as gold and silver, colloidal non-metal particles such as selenium, tellurium and sulfur and also organic polymer latex particles. ' Detection reagents preferred for use' in the present invention are the visually detectable coloured particles, such as colloidal metals, particularly colloidal gold.
In a first embodiment of the present invention, a buffer is added to reconstitute dried mouse monoclonal • anti-human IgG4 antibody conjugated to colloidal gold
.in a microwell. Then, a biological sample such as blood, serum, plasma, urine or tears is introduced to the sample receiving end of an absorbent nitrocellulose membrane and ■ is allowed to migrate laterally via capillary action towards the reaction ■ zone of the membrane. The anti-filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen -Complex or immune-complex. Next, the absorbent nitrocellulose membrane is placed in the microwell containing the reconstituted mouse monoclonal anti-lruman IgG4 antibody conjugated to colloidal gold. The mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold absorbs through the membrane and migrates to the reaction zone and binds to the
antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse inonoclonal anti-human IgG4 antibody conjugated to colloidal gold. The presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested. The unbound mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold from the reaction ^ zone will further migrate to the control zone and bind with the anti-mouse IgG antibody incorporated therein, forming a red-purplish line in the control zone. This control zone serves as an . internal control to ensure the stability of the gold conjugated reagent.
In a second embodiment of the present invention, a biological sample such as blood, seruτti, plasma, urine or tears is firs-t introduced to tiαe sample receiving end of the absorbent nitrocellulose membrane and is allowed to migrate laterally via capillary, action towards the reaction zone of the membrane. The anti-filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen complex or immunocotnplex. A buffer is then introduced to the reagent releasing end to reconstitute dried mouse monoclonal anti-lnuman IgG4 antibody conjugated to colloidal gold incorporated therein. The mouse monoclonal anti-human IgG4- antibody conjugated -to colloidal gold migrates from the reagent • releasing end to the reaction zone and binds to the
antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold. The presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested. The unbound mouse monoclonal anti-human IgG4 antibody conjugated to colloidal go'ld from the reaction zone will further migrate to the control zone and bind with the anti-mouse IgG antibody, forming a rsd-purplish line in the control zone .
In a third embodiment of the present invention a biological sample such as blood, serum, plasma, urine or tears is first introduced to the sample receiving end of the absorbent nitrocellulose membrane and is allowed to migrate laterally via capillary action towards the reaction zone of the membrane. The anti- filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen complex or imtnunocomplex. A buffer is then introduced to the reagent releasing end to reconstitute dried mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold incorporated therein. The mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold migrates from the reagent releasing end to the control zone and binds with the anti-mouse IgG antibody, forming a red-purplish line in the control zone. The unbound mouse monoclonal anti-human IgG4
antibody conjugated to colloidal gold will further migrate to the reaction zone and binds to the antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti -human IgG4. antibody conjugated to colloidal gold. The presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested.
The rapid imraunochromatography assay was then evaluated using various categories of serupi samples . The results of the evaluation as tabulated in Table 1 and 2 demonstrate the sensitivity and specificity of the immunochromatography assay of thg present invention:
(a) Sensitivity:
Table 1 showing- the sensitivity of the pres ent method to detect Wuchereria. bancrofti infection was 75/76 = 98 . 6 % .
(b) Specificity
Table 2 showing the specificity of the present method was 437/438 = 99.8%.
Further included in this invention is a diagnostic kit for rapid detection of lymphatic filariasis employing the methods described above. In one embodiment of the present invention, the kit comprises a detection device wherein the detection device contains an absorbent nitrocellulose membrane. The nitrocellulose membrane has three zones : a sample receiving end, a reaction zone and a control zone characterized by the SXP/SXP-1 recombinant antigen
immobilized within the reaction zone. Anti -mouse IgG antibody is immobilized, within the control zone- The detection device preferably is in a dipstick format . The kit further provides a microwell containing dried mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold,. The kit also comprises of. a buffer wherein the buffer comes in a separate container from the detection device .
In another embodiment of the kit which comprises a detection device wherein the detection device contains an absorbent nitrocellulose membrane. The nitrocellulose membrane has four zones; a sample receiving end, a reagent releasing end, a reaction zone and a control zone characterized by the SXP/SXP-1 recombinant antigen immobilized within the reaction zone. Mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold is incorporated within the reagent releasing end and anti-mouse IgQ antibody is immobilized within the control zone of the membrane. The detection device may be in a dipstick or cassette format. The kit also comprises of a buffer wherein the buffer comes in a separate container from the detection device.
. A volume of sample ranging from 10 to 50 jJl is required for the rapid immunochromatography assay and rasult is obtained within 15 to 20 minutes. If a sample contains anti-filarial IgG4 antibodies specific to SXP/SXP-1 recombinant antigen, two red-purplish lines will be observed, each in the reaction, zone and control zone. If the sample does not contain anti-
filarial IgG4 antibodies specific to SXP/SXP-1 recombinant antigen, the complex SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold will not be formed, thus no red- purplish line will be seen in the reaction zone, resulting in the final appearance of only one red- purplish line in the control zone. In cone Lusion two red-purplish lines on the nitrocellulose membrane denotes a positive test result and oμe line denotes a negative test result as shown in Figure 3.
While particular embodiments of the subject invention have been described, it will be obvious to
'those skilled in the art that various changes and modifications to the subject invention cart be made without departing from the scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention.