Patent Application: US-51874207-A

Abstract:
the present invention refers to an immuno - chromatographic detection cup and its use for simultaneously detecting at least one antigen , e . g . tuberculosis antigen , malaria antigen and pneumonia antigen , and at least one antibody , e . g . hiv antibody , hcv antibody and h . pylori antibody , in a urine sample . the urinary detection cup comprises a sample - collecting container ; a conjugate releasing pad comprising a gold labelled antibody x and an oligo - nucleotide linked antibody x ′, wherein both antibodies are directed against the same antigen , and a gold labelled antigen y and an oligo - nucleotide linked antigen y ′, wherein both antigens are recognized by the same antibody ; a test means inside the container separated from the conjugate releasing pad , which test means comprises a region comprising an oligo - nucleotide complementary to the oligo - nucleotide linked to antibody x ′, a region comprising an oligo - nucleotide complementary to the oligo - nucleotide linked antigen y ′, and at least one region comprising a control antibody and / or a control antigen ; and at least two sample absorbent pads linked to the test device at different positions .

Description:
fig1 shows top and side views of a typical rapid - flow immuno - chromatographic test device in the form of a test strip 101 including a sample pad 102 , a conjugate pad 103 , a membrane 104 , an absorbent pad 105 , an adhesive 106 , a supporting backing 107 , a test zone 108 , and a control zone 109 . fig2 a shows an assembly comprising a test strip 201 , an absorbent sample pad 202 , and an absorbent pad 205 . fig2 b shows an immuno - chromatographic detection cup comprising a sample - collecting container 210 , a cap 211 , and the assembly shown in fig2 a . fig3 a shows an assembly comprising a test strip 101 , an absorbent sample pad 102 , a nitrocellulose membrane 104 , a conjugate pad 103 , an adhesive 106 , a supporting backing 107 , a control zone 109 , and an absorbent pad 105 . the conjugate pad 103 contains the gold - labelled antibody 302 , oligonucleotide 1 labelled antibody 304 , gold labelled antigen 303 , oligonucleotide 2 labelled antigen 301 . capturing complementary oligo - nucleotide 1 ′ and 2 ′ are immobilized within sample zone ( s ) 305 and 306 , respectively , onto the nitrocellulose membrane 104 . fig3 b shows the detection test strip of fig3 a after the application of a sample 307 that contains a target antibody and a target antigen . one embodiment of the rapid immuno - chromatographic detection system of the present invention is shown in fig2 a , b . the immuno - chromatographic detection cup comprises a sample - collecting container 210 , actually the “ cup ”, a cap 211 for closing the container , and a detection test strip 201 inserted into the container 210 . the test strip 201 may be placed inside the transparent container wall , and the test result can be read on the outer surface . the test strip 201 comprises a nitrocellulose membrane 204 and is linked via an absorbent pad 202 on the test strip 201 to an absorbent sample pad 202 placed on the bottom of the container 210 . the absorbent sample pad 202 is designed to cover the inner surface of the container bottom . at the opposite end , the test strip 201 is linked via an absorbent pad 205 on the test strip to an absorbent pad 205 which is fitted into the cap 211 of the container and is designed to be thick enough to absorb more than 15 ml of sample . a gold conjugate releasing pad 203 is fixed to the internal surface of the container wall which pad 203 comprises ( 1a ) colloidal gold - labelled antibodies and ( 1b ) oligo - nucleotide - linked antibodies , and ( 2a ) colloidal gold - labelled antigens and ( 2b ) oligo - nucleotide - linked antigens . the gold conjugates will begin releasing from the pad 203 during the urine sample is streaming into the container 210 . release into the sample will increase the possibility of interaction between the conjugates and the analytes , e . g . antibodies and / or antigens . capturing complementary oligo - nucleotides are immobilized within one or more sample zones 208 onto the nitrocellulose membrane 204 ( see also fig3 a and fig3 b where it is referred to as 305 , 306 , i . e . “ capturing complementary oligo - nucleotide 1 or 2 sample zones ”). if the target antigen is present in the sample , the colloidal gold - labelled antibody and oligo - nucleotide - linked antibody will form a complex with the antigen . if the target antibody is present in the sample , the colloidal gold - labelled antigen and oligo - nucleotide - linked antigen will form a complex with the antibody . subsequently , the formed complexes migrate to the fixed complementary oligo - nucleotides located at the sample zones 208 and are bound to the solid phase via nucleotide - nucleotide interaction . as a result , at least two types of reactions can be detected on a single test device by the combination of ( 1 ) colloidal gold - labelled and oligo - nucleotide - linked antibodies and capturing complementary oligo - nucleotides for detecting an antigen , and ( 2 ) colloidal gold - labelled and oligo - nucleotide - linked antigens and capturing complementary oligo - nucleotides for detecting antibodies , wherein antibodies , antigens and complementary oligo - nucleotides are immobilized on a nitrocellulose membrane . non - specific antibody and / or antigen is immobilized as a control zone 209 onto the nitrocellulose membrane 204 for non - specific capturing of the gold conjugates ( see also fig3 ). the complementary oligo - nucleotides within the sample zones 208 are for specific capturing of the specific colloidal gold - labelled antibody or antigen - linked oligo - nucleotides . the antigens used may be synthetic or recombinant . the antibodies used may be polyclonal or monoclonal . in the embodiment shown in fig2 a , b the sample zone 208 is realized by a sample line , and the control zone 209 is realized by a control line . preferably , one sample zone is provided for each parameter . more than one sample zone 208 for each parameter and / or more than one control zone 209 are also contemplated . the preparation of oligo - nucleotide - labelled antibody fab ′ comprises the following steps 6 : the preparation of oligo - nucleotide - labelled protein comprises the following steps 7 : the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled recombinant envelop protein gp160 , oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 1 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hiv antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , and the second sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hiv antibody and tuberculosis antigen free sample . the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - hrp - ii , oligo - nucleotide 3 linked anti - hrp - ii , gold labelled recombinant envelop protein gp160 , oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( malaria antigen = hrp - ii specific detection zone ) comprises complementary oligo - nucleotide 3 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hiv antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that malaria antigen ( hrp - ii ) is present in the sample , and the second sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hiv antibody and malaria antigen free sample . the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled anti - hrp - ii , oligo - nucleotide 3 linked anti - hrp - ii , gold labelled recombinant envelop protein gp160 , and oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 5 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( malaria antigen = hrp - ii specific detection zone ) comprises complementary oligo - nucleotide 3 ′ immobilized onto the nitrocellulose membrane . a third sample zone 208 ( hiv antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , the second sample zone 208 and control zone 209 turn into purple colour in case that malaria antigen ( hrp - ii ) is present in the sample , and the third sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hiv antibody , tuberculosis antigen and malaria antigen free sample . the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - hrp - ii , oligo - nucleotide 3 linked anti - hrp - ii , gold labelled recombinant hcv ns3 antigen , oligo - nucleotide 4 linked hcv ns3 antigen . a first sample zone 208 ( malaria antigen = hpr - ii specific detection zone ) comprises complementary oligo - nucleotide 3 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hcv ns3 antibody specific detection zone ) comprises complementary oligo - nucleotide 4 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg and goat anti - hcv ns3 antigen . the first sample zone 208 and control zone 209 turn into purple colour in case that malaria antigen ( hrp - ii ) is present in the sample , and the second sample zone 208 and control zone 209 turn into purple colour in case that hcv antibody ( anti - ns3 ) is present in the sample ; only the control zone turns into purple colour in case of hcv ns3 antibody and malaria antigen free sample . the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled recombinant hcv ns3 antigen , oligo - nucleotide 4 linked hcv ns3 antigen . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 1 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hcv ns3 antibody specific detection zone ) comprises complementary oligo - nucleotide 4 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg and goat anti - hcv ns3 antigen . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , and the second sample zone 208 and control zone 209 turn into purple colour in case that hcv ns3 antibody is present in the sample ; only the control zone turns into purple colour in case of hcv ns3 antibody and tuberculosis antigen free sample . urinary hiv antibody , hcv antibody and malaria antigen detection cup ( three - parameters ) the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - hrp - ii , oligo - nucleotide 3 linked anti - hrp - ii , gold labelled recombinant hcv ns3 antigen , oligo - nucleotide 4 linked hcv ns3 antigen , gold labelled recombinant envelop protein gp160 , and oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( malaria antigen = hrp - ii specific detection zone ) comprises complementary oligo - nucleotide 3 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hcv ns3 antibody specific detection zone ) comprises complementary oligo - nucleotide 4 ′ immobilized onto the nitrocellulose membrane . a third sample zone 208 ( hiv gp160 antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg , goat anti - hcv ns3 antigen and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that malaria antigen ( hpr - ii ) is present in the sample , the second sample zone 208 and control zone 209 turn into purple colour in case that hcv antibody ( anti - ns3 ) is present in the sample , and the third sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hcv ns3 antibody , hiv gp160 antibody and malaria antigen ( hrp - ii ) free sample . urinary hiv antibody , hcv antibody and tuberculosis antigen detection cup ( three - parameters ) the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled recombinant hcv ns3 antigen , oligo - nucleotide 4 linked hcv ns3 antigen gold labelled recombinant envelop protein gp160 , and oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 1 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( hcv ns3 antibody specific detection zone ) comprises complementary oligo - nucleotide 4 ′ immobilized onto the nitrocellulose membrane . a third sample zone 208 ( hiv gp160 antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg , goat anti - hcv ns3 antigen and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , the second sample zone 208 and control zone 209 turn into purple colour in case that hcv antibody ( anti - ns3 ) is present in the sample , and the third sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hcv antibody ( anti - ns3 ), hiv antibody ( anti - gp160 ) and tuberculosis antigen ( lam ) free sample . urinary hiv antibody , hcv antibody , malaria antigen and tuberculosis antigen detection cup ( four - parameters ) the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled anti - hrp - ii , oligo - nucleotide 3 linked anti - hrp - ii , gold labelled recombinant hcv ns3 antigen , oligo - nucleotide 4 linked hcv ns3 antigen , gold labelled recombinant envelop protein gp160 , and oligo - nucleotide 2 linked recombinant envelop protein gp160 . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 1 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( malaria antigen = hrp - ii specific detection zone ) comprises complementary oligo - nucleotide 3 ′ immobilized onto the nitrocellulose membrane 204 . a third sample zone 208 ( hcv ns3 antibody specific detection zone ) comprises complementary oligo - nucleotide 4 ′ immobilized onto the nitrocellulose membrane . a fourth sample zone 208 ( hiv gp160 antibody specific detection zone ) comprises complementary oligo - nucleotide 2 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg , goat anti - hcv ns3 antigen and goat anti - gp160 . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , the second sample zone 208 and control zone 209 turn into purple colour in case that malaria antigen ( hrp - ii ) is present in the sample , the third sample zone 208 and control zone 209 turn into purple colour in case that hcv antibody ( anti - ns3 ) is present in the sample , and the fourth sample zone 208 and control zone 209 turn into purple colour in case that hiv antibody ( anti - gp160 ) is present in the sample ; only the control zone turns into purple colour in case of hcv antibody , hiv antibody , malaria antigen and tuberculosis antigen free sample . urinary tuberculosis antigen , pneumonia antigen and h . pylori antibody detection cup ( three - parameters ) the conjugate releasing pad 203 immobilized onto the internal surface of the sample - collecting container 210 comprises gold labelled anti - lam , oligo - nucleotide 1 linked anti - lam , gold labelled anti - pneumolysin ( ply ), oligo - nucleotide 5 linked anti - pneumolysin ( ply ), and gold labelled h . pylori antigen , oligo - nucleotide 6 linked h . pylori antigen . a first sample zone 208 ( tuberculosis antigen = lam specific detection zone ) comprises complementary oligo - nucleotide 1 ′ immobilized onto the nitrocellulose membrane 204 . a second sample zone 208 ( pneumonia antigen = ply specific detection zone ) comprises complementary oligo - nucleotide 5 ′ immobilized onto the nitrocellulose membrane . a third sample zone 208 ( h . pylori antibody specific detection zone ) comprises complementary oligonucleotide 6 ′ immobilized onto the nitrocellulose membrane . the control zone 209 comprises a mixture of anti - mouse igg , and goat anti - h . pylori antigen . the first sample zone 208 and control zone 209 turn into purple colour in case that tuberculosis antigen ( lam ) is present in the sample , the second sample zone 208 and control zone 209 turn into purple colour in case that pneumonia antigen ( ply ) is present in the sample , and the third sample zone 208 and control zone 209 turn into purple colour in case that h . pylori antibody is present in the sample ; only the control zone turns into purple colour in case of h . pylori antibody , tuberculosis antigen and pneumonia antigen free sample . the colloidal gold conjugate colour intensity was amplified using water - soluble chitosan ( or modified water - soluble chitosan ) as a colour intensity modification agent . the action of water - soluble chitosan ( or modified water - soluble chitosan ) in connection with the antibodies or antigens is on the colour intensity of the colloidal gold . chitosan is added during the preparation of colloidal gold , but prior to the conjugation of colloidal gold with the antibodies or the antigens . water - soluble chitosan ( or modified water - soluble chitosan ) affect the colour intensity of colloidal gold and so increases the ability of the human eye to identify the colour , and , thus , enables to detect very low concentrations of the analyte . signal amplification lies in the range of up to 10 folds . colloidal gold could be prepared by the reduction of 1 % aqueous solution of tetra - chloroauric acid ( haucl 4 ) using tri - sodium citrate aqueous solution to produce spheroidal gold particles . after colloidal gold preparation , water - soluble chitosan ( or modified water - soluble chitosan ) aqueous solution was added with a suitable volume and concentration to convert colour from purple to violet pending on the volume and concentration of the added modification solution . 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