Multiplex immuno screening assay
The present invention provides an immunoassay leading to the rapid and simultaneous detection of antibodies to a wide range of infectious pathogens in biological fluids of infected patients. This immunoassay involves the covalent and oriented coupling of fusion proteins comprising an AGT enzyme and a viral antigen on an identifiable solid support (e.g. fluorescent microspheres). The thus obtained antigen-coupled microspheres show enhanced capture of specific antibodies as compared to antigen-coupled microspheres produced by standard amine coupling procedures.
1. A method for detecting at least two target antibodies in a biological sample comprising:
(a) contacting said biological sample selected from the group consisting of whole blood, serum, plasma, urine, seminal fluid, cerebrospinal fluid and saliva with at least one first microparticle bound to a 6-alkylguanine-DNA-alkyltransferase (AGT) substrate covalently coupled to a first fusion protein comprising:
a 6-alkylguanine-DNA-alkyltransferase (AGT) polypeptide having a O6-alkylguanine-DNA alkyltransferase activity and
a first epitope that is recognized by a first target antibody;
(b) contacting said biological sample with at least one second microparticle bound to a 6-alkylguanine-DNA-alkyltransferase (AGT) substrate covalently coupled to a second fusion protein comprising:
a 6-alkylguanine-DNA-alkyltransferase (AGT) polypeptide having a O6-alkylguanine-DNA alkyltransferase activity and
a second epitope that is recognized by a second target antibody, but not by said first target antibody; and
(c) detecting the presence or absence of the two target antibodies by detecting the binding or lack of binding of the two target antibodies to the two fusion proteins.
2. The method of claim 1 , wherein each of said microparticles is covalently coupled to said substrate of 6-alkylguanine-DNA-alkyltransferase (AGT) polypeptide.
3. The method of claim 2 , wherein each of said microparticles is a magnetic microparticle internally labeled with a fluorescent dye.
4. The method of claim 3 , wherein said biological sample is serum.
5. The method of claim 1 , wherein each of said microparticles is magnetic.
6. The method of claim 1 , wherein each of said microparticles is labeled with a label selected from the group consisting of a fluorochrome, a chromophore, a radioisotope, and a mass tag.
7. The method of claim 1 , wherein each of said microparticles is a microparticle internally labeled with a fluorescent dye with magnetite encapsulated in a functional polymer outer coat containing surface carboxyl groups for covalent coupling of ligands.
8. The method of claim 1 , comprising contacting said biological sample with at least 10 differently coupled microparticles.
9. The method of claim 1 , further comprising detecting the presence or absence of the two target antibodies with secondary antibodies recognizing the constant part of the target antibodies.
10. The method of claim 1 , wherein said first and second fusion proteins comprise a 6-alkylguanine-DNA-alkyltransferase (AGT) polypeptide having at least 85% identity with the amino acid sequence of SEQ ID NO:2.
11. The method of claim 1 , wherein said first and second fusion proteins comprise a 6-alkylguanine-DNA-alkyltransferase (AGT) polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO:2.
12. The method of claim 1 , wherein said first or second epitope is selected from the group consisting of the amino acid sequences encoded by nucleotide sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58.
13. The method of claim 1 , wherein said first and second fusion proteins that are coupled with said first and second microparticles are selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149 and SEQ ID NO:151.