Immunochromatography devices, methods and kits
A technology of immunochromatography and analytes, which is applied in the field of immunochromatography devices and assays, and can solve problems such as insufficient diagnosis
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[0076]One of ordinary skill in the art can determine the nucleic acid sequence of a monoclonal antibody using a variety of techniques known in the art. Nucleotides encoding monoclonal antibodies can be cloned to produce "recombinant" monoclonal antibodies. Any recombinant cloning technique can be used, including the use of polymerase chain reaction (PCR) to prime the synthesis of antibody-encoding nucleotide sequences. Accordingly, methods for producing monoclonal antibodies include methods comprising: obtaining at least a first suitable anti-Z-AAT antibody-encoding nucleotide molecule or segment from a suitable anti-Z-AAT antibody-producing cell (preferably a hybridoma); and The nucleic acid molecules or segments are expressed in recombinant host cells to obtain recombinant anti-Z-AAT monoclonal antibodies.
[0077] Other recombinant techniques are known in the art, such as phage library-based methods. For example, the method may comprise: (a) administering to the animal at...
Embodiment 1
[0169] Monoclonal antibodies
[0170] Hybridomas LG96 and MG97 were prepared by immunizing BALB / c mice with multimerized human α1-antitrypsin (AAT) in complete Freund's adjuvant. Mice were immunized intraperitoneally with an interval of 7 to 8 days between immunizations. Splenocytes from immunized mice were fused with the plasmacytoma cell line NSW. The presence of monoclonal antibodies was screened by ELISA using microtiter plates coated with multimerized hAAT or sera from AAT-deficient PiZZ patients.
[0171] Selected hybridomas are cloned and screened again to select for those that produce against multimeric AAT but not native AAT. Monoclonal antibodies from hybridomas LG96 and MG97 were shown to recognize multimeric AAT and react specifically with PiZ sera.
[0172] Hybridomas were frozen in a specific medium (DMED with 20% FCS and 10% DMSO) at a cell concentration of 2 x 106 cells / ml per vial. Store cells under nitrogen. Cells can be recovered using DMED-10 medium or...
Embodiment 2
[0174] Test of antibodies LG96 and MG97 against PiZZ-type AAT
[0175] The ability of monoclonal antibodies LG96 and MG97 to specifically bind native PiZZ was tested in a sandwich ELISA. A small amount of cell culture supernatant containing the antibodies was obtained and both antibodies were partially purified with CANDOR Bioscience GmbH (Wangen, Germany).
[0176] Pair test, sandwich ELSA
[0177] All immunoassay buffers used were supplied by CANDOR Bioscience. Partially purified antibodies LG96 or MG97 were used to coat microtiter plates (MaxiSorp TM , Nunc, Langenselbold, Germany), and incubated for 3 hours at room temperature with shaking. After removal of the coating buffer, the plate was blocked by adding 300 μl of blocking solution (product code 110) to each well and incubated overnight at 4°C. Plates were washed three times with Wash Buffer (Product #140). Afterwards, human serum (mixed genotype ZZ serum or MM serum) was diluted 1:20 and 1:80 (5% and 1.25% serum)...
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