Description

·         Used antigen:

o   Group 1 (ANA): Jo-1, PL-7, PL-12, EJ, OJ, KS, YARS, ZoA, ZoB, SAE-1, SAE-2, SRP54, Mi-2, TIF1γ, MDA5, NXP2, PMScl 75, PMScl 100, CENP A, CENP B, Scl70, POLR3A, NOR90, PDGFR-β, Fibrillarin, Th/To, Ro52, Ro60, La, SmB, SmD, RNP A, RNP C, RNP 68/70, P0, Ku, Nucleolin, dsDNA, Histone, Nucleosome, PCNA; Supplementary antigens: M2, DFS70

o   Group 2 (Myositis): Jo-1, PL-7, PL-12, EJ, OJ, KS, YARS, ZoA, ZoB, SAE-1, SAE-2, SRP54, Mi-2, TIF1γ, MDA5, NXP2; Supplementary antigens: Ro52, PMScl 75, PMScl 100, Ku

o   Group 3 (Scleroderma): CENP A, CENP B, Scl70, POLR3A, NOR90, PDGFR-β, Fibrillarin, Th/To, PMScl 75, PMScl 100, RNP A, RNP C, RNP 68/70; Supplementary antigens: Ro52, Ku, M2

o   Group 4 (SLE and other connective tissue diseases): dsDNA, Histone, Nucleosome, PCNA, SmB, SmD, RNP A, RNP C, RNP 68/70, P0, Ku, Nucleolin, Ro52, Ro60, La, NOR90

·         Antigens are applied in the form of microdots (spots) in triplets to a nitrocellulose membrane in a microtiter well format

·         If specific antibodies are present in the sample, they will bind to the respective separate antigens

·         The complex is labelled with Conjugate and detected through a colour reaction with Substrate (BCIP/NBT)

·         The kit enables 96 tests

·         Short incubation periods, total assay time: approximately 1.5 h.

·         Each individual test includes control spots, which serves to check functionality and sensitivity of the sets, and also the spots for checking the presence of the conjugate

·         Included positive controls enable validation of the tests

·         For quantitative evaluation the wells contain calibration spots

·         All reagents are supplied ready to use

·         The kit may be used sequentially for smaller batches of samples (reagents are provided in sufficient quantities)

·         Evaluation using Microblot-Array reader and the appropriate software

Applications

·         Confirmation for ELIA or other screening tests

·         Identification of individual antibodies types, differentiation of individual systemic autoimmune diseases

Brief assay procedure

·         Dilute samples of serum/plasma (1:51)

·         Pipette diluted samples

·         Incubate at room temperature for 30 min.

·         Wash 3 times for 5 min.

·         Add Conjugate

·         Incubate at room temperature for 30 min.

·         Wash 3 times for 5 min.

·         Add Substrate (BCIP/NPT)

·         Incubate at room temperature for 15 min.

·         Wash 2 times for 5 min. in distilled water

·         Dry and evaluate test