BioScience Slovakia s.r.o., Dúbravská cesta 9, Bratislava 841 04 mail: PO BOX 135, Bratislava 840 05, web: www.bioscience.sk IČO: 35898241 | IČ DPH: SK2021885888 | Bank: 2629034215/1100 Code: Product Name: B2EL Bulk antigen for diagnosing Chlamydia by ELISA Specifications A white or slightly yellow suspension of highly purified Chlamydia* cells in phosphate buffered saline with thiomersal as a preservative. A sediment is formed after a longer period of storage. The sediment can easily be resuspended by shaking. The antigen has a titer of 40. * Chlamydophila abortus, Chlamydophila psittaci and Chlamydia trachomatis are used as the source of antigen according to the customer’s request. Indication The antigen is used for the detection of antibodies against Chlamydia in the blood serum samples of humans and animals by the enzyme-­‐linked immunosorbent assay (ELISA). It is very useful for the serological screening and surveillance. Testing Shake the antigen well before use and prepare its working dilution of 1:20 using the carbonate buffer (pH 9.6). A working dilution 1:40 can also be used. It is recommended to perform a control test close to the antigen expiration date to prove whether both antigen dilutions give similar results. The method is performed with the microtiter plates type P. The plates are coated with the antigen having the working dilution of 1:20 (in a volume 50 μl per well) and kept covered with a cover slide at 4°C overnight. The coated plates are washed three times with distilled water and blocked with 2.5% casein solution (150 μl per well) for 1 h at 37°C. The plates are then washed once with distilled water and three times with phosphate buffered saline (PBS), and the sera diluted gradually from 1:100 in 2.5% casein solution containing 1.7% of bovine serum albumin (BSA)** are added (50 μl per well) and incubated for 1 h at 37°C. After threefold washing of the plates with PBS, the respective peroxidase-­‐conjugated anti IgG, IgM or IgA immunoglobulin diluted according to the manufacturer’s instructions in 2.5% casein solution is added (50 μl per well) and the plates are incubated for 1 h at 37°C. After washing in PBS (three times), a freshly prepared substrate solution composed of 0.05% 1,2-­‐phenylene diamine in 2.9% sodium citrate (pH 5.0) containing 0.04% hydrogen peroxide is added (50 μl per well). After 20 min at 37°C in dark, the reaction is stopped by the addition of 2.3 M sulphuric acid (50 μl per well) and the A492 values are read in a microplate ELISA reader (492 nm). Standard negative and positive sera are used as controls. ** BSA is not added to 2.5% casein solution when bovine sera are examined. Test evaluation A serum dilution is considered to be positive when it gives the same or a higher A492 value than that representing the A492 value of negative control serum plus three standard deviations. Storage Store in a dark and dry place at the temperature of 2 – 8°C. Expiration date One year from the date of manufacture. BioScience Slovakia s.r.o., Dúbravská cesta 9, Bratislava 841 04 mail: PO BOX 135, Bratislava 840 05, web: www.bioscience.sk IČO: 35898241 | IČ DPH: SK2021885888 | Bank: 2629034215/1100 Solutions for ELISA test: Carbonate buffer Add 0.4 g Na2CO3 and 0.73 g NaHCO3 to 200 ml of distilled water Adjust pH to 9.5 with NaOH Add 2.5 ml of 1% thiomersal Fill to 250 ml with distilled water Phosphate buffered saline (PBS) Add 40 g NaCl and 14.28 g NaHPO4 x 12 H2O and 1 g KCl to 1000 ml of distilled water Adjust pH to 7.2 with 0.5 M KH2PO4 Fill to 5000 ml with distilled water Casein solution Dissolve 50 g of casein in 800 ml of 0.3 M NaOH Stir overnight at room temperature Adjust pH to 7.5 with HCl (25% HCl ca. 27 ml) During adjustment of pH with HCl aggregates are formed that are difficult to dissolve. Therefore, titration with HCl should proceed slowly. HCl is added dropwise always after dissolving of aggregate. Add 5.84 ml of 0.5 M KH2PO4 Add 32 ml of 0.5 M Na2HPO4 Adjust pH to 7.3 with 25% HCl During adjustment of pH with HCl aggregates are formed that are difficult to dissolve. Therefore, titration with HCl should proceed slowly. HCl is added dropwise always after dissolving of aggregate. Add 20 ml of 1% thiomersal Fill to 2000 ml with distilled water Substrate solution Add 2.94 g sodium citrate to 50 ml of distilled water Adjust pH to 5.0 with H3PO4 Fill to 100 ml with distilled water