Rat SIgA ELISA Kit

Product No.: eia05548 read the manual thoroughly before use Based on the principle of classical enzyme-linked sandwich technology, this ELISA kit can measure rat secretory immunoglobulin A (sIgA), which can only be used for research purposes, not for medical diagnosis Working principle secretory immunoglobulin A (sIgA) is an IgA antibody first found in human exocrine fluid by chodircker and Tomas in the early 1960s It is different from IgA in physical and chemical properties or immunological properties in serum SIgA is the main effector molecule of mucosal immune system, which mainly exists in tears, saliva, milk, respiratory tract, digestive tract and urogenital tract There is secretory immunity on the surface of mucous membrane, and IgA on the surface of mucous membrane provides * immune defense line SIgA is a characteristic of mucosal immune response SIgA antibody can provide a special immune barrier to the mucosal surface by blocking the mucosal contact with bacteria and viruses The gastrointestinal tract is the central organ of SIgA SIgA is the immunoglobulin with more Zui, which can be secreted as high as 30-100 mg / D · kg SlgA is a polymer, so it can cross connect with viruses, bacteria, parasites or their toxins to form a macromolecular complex The provided ELISA kit is a typical sandwich ELISA kit The prepackaged antibody is polyclonal antibody The antibody in the detection phase was also polyclonal and labeled with biotin The sample and biotin labeled antibody were added to the enzyme plate pore successively and then washed by PBS or TBS After that, we added the peroxidase labeled avidin reaction, washed PBS or TBS thoroughly, and then developed the color with the substrate TMB TMB was converted to blue under the catalysis of peroxidase, and to Zui yellow under the action of acid There was a positive correlation between the color depth and the factors to be measured Content of each kit (96T) material quantity standard 96T (2vial) pre coated 96 pore plate 96T (8 × 12) biotin labeled antibody 96T (1:100) ABC (avidin peroxidase complex) 96T (1:100) sample diluent 96T × 2 antibody diluent 96T × 1 ABC diluent 96T × 1 TMB developer a 96T × 1 TMB developer B 96T × 1 TMB termination solution 96T × 1 1.37 ℃ incubator for reagents and equipment not provided but needed for ELISA special detergent 96T × 1 (1:25) 2 Standard Specification enzyme labeling instrument 3 Automatic washing machine 4 Single channel or multi-channel adjustable pipette and its suction head 5 Distilled water, volumetric flask, etc 6 Clean test tube and eppendof tube Note 1 Balance at room temperature for at least 30 minutes before opening the kit 2 When preparing various reagents, remember to mix them well! 3 When using the kit for the first time, the user should centrifuge all kinds of reagent tubes for several minutes so that the reagent can be concentrated at the bottom of the tube 4 It is recommended that all standards and samples be tested in duplicate 5 Strictly avoid the drying of enzyme plate during operation Drying will make the biological components on the enzyme plate inactivate quickly! 6 In order to avoid cross contamination, it is necessary to avoid reusing the suction head and test tube in hand Washing method: manually wash the board: suck (do not touch the board wall) or shake off the liquid in the enzyme standard board; lay several layers of absorbent paper on the experimental platform, and pat the enzyme standard board downward for several times; inject at least 0.35ml of recommended PBS or TBS washing buffer into the hole and soak for 1-2 minutes Repeat the process as many times as necessary Automatic plate washing: if there is an automatic plate washing machine, it shall be used in the formal experiment process after skilled use If the preparation and preservation of samples are not analyzed immediately, they shall be refrigerated and preserved after sub packaging, and repeated freezing and thawing shall be avoided Serum: collect the blood with a clean tube, coagulate at room temperature for 2 hours or overnight at 4 ℃, centrifugate 1000 × g for 10 minutes, and collect the serum Immediately analyze or repack and freeze at - 20 ℃ Cell culture, tissue homogenate, body fluid - remove the precipitate by centrifugation, analyze it immediately or freeze it at - 20 ℃ after repacking The general principle of sample dilution is that the user should consult the literature to understand the content of the factors to be measured in the sample, and determine the appropriate dilution ratio, so that the concentration of the factors to be measured in the diluted sample is within the Zui optimal detection range of the ELISA kit The dilution of samples shall be recorded in detail Preparation and preservation of reagents A dilution and use of standards: prepare within 2 hours before use Add 1ml of sample diluent into the lyophilized tube, dissolve it thoroughly, and then dilute it by multiple ratio The diluted standard should be used within 2 hours B Biotin labeled antibody working solution: prepare within 2 hours before use 1 Calculate the total dosage according to the requirement of 0.1ml per hole (0.1-0.2ml more should be prepared in actual preparation) 2 Prepare the working solution according to the ratio of 10ul biotin labeled antibody and 990ul antibody diluent Mix gently C Preparation of ABC working solution: prepare within 1 hour before use 1 Calculate the total dosage according to the requirement of 0.1ml for each hole (0.1-0.2ml more should be prepared in actual preparation) 2 The working solution was prepared according to the ratio of 10ul avidin peroxidase complex (ABC) and 990ul ABC diluent Mix gently D Preparation of TMB working solution: 30 minutes before use, prepare TMB working solution according to the proportion of 9 parts of TMB developing solution a and 1 part of TMB developing solution B Operation procedure 1 Determine the number of enzyme plate holes coated with antibody required for this test 2 Add 0.1ml of diluted standard into a row of 7 holes in turn, and only add the sample diluent into one hole as the control The treated samples were added as 100ul per hole 3 Enzyme plate and cover, reaction at 37 ℃ for 90 minutes 4 After reaction, use the automatic washing machine to absorb the liquid in the enzyme standard plate; or shake off the liquid in the enzyme standard plate, and then pat it on the absorbent paper for several times Wash twice 5 Add the prepared biotin antibody working solution in order of 0.1ml per pore Reaction at 37 ℃ for 60 minutes Wash 6.0.01m TBS or 0.01M PBS for 3 times and soak for about 1 minute each time 7 Add 0.1ml ABC working solution into each hole in turn 37 ℃ for 30 minutes Wash 8.0.01m TBS or 0.01M PBS for 5 times and soak for about 1-2 minutes each time 9 Add 0.09mL TMB chromogenic solution which has been balanced at 37 ℃ for 30 minutes in turn according to each hole, and avoid light reaction at 37 ℃ During the reaction, observe frequently When the first 3-4 holes of the standard have obvious gradient blue color, and the last 3-4 holes have no obvious difference, add 0.1ml/hole TMB termination solution (the color reaction Zui should not be longer than 30 minutes) 10 The O.D value was measured at 450 nm by enzyme labeling instrument 11 Find out the corresponding concentration on the coordinate according to the absorption value of the sample Users can also use a variety of applications to calculate It should be remembered that as the sample is diluted by N times, its actual concentration should be x n Summary of operation procedures: prepare reagents, add samples and standards to the prepared samples and standards, wash the plate twice in 90 minutes at 37 ℃, add biotinylated antibody working solution, wash the plate three times in 60 minutes at 37 ℃, add ABC working solution, wash the plate five times in 30 minutes at 37 ℃, add TMB developer, add TMB termination solution at 37 ℃ and read OD value within 30 minutes The detection range of the factors to be measured was calculated: 100ng / ml → 1.56ng/ml Sensitivity: < 0.5ng/ml specificity: no cross reaction between the system and other cytokines Storage: - 20 ℃ [4 ℃ for a short period (such as two weeks)] validity period: 12 months (- 20 ℃) Purpose: for quantitative analysis of liquid samples in vitro REFERENCES 1． Avwenagha O,Campbell G,Bird MM.The outgrowth response of the axons of developing and regenerating rat retinal ganglion cells in vitro to neurotrophin treatment[J].J Neurocytol 2003;32（9）:1055-1075 2． Garcia M,Forster V,Hicks D, Vecino E.In vivo expression of neurotrophins and neurotrophin receptors is conserved in adult porcine retina in vitro[J].Invest Ophthalmol Vis Sci 2003;44（10）:4532-4541 3． von Bartheld CS, Butowt R.Expression of neurotrophin-3（NT-3） and anterograde axonal transport of endogenous NT-3 by retinal ganglion cells in chick embryos[J].J Neurosci 2000;20（2）:736-748 4． Bennett JL,Zeiler SR,Jones KR.Patterned expression of BDNF and NT-3 in the retina and anterior segment of the developing mammalian eye[J].Invest Ophthalmol Vis Sci 1999;40（12）:2996-3005