Protocol for ELISA Kit

Transcription

1 Prtcl fr ELISA Kit Preparatin Sample Preparatin and Strage Stre samples t be assayed within 24 hurs at 2-8 C. Fr lng-term strage, aliqut and freeze samples at -20 C. Avid repeated freeze-thaw cycles. Cell culture supernate, e, tissue lysate r bdy fluids: Remve particulates by centrifugatin, analyze immediately r aliqut and stre at -20 C Serum: Allw the serum t clt in a serum separatr tube (abut 2 hur) at rm temperature. Centrifuge at apprximately 1000 X g fr15min. Analyze the serum immediately r aliqut and stre frzen at -20 C. Plasma: Cllect plasma using heparin r EDTA as an anticagulant. Centrifuge fr 15 min at 2-8 at 1000 x g within 30 min f cllectin. Analyze immediately r aliqut and stre frzen at -20 C. Sample Dilutin Guideline The user needs t estimate the cncentratin f the target prtein in the sample and select a prper dilutin factr s that the diluted target prtein cncentratin falls near the middle f the linear regime in the standard curve. Dilute the sample using the prvided diluent buffer. The fllwing is a guideline fr sample dilutin. Several trials may be necessary in practice. The sample must be well mixed with the diluents buffer. High target prtein cncentratin ( ng/ml). The wrking dilutin is 1:100. i.e. Add 3 l sample int 297 l sample diluent buffer. Medium target prtein cncentratin ( ng/ml). The wrking dilutin is 1:10. i.e. Add 25 l sample int225 l sample diluent buffer. Lw target prtein cncentratin ( pg/ml). The wrking dilutin is 1:2. i.e. Add 100 l sample t 100 l sample diluent buffer. Very Lw target prtein cncentratin ( pg/ml). N dilutin necessary, r the wrking dilutin is 1:2. Reagent Preparatin and Strage A. Recnstitutin f the human BAFF standard BAFF standard slutin shuld be prepared n mre than 2 hurs prir t the experiment. Tw tubes f BAFF standard (10ng per tube) are included in each kit. Use ne tube fr each experiment. a. 10,000pg/ml f human BAFF standard slutin: Add 1 ml sample diluent buffer int ne tube, keep the tube at rm temperature fr 10 min and mix thrughly. b. 4000pg/ml f human BAFF standard slutin: Add 0.4 ml f the abve 10ng/ml BAFF standard slutin int 0.8 ml sample diluent buffer and mix thrughly. c. 2000pg/ml 62.5pg/ml f human BAFF standard slutins: Label 6 Eppendrf tubes with 2000pg/ml, 100pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, respectively. Aliqut 0.3 ml f the sample diluent buffer int each tube. Add 0.3 ml f the abve 2000pg/ml BAFF standard slutin int 1st tube and mix. Transfer 0.3 ml frm 1st tube t 2nd tube and mix. Transfer 0.3 ml frm 2nd tube t 3rd tube and mix, and s n. Nte: The standard slutins are best used within 2 hurs. The 10 ng/ml standard slutin may be stred at 4 C fr up t 12 hurs, r at -20 C fr up t 48 hurs. Avid repeated freeze-thaw cycles. B. Preparatin f bitinylated anti-human BAFF antibdy wrking slutin: The slutin shuld be prepared n

2 Prtcl fr ELISA Kit mre than 2 hurs prir t the experiment. a. The ttal vlume shuld be: 0.1ml/well x (the number f wells). (Allwing ml mre than ttal vlume) b. Bitinylated anti-human BAFF antibdy shuld be diluted in 1:100 with the antibdy diluent buffer and mixed thrughly. (i.e. Add 1 l Bitinylated anti-human BAFF antibdy t 99 l antibdy diluent buffer.) C. Preparatin f Avidin-Bitin-Perxidase Cmplex (ABC) wrking slutin: The slutin shuld be prepared n mre than 1 hur prir t the experiment. a. The ttal vlume shuld be: 0.1ml/well x (the number f wells). (Allwing ml mre than ttal vlume) b. Avidin- Bitin-Perxidase Cmplex (ABC) shuld be diluted in 1:100 with the ABC dilutin buffer and mixed thrughly. (i.e. Add 1 l ABC t 99 l ABC diluent buffer.) Assay Prcedure The ABC wrking slutin and TMB clr develping agent must be kept warm at 37 C fr 30 min befre use. When diluting samples and reagents, they must be mixed cmpletely and evenly. Standard BDNF detectin curve shuld be prepared fr each experiment. The user will decide sample dilutin fld by crude estimatin f BAFF amunt in samples. 1. Aliqut 0.1ml per well f the 4000pg/ml, 2000pg/ml,1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml human BAFF standard slutins int the precated 96-well plate. Add 0.1ml f the sample diluent buffer int the cntrl well (Zer well). Add 0.1ml f each prperly diluted sample f muse sera, plasma, bdy fluids, tissue lysates r cell culture supernatants t each empty well. See Sample Dilutin Guideline abve fr details. We recmmend that each human BAFF standard slutin and each sample is measured in duplicate. 2. Seal the plate with the cver and incubate at 37 C fr 90 min. 3. Remve the cver, discard plate cntent, and blt the plate nt paper twels r ther absrbent material. D NOT let the wells cmpletely dry at any time. 4. Add 0.1ml f bitinylated anti-human BAFF antibdy wrking slutin int each well and incubate the plate at 37 C fr 60 min. 5. Wash plate 3 times with 0.01M TBS r 0.01M PBS, and each time let washing buffer stay in the wells fr 1 min. Discard the washing buffer and blt the plate nt paper twels r ther absrbent material. Plate Washing Methd: Discard the slutin in the plate withut tuching the side walls. Blt the plate nt paper twels r ther absrbent material. Sak each well with at least 0.3 ml PBS r TBS buffer fr 1~2 minutes. Repeat this prcess tw additinal times fr a ttal f THREE washes. Nte: Fr autmated washing, aspirate all wells and wash THREE times with PBS r TBS buffer, verfilling wells with PBS r TBS buffer. Blt the plate nt paper twels r ther absrbent material. 6. Add 0.1ml f prepared ABC wrking slutin int each well and incubate the plate at 37 C fr 30 min. 7. Wash plate 5 times with 0.01M TBS r 0.01M PBS, and each time let washing buffer stay in the wells fr 1-2 min. Discard the washing buffer and blt the plate nt paper twels r ther absrbent material. (See Step 5 fr plate washing methd). 8. Add 90 l f prepared TMB clr develping agent int each well and incubate plate at 37 C in dark fr 15-20min (Nte: Fr reference nly, the ptimal incubatin time shuld be determined by end user. And the shades f blue can be seen in the wells with the fur mst cncentrated human BAFF standard

3 Prtcl fr ELISA Kit slutins; the ther wells shw n bvius clr). 9. Add 0.1ml f prepared TMB stp slutin int each well. The clr changes int yellw immediately. 10. Read the O.D. absrbance at 450nm in a micrplate reader within 30 min after adding the stp slutin. Fr calculatin, (the relative O.D.450) = (the O.D.450 f each well) (the O.D.450 f Zer well). The standard curve can be pltted as the relative O.D.450 f each standard slutin (Y) vs. the respective cncentratin f the standard slutin (X). The muse BDNF cncentratin f the samples can be interplated frm the standard curve. Nte: if the samples measured were diluted, multiply the dilutin factr t the cncentratins frm interplatin t btain the cncentratin befre dilutin. Summary 1. Add samples and standards and incubate the plate at 37 C fr 90 min. D nt wash. 2. Add bitinylated antibdies and incubate the plate at 37 C fr 60 min. Wash plate 3 times with 0.01M TBS. 3. Add ABC wrking slutin and incubate the plate at 37 C fr 30 min. Wash plate 5 times with 0.01M TBS. 4. Add TMB clr develping agent and incubate the plate at 37 C in dark fr min. 5. Add TMB stp slutin and read. Typical Data Obtained frm Human BAFF (TMB reactin incubate at 37 C fr 16 min) Cncentratin 0.0pg/ml 62.5pg/ml 125pg/ml 250pg/ml 500pg/ml 1000pg/ml 2000pg/ml 4000pg/ml O.D Typical Standard Curve This standard curve was generated at GenWay fr demnstratin purpse nly. A standard curve must be run with each assay O.D Cncentratin(pg/ml)

4 Prduct Infrmatin Sheet Catalg N. GWB-ZZD169 Size 96T Range 62.5pg/ml-4000pg/ml Sensitivity < 2pg/ml Specificity N detectable crss-reactivity with any ther cytkine. Strage Stre at 4 fr frequent use, at -20 fr infrequent use. Avid multiple freeze-thaw cycles (Shipped with wet ice.) Expiratin Tw mnths at 4 and fur mnths at -20. Applicatin Fr quantitative detectin f human BAFF in sera, plasma, bdy fluids, tissue lysates r cell culture supernates. Principle GenWay s human BAFF ELISA Kit was based n standard sandwich enzyme-linked immune-srbent assay technlgy. Human BAFF specific-specific plyclnal antibdies were precated nt 96-well plates. The human specific detectin mnclnal antibdies were bitinylated. The test samples and bitinylated detectin antibdies were added t the wells subsequently and then fllwed by washing with PBS r TBS buffer. Avidin-Bitin-Perxidase Cmplex was added and unbund cnjugates were washed away with PBS r TBS buffer. HRP substrate TMB was used t visualize HRP enzymatic reactin. TMB was catalyzed by HRP t prduce a blue clr prduct that changed int yellw after adding acidic stp slutin. The density f yellw is prprtinal t the human BAFF amunt f sample captured in plate. Kit Cmpnents 1. Lyphilized recmbinant human BAFF standard: 10ng/tube One 96-well plate precated with anti- human BAFF antibdy. 3. Sample diluent buffer: 30 ml 4. Bitinylated anti- human BAFF antibdy: 130 l, dilutin 1: Antibdy diluent buffer: 12ml. 6. Avidin-Bitin-Perxidase Cmplex (ABC): 130 l, dilutin 1: ABC diluent buffer: 12ml. 8. TMB clr develping agent: 10ml. 9. TMB stp slutin: 10ml. Material Required But Nt Prvided 1. Micrplate reader in standard size. 2. Autmated plate washer. 3. Adjustable pipettes and pipette tips. Multichannel pipettes are recmmended in the cnditin f large amunt f samples in the detectin. 4. Clean tubes and Eppendrf tubes. 5. Washing buffer (neutral PBS r TBS). Preparatin f 0.01M TBS: Add 1.2g Tris, 8.5g Nacl; 450 l f purified acetic acid r 700 l f cncentrated hydrchlric acid t 1000ml H 2O and adjust ph t Finally, adjust the ttal vlume t 1L. Preparatin f 0.01 M PBS: Add 8.5g sdium chlride, 1.4g Na 2HPO 4 and 0.2g NaH 2PO 4 t 1000ml distilled water and adjust ph t Finally, adjust the ttal vlume t 1L.

5 Prduct Infrmatin Sheet Ntice fr Applicatin f Kit 1 Befre using Kit, spin tubes and bring dwn all cmpnents t bttm f tube. 2 Duplicate well assay was recmmended fr bth standard and sample testing. 3 Dn t let 96-well plate dry, dry plate will inactivate active cmpnents n plate. 4 In rder t avid marginal effect f plate incubatin due t temperature difference (reactin may be strnger in the marginal wells), it is suggested that the diluted ABC and TMB slutin will be pre-warmed in 37 fr 30 min befre using. -1X96 Well Plate Image O.D Cncentratin(pg/ml) Backgrund BAFF was regularly detected by enzyme-linked immunsrbent assay in brain tissue lysates and in nrmal spinal fluid, and in astrcytes by duble flurescence micrscpy. BAFF was lcalized in astrcytes clse t BAFF-R-expressing immune cells. BAFF receptrs were strngly expressed in situ in primary central nervus system (CNS) lymphmas. 1 The TNF superfamily member B cell-activating factr (BAFF) plays an imprtant rle in humral immunity and in autimmune diseases, including RA.Lcal BAFF gene targeting inhibited prinflammatry cytkine expressin, suppressed generatin f plasma cells and Th17 cells, and markedly amelirated jint pathlgy. 2 The B cell activating factr BAFF (BlyS/TALL-1/zTNF4) is a tumr necrsis factr (TNF)-related ligand that prmtes B cell survival and binds t three receptrs (BCMA, TACI, and the recently described BAFF-R). 3 Human BAFF was mapped t chrmsme 13q The standard used in this kit is recmbinant sluble human BAFF (A134-L295) with the mlecular mass f 19.6KDa. Reference 1. Krumbhlz, M., Theil, D., Derfuss, T., Rsenwald, A., Schrader, F., Mnranu, C.-M., Kalled, S. L., Hess, D. M., Serafini, B., Alisi, F., Wekerle, H., Hhlfeld, R., Meinl, E. BAFF is prduced by astrcytes and up-regulated in multiple sclersis lesins and primary central nervus system lymphma. J. Exp. Med. 201: , Lam, Q. L. K., K, O. K. H., Zheng, B.-J., Lu, L. Lcal BAFF gene silencing suppresses Th17-cell generatin and amelirates autimmune arthritis. Prc. Nat. Acad. Sci. 105: , Schiemann, B., Gmmerman, J. L., Vra, K., Cacher, T. G., Shulga-Mrskaya, S., Dbles, M., Frew, E., Sctt, M. L. An essential rle fr BAFF in the nrmal develpment f B cells thrugh a BCMA-independent pathway. Science 293: , Schneider, P., MacKay, F., Steiner, V., Hfmann, K., Bdmer, J. L., Hller, N., Ambrse, C., Lawtn, P., Bixler, S., Acha-Orbea, H., Valmri, D., Rmer, P., Werner-Favre, C., Zubler, R. H., Brwning, J. L., Tschpp, J. BAFF, a nvel ligand f the tumr necrsis factr family, stimulates B cell grwth. J. Exp. Med. 189: , 1999.