ELISA: Direct and Indirect “Sandwich” ELISA General Protocols

Sandwich ELISA (“multiple antibodies with antigen trapped in between”) has several advantages over Direct and Indirect ELISA.

                                  

Primarily, there is no need to pre-purify the sample for detection and specificity of antigen recognition is greatly enhanced by combining the selectivity of two antibodies, even in the presence of some contamination. Both polyclonal and monoclonal antibodies can be used and should be of very high purity. The key requirement of the system is that the primary “capture” antigen epitope (binding site) not interfere with or overlap the “secondary” or detection antibody epitope.

The Methods for ELISA Kits Coating

The process of set antigen and antibody called coating. In another word, coating is the process of combine antigen or antibody with solid phase surface. Protein and the polystyrene solid phase are combined by physical adsorption. It relies on the effect between hydrophobic groups on protein molecular and solid phase. This unspecific physical absorption is effect by molecular weight, isoelectric point and concentration of protein. Vector has different adsorption capacity for different proteins. Because macromolecule protein has more hydrophobic groups than small molecule protein has. It easier to absorbed on solid phase surface. IgG has strong adsorption for polystyrene solid, it usually happens on Fc parts. The connection point of antibody usually expose outside, so direct adsorption method usually using for coat antibody. Most of protein antigen can use similar method for coating. When the epitope exists in or near the hydrophobic region, the direct adsorption of antigen and solid phase can avoid epitopes over exposed. Under this situation, direct coating is not good, which can be replaced by indirect capture coating. The indirect coating is coating according to antigens specific antibody and solid the antigen through antigen-antibody reaction. This antigen is far from phase surface, the eptiope can be fully exposed. The indirect antigen coated solid phase antibody affinity chromatography role, the purity of antigen is increased largely. Because of this, antigen with lots of impurities can use capture coating method. This method can improve test specificity, sensitivity and repeatedly. The other advantage of indirect coating is small usage of antigen (0.1%~1% of direct coating used). Non-protein antigen which is hard to adsorb on polystyrene phase can use special coating method. For example, DNA is required as coating antigen to determine anti-DNA antibody. However, common solid phase can not combine with nuclear acid directly. At first, irradiate polystyrene plate......

Clinical Quality Assessment for ELISA Kits

There are two parts to evaluate ELISA Kits. The first one is evaluate reagents quality. The reagents can be produce if it meets certain requirements. The other is evaluating reagents clinical value. Take the hepatitis ELISA Kit as example. It can get the production license after it passes the Chinese Pharmaceutical and biological products determination. Testing items contain not only packaging, labeling, instructions but also reagents performance. The performance of reagent such as specificity, sensitivity, precision and linearity, should be check one by one. The reagents which pass the detection of reference materials are qualified productions. The clinical quality evaluation of ELISA Kit is using reagent to test clinical sample. The clinical text center do exam on hepatitis ELISA Kit. This can increase the quality of ELISA Kit.   A.      Clinical quality assessment points Whether can distinguish health and disease is the basic requirement for ELISA Kits clinical evaluation. At present, 100% suitable reagent is difficult to find, any experiment will produce false positive or false negative result. Evaluate reagents reliability usually take its sensitivity and specificity as assessment standard. Clinical sensitivity is display by percentage of patients positive test result. The specificity means health peoples negative exam result.   In order to process this evaluation, patients serum should be collected and exam by most reliability reagent to determine its positive or negative.   The series serum which can display the positive or negative of determine called serum panel. The relationship of reagent determination result and serum panels result is display in the following table.   Serum Panel Result Total + - Determination Result   + a b a+b _ c d c+d Total a+c b+d a+b+c+d In the table, a means true positive, b means false pos......

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人补体因子H前体,pre-CFH, Human complement factor H precursor,pre-CFH ELISA Kit 
人补体因子B前体,pre-CFB, Human complement factor B precursor,pre-CFB ELISA Kit 
猪载脂蛋白B,Apo-B, Porcine apolipoprotein B,Apo-B ELISA kit 
微囊藻毒素LR,MC-LR, Microcystin-LR,MC-LR ELISA Kit 
人CD98分子,CD98, Human Cluster of differentiation 98,CD98 ELISA Kit 
大鼠CXC趋化因子受体2,CXCR2, Rat CXC-chemokine receptor 2,CXCR2 ELISA Kit 
人白介素34,IL-34, Human Interleukin 34,IL-34 ELISA Kit 
小鼠白介素34,IL-34, Mouse Interleukin 34,IL-34 ELISA Kit 
人白介素35,IL-35, Human Interleukin 35,IL-35 ELISA Kit 
小鼠白介素35,IL-35, Mouse Interleukin 35,IL-35 ELISA Kit 
大鼠游离胆固醇,fCHOL, Rat free cholesterol,fCHOL ELISA kit 
人补体因子B,CFB, Human complement factor B,CFB ELISA Kit 
人二氢嘧啶脱氢酶,DPYD, Human Dihydropyrimidine Dehydrogenase,DPYD ELISA Kit 
人切除修复交叉互补因子,ERCC2, Human Excisition Repair Cross Complement 2,ERCC2 ELISA Kit 
人CC趋化因子受体6,CCR-6, Human CC Chemokine receptor 6,CCR-6 ELISA Kit 
人17-α-羟化酶,,17-α-OH, Human 17-α-hydroxylase,17-α-OH ELISA Kit 
人Ⅱ型前胶原,PCⅡ, Human Type Ⅱ Procollagen,PCⅡ ELISA Kit 
人硫酸软骨素3B3表位,CS3B3, Human Chondroitin Sulfate Epitope 3B3,CS3B3 ELISA Kt 
大鼠硫酸软骨素3B3表位,CS3B3, Rat Chondroitin Sulfate Epitope 3B3,CS3B3 ELISA Kt 
人血小板活化因子乙酰水解酶,PAFA, Human platelet-activating factor acetylhydrolase,PAFA ELISA ......

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大鼠促肾上腺皮质激素释放因子,CRF, Rat corticotropin-releasing factor,CRF ELISA Kit
小鼠胰岛素降解酶,IDE, Mouse insulin-degrading enzyme,IDE ELISA Kit 
人氧鲨烯环化酶,OSC, Human oxidosqualene cyclase,OSC ELISA Kit 
大鼠氧鲨烯环化酶,OSC, Rat oxidosqualene cyclase,OSC ELISA Kit 
人前胰淀粉样蛋白/前胰淀素,proIAPP, Human pro-islet amyloid polypeptide/Pro-amylin,proIAPP ELISA Kit 
人抗γ干扰素诱导蛋白16抗体,IFI16, Human interferon-inducible protein 16 autoantibody,IFI16 ELISA Kit 
人活化素受体样激酶1,ALK1, Human Activin receptor-like kinase 1,ALK1 ELISA Kit 
人H-钙粘着蛋白/H-钙粘连蛋白,H-Cad/CDH13, Human E-Cadherin,H-Cad/CDH13 ELISA Kit 
人纤维胶凝蛋白3,FCN3, Human ficolin 3,FCN3 ELISA Kit 
人血小板膜糖蛋白ⅠbⅨα,GP-ⅠbⅨα/CD42b+CD42a, Human platelet membrane glycoprotein ⅠbⅨα,GP-ⅠbⅨαELISA Kit 
大鼠可溶性白介素7受体,sIL-7R/CD127, Rat soluble Interleukin 7 receptor,sIL-7R/CD127 ELISA Kit 
大鼠可溶性白介素8受体,sIL-8R/CD128, Rat soluble Interleukin 8 receptor,sIL-8R/CD128 ELISA Kit 
人纤维胶凝蛋白2,FCN2, Human ficolin 2,FCN2 ELISA Kit 
人聚集蛋白聚糖,Agc, Human aggrecan,Agc ELISA Kit 
人p53上调细胞凋亡调控因子,PUMA, Human p53 up-regulated modulator of apoptosis,PUMA ELISA Kit 
人激肽释放酶7,KLK 7, Human Kallikrein 7,KLK 7 ELISA Kit 
人视网膜可溶性抗原,Sag, Human retinal soluble antigen,Sag ELISA Kit 
人α-防御素/隐窝素,α-DF/Crps, Human α-Defensins/cryptdins,α-DF/Crps ELISA Kit 
植物α-防御素,α-DF, Plant α-Defensins,α-DF ELISA Kit 
人T细胞受体γδ,TCRγδ, Human ......