
ELISA 2.0 kit for measuring Arachis hypogaea allergen, Ara h 1. | |||||||
Antibodies: | 2C12/2F7 | ||||||
Standard Range: | 2,000-4.0 ng/mL | ||||||
Limit of Detection: | 31.5 ng/mL | ||||||
Background: | OD<0.08 at 450nm | ||||||
Coefficient of Determination: | R-squared >0.98 | ||||||
Contents: | Plate: Pre-coated with anti-Ara h 1 monoclonal antibody 2C12Vial 1: (white top) Ara h 1 allergen standardConcentration: 20,000 ng/mlVial 2: (brown) anti-Ara h 1 antibody 2F7Vial 3: (blue top) Streptavidin-PeroxidaseBottle 1: Wash buffer, (10x concentrate)Bottle 2: Assay buffer, (10x concentrate)Bottle 3: TMB developing substrateBottle 4: Stop solution (0.5N sulfuric acid) | ||||||
Storage: | The ELISA 2.0 kit should be stored at 2-8°C. | ||||||
Expiry: | 6 months from date of receipt | ||||||
Not Provided: | Type I ultrapure water or 18.2MΩ de-ionized waterVolumetric measuring equipment (e.g. serological pipette, graduated cylinder)Clean containers for buffer and reagent preparationCalibrated single and multi-channel micropipettes and tipsVortex mixerPlate reader capable of reading absorbance at 450nmAnalysis software (recommended, but not required) | ||||||
Notes: | The allergen standard is recommended for immunoassay calibrationpurposes only.A list of frequently asked questions and troubleshooting guide can be found under the ‘Support’ tab on our website. | ||||||
Product Resources: | EPC-AH1-1 Certificate of Analysis Validation Performance Data | ||||||
For research and commercial use in vitro:not for human in vivo or therapeutic use. | |||||||
Certificate of Analysis | |||||||
Pre-coated Plate: | 96-well polystyrene microtiter plate coated withmonoclonal antibody 2C12 and treated with stabilizingagent. Sealed in foil pouch with desiccant. | ||||||
Monoclonal Antibody: | 2C12 | ||||||
Immunogen: | Ara h 1 | ||||||
Isotype: | Mouse IgG1 | ||||||
Specificity: | Binds to species specific epitope present on Arachis hypogaea allergen, Ara h 1. | ||||||
Purification: | Produced in vitro by BioVectra dcl bioreactors and purified by chromatography using Protein G. Single heavy and light chain bands on SDS-PAGE. | ||||||
Certificate of Analysis | |||||||
Detection Antibody: | 2F7 | ||||||
Immunogen: | Ara h 1 | ||||||
Isotype | Mouse IgG1 | ||||||
Specificity: | Binds to species specific epitope present onArachis hypogaeaallergen, Ara h 1. | ||||||
Activity: | Biotinylated and titrated for use in ELISA at 1/1000 dilution. Prepared in 1% BSA/50% glycerol/PBS, pH 7.4, 0.22 μm filtered, preservative free. | ||||||
Certificate of Analysis | |||||||
Allergen Standard: | Purified natural Ara h 1 prepared in 1% BSA/50%glycerol/PBS, pH 7.4. | ||||||
Concentration/Calibration: | 20,000 ng/mL (based on amino acid analysis) | ||||||
References: | |||||||
1. Pomés A, Helm RM, Bannon GA, Burks AW, Tsay A, Chapman MD. Monitoring peanut allergen in food products by measuring Ara h 1. J Allergy Clin Immunol 2003;111:640-5. | |||||||
2. Pomés A, Vinton R, Chapman MD. Peanut allergen (Ara h 1) detection in foods containing chocolate. J Food Prot. 2004 Apr:67 (4):793-8. 3. Perry TT, Conover-Walker MK, Pomés A, Chapman MD, Wood RA. Distribution of peanut allergen in the environment. J Allergy Clin Immunol. 2004 May;113(5):973-6. 4. Maloney JM, Chapman MD, Sicherer SH. Peanut allergen exposure through saliva: assessment and interventions to reduce exposure. J Allergy Clin Immunol. 2006 Sep;118(3):719-24. | |||||||
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GM实验:试剂说明书上要求20分钟显色,10分钟的时候看一下,差不多就可以加终止液,但是我的标曲显色特别快,2分钟最低浓度蓝色就就深了,此时样本颜色还很浅很浅,我只能一块儿加终止液。所以,标曲显色过快,可能是什么原因?
(ps:第一列是标曲,后面是样本)
1、直接竞争,标记抗原,与检测样品中的抗原竞争抗体。
2、间接竞争,标记抗体,固相抗原与液相抗原(样品)竞争标记抗体。
3、定义
间接竞争法的模型:包被抗原,用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab,固相吸附的HRP-Ab与样本中的Ag浓度成反比。
直接竞争法的模型:包被抗体,用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab,固相吸附的HRP-Ag与样本中的Ag浓度成反比。
4、竞争法的理论基础:是限量抗体。只有在限量抗体基础上,两种抗原才会形成竞争关系。
5、、间接竞争法具备较高灵敏度原因。
直接竞争法里,标记抗原与待测抗原均是液相,与抗体的结合机会是一样的,例如有1份标记抗原与1份待测抗原竞争1份抗体,那么有50%的标记抗原能与抗体结合,所以标记抗原的相对结合率为50%。间接法里,固相抗原与抗体的接触面积较小,固相抗原与待测抗原的结合抗体机会是不平等的,接近顺序饱和法,即只有与待测抗原结合剩余的抗体才会与固相抗原结合,同样有1份固相抗原与1份待测抗原竞争1份抗体时,基本上抗体会被待测抗原中和掉,与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此,间接法的抑制曲线斜率会大于竞争法。
因为抑制率越大则斜率越大,从而灵敏度越大。(假设零管变异5%,以两倍SD为灵敏度限,则为90%相对结合率,则间接法可以在较低的待测抗原浓度达到这一相对结合率,因此灵敏度要高。)
在进行系统放大时,间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位,所以存在放大效应,从而能提高间接法的灵敏度。直接法一般难以进行放大,常用的有生物 素化抗原与酶标亲和素,但模式上似乎不存在放大效应。
6、间接法的高灵敏度难以实现的原因:
双抗体 夹心的免放(IRMA)模式刚出现时,也被模型证明灵敏度优于竞争法的放免(RIA),原因也是较大的斜率,但是IRMA的高灵敏度一直到单抗发展后才得以实现。
采用抗-HBe抗体包被反应板,加入校准品及被测样本,同时加入定量HBeAg中和抗原,经过振荡孵育,洗板后再加入铕标记的抗-HBe,若标本中抗-HBe浓度高,HBeAg将被大量中和,使最后形成的抗-HBe-HBeAg-铕标记抗-HBe复合物减少。增强液(β-NTA)将标记在抗体上的Eu3+解离到溶液中,Eu3+和增强液中的有效成分形成高荧光强度的螯合物,荧光强度和样本中的抗-HBe浓度成反比。

