ELISA 2.0 kit for measuring Arachis hypogaea allergen, Ara h 2. | |||||||
| Antibodies: | 1C4/pAb | ||||||
| Standard Range: | 250-0.5 ng/mL | ||||||
| Limit of Detection: | 2.0ng/mL | ||||||
| Background: | OD<0.1 at 450nm | ||||||
| Coefficient of Determination: | R-squared>0.98 | ||||||
| Contents: | Plate: Pre-coated with anti-Ara h 2 monoclonal antibody 1C4Vial 1: (white top) Ara h 2 allergen standardConcentration: 2,500 ng/mlVial 2: (brown) Rabbit anti Ara h 2 antibodyVial 3: (blue top) Peroxidase-conjugated Goat Anti-RabbitBottle 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-AH2-1 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 1C4 and treated with stabilizingagent. Sealed in foil pouch with desiccant. | ||||||
| Monoclonal Antibody: | 1C4 | ||||||
| Immunogen: | Ara h 2 | ||||||
| Isotype: | Mouse IgG1 | ||||||
| Specificity: | Binds to a species specific epitope present on Arachis hypogaea allergen, Ara h 2. | ||||||
| Purification: | Produced in ascites and purified by affinity chromatography using Protein G. Single heavy and light chain bands on SDS-PAGE. | ||||||
| Certificate of Analysis | |||||||
| Detection Antibody: | Rabbit polyclonal antiserum | ||||||
| Immunogen: | Polyclonal rabbit antiserum raised against natural purified Ara h 2 | ||||||
| Isotype | Multiple | ||||||
| Specificity: | The antiserum contains IgG antibodies to peanut Arachis hypogaea allergen, Ara h 2. | ||||||
| Activity: | 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 2 prepared in 1% BSA/50%glycerol/PBS, pH 7.4. | ||||||
| Concentration/Calibration: | 2,500 ng/mL (based on amino acid analysis) | ||||||
| References: | |||||||
| 1. 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. | |||||||
| 2. 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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需要的仪器有:酶标仪、恒温箱、枪、枪头、烧杯、滤纸.当然有自动洗板机最好.
复习了很多文献在测定方法里面均提到了CV
例如:
Theintra-assaycoefficientofvariationfortheassaywas4-6%.
Theanalyticcoefficientofvariationwas10.2%
问题是:
我是否可以直接使用说明书里的CV值?
如果不可以,我该如何操作进行计算
DAS-ELISA试剂盒中抗体有包被抗体和酶标抗体,这两种抗体是一种抗体,前者没有酶标,后者用酶进行了标记。
NCM-ELISA一般有一抗和二抗,这两种抗体不一样,前者(一抗)一般是鼠抗体或兔抗体,没有酶标,相对应的二抗一般是羊抗鼠或羊抗兔抗体,用酶进行标记。也有用马大规模备抗体的。
TDS-ELISA是三抗体ELISA,第三级的抗体是将信号级联放大。
以上三种是比较常见的,还有其他一些试剂盒,可参考免疫学的内容。
现在国内最差也是用3代试剂,有些地方会用4代试剂。
4代试剂(检查抗原+抗体)——窗口期为4周。因为抗原于3-4周达到复制的峰值,此时通过4代试剂检查,如果感染了HIV,抗原/抗体至少有一个为阳性,如果都是阴就排除了。
3代试剂(只查抗体)——窗口期为6周。
以上为理论分析+临床经验的结果,可以说是99.9%的准确度。
但是目前FDA、CDC和试剂生产商统一达成的共识,也就是针对普通人,最保守的窗口期是3个月。无论什么试剂,3个月都100%排除。
1.取出试剂盒室温平衡30min,取出血样放至室温。
2.配标准品:取150uL标准品加入150uL标准品稀释液稀释,依次稀释5次。
3.加样:分别于各反应孔中加入标准品50uL,样品40UL,标准品做复孔,样品做3孔。
4.分别于样品孔中加入10UL抗体。
5.标准品和样品孔中分别加入50uL链酶亲和素-HRP,盖上封板膜,轻轻震荡混匀,37℃温育60min。
6.配洗涤液:将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
7.洗涤:小心揭开封板膜,弃去液体,甩干,每孔加200uL洗涤液,静置30s后弃去,如此重复5次,拍干。
8.显色:每孔先加入显色剂A 50uL,再加显色剂B 50uL,轻轻震荡混匀,37℃避光显色6min。
9. 终止:每孔加入终止液50uL,终止反应(此时蓝色立即转为黄色)。
10.测定:以空白孔调零,450nm波长依序测量各孔的吸光度。测定应在加终止液10min之内进行。
11.保存结果,收拾桌面。
12.分析处理数据
注意事项
1. 取出板条前恢复到室温后再打开外包装袋,实验中不用的板条立即放回包装中,密闭封口,其余不用试剂应盖好。
2. 实验操作中请使用一次性的吸头,避免交叉污染。
3.实验板孔加入试剂的顺序应一致,以保证所有反应孔的孵育时间一致。
4.洗涤过程中反应孔中残留的洗涤液应在滤纸上充分拍干,勿将滤纸直接放入反应孔中吸水。
5.试剂盒内试剂请在保质期内使用,不同批号试剂不要混用。
6.1000pg/ml以上的结果为非线性的,根据此标准曲线无法得到精确的结果。大于1000pg/ml 的样品应以标准稀释缓冲液稀释后重做。在结果分析时,结合考虑相应的稀释度。

