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小木虫论坛-学术科研互动平台 » 生物医药区 » 分子生物 » 存档旧帖 » ELISA protocol
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yyc12596

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[交流] ELISA protocol 已有1人参与

Step 1: Coat Capture Antibody (CA), 4ug/ml*100ul (720ug/ml*17.8ul, diluted with 1xPBS  3182.2ul), overnight.

Step 2: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes.  

Step 3: Block Buffer (1% BSA in 1xPBS, 0.3g BSA in 30ml 1xPBS),
           300ul/well, stand for 1h.  

Step 4: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes.  

Step 5: Standards or Samples, diluted with Reagent Diluent (2:1 dilute, 7 point, 1 Blank), 100ul/well, stand for 2h.

Step 6: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes.  

Step 7: Detection Antibody (DA), 1.5ug/ml*100ul (270ug/ml*17.8ul, diluted with Reagent Diluent to 3182.2ul), 100ul/well, stand for 2h.  

Step 8: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes.  

Step 9: Sreptavidin-HRP, 1:200, diluted with Reagent Diluent (16ul in 3184ul), 100ul/well, stand for 20 min.

Step 10: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes.  

Step 11: Glo A:B, 1:2, A 1066.7ul,  B 2133.3ul), 100ul/well, stand for 5 min.

Step 12: Read.
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1楼 2013-10-09 04:17:41
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yyc12596

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REAGENT PREPARATION
Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. Working dilutions should be prepared and used immediately.
A.        Capture Antibody: Reconstitute with 1ml PBS, as recommended in the product datasheet. After reconstitution, store at 2 °C to 8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.* Dilute to the working concentration as recommended in the product datasheet.
B.        Biotinylated Detection Antibody: Reconstitute with 1ml Reagent Diluent, as recommended in the product datasheet. After reconstitution, store at 2 °C to 8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.* Dilute to the working concentration as recommended in the product datasheet.
C.        Standard: Reconstitute the standard with 0.5ml Reagent Diluent, as recommended in the product datasheet . Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Aliquot and store reconstituted standard at -70 °C. Refer to the product datasheet for the length of time the reconstituted standard may be stored. A seven point standard curve using 2-fold serial dilutions in Reagent Diluent. Refer to the product datasheet for the concentration of the high standard recommendation.
D.        Streptavidin-HRP (1 vial): 1.0 mL of streptavidin conjugated to horseradish-peroxidase. Store at 2 °C to 8 °C for up to 6 months after initial use.* DO NOT FREEZE. Dilute to the working concentration specified on the vial label using Reagent Diluent.

Plate Preparation
1.        Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
2.        Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
3.        Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.

Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
4.        Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
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2楼2013-10-09 04:19:18
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yyc12596

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Standards curve is not accurate these days (duplicated wells had quite different results).

Tests.

Set:
        1        2        3        4
A        Standards-125pg/ml        125pg/ml        125pg/ml        125pg/ml
B        125pg/ml        125pg/ml        125pg/ml        125pg/ml
C        125pg/ml        125pg/ml        125pg/ml        125pg/ml
D        125pg/ml        125pg/ml        125pg/ml        125pg/ml
E        125pg/ml        125pg/ml        125pg/ml        125pg/ml
F        125pg/ml        125pg/ml        125pg/ml        125pg/ml
G        125pg/ml        125pg/ml        125pg/ml        125pg/ml
H        125pg/ml        blank        air        air

Repeat 1
        1        2        3        4
A        75420        104800        100610        171050
B        226310        89770        102150        127120
C        91280        85810        92720        133330
D        75140        97260        188420        108070
E        68740        93630        81660        123240
F        73220        118040        116920        134710
G        69340        102640        104190        141420
H        69360        36530        30        10

Repeat 2
        1        2        3        4
A        72320        101410        97890        164680
B        227440        88230        98810        122710
C        91480        83770        90490        129730
D        74100        96000        182870        106370
E        67740        92070        79970        121070
F        70800        112810        113280        132400
G        66750        99510        101360        137430
H        68430        30920        20        20

Repeat 3
        1        2        3        4
A        73510        100150        96550        161280
B        214830        85440        94580        120140
C        87440        82070        88510        128410
D        72690        94990        179210        104840
E        65640        89340        78720        118330
F        69700        111700        112570        128420
G        66150        96670        100200        134860
H        66430        32610        0        20
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3楼2013-10-09 23:30:21
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dampit
4楼2014-08-05 08:46:36
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yyc12596

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更新一下最新信息，免得误导别人。当时测125pg/ml的孔，每孔读数差别都极大，证明不是操作误差原因。

后来发现是我的Triza base配出来的Reagent Diluent没有用12M 的浓盐酸调PH值。
拿出来用PH计测了一下，PH9.98，怪不得ELISA结果简直不能看。

后来所有做ELISA的试剂都把PH调成7.2-7.4之间，实验就没有问题了。
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5楼2014-08-05 23:03:16
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yyc12596

新虫 (著名写手)
抗体对于碱性环境反应性很差，而且不稳定。

这就是我对于PH9.98得到的结论。
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6楼2014-08-05 23:04:25
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