ELISA检测样品处理方法
通常我们可用于ELISA检测的样本是有多种类型的，如血清、血浆、尿液、细胞培养上清或组织匀浆液等，不同类型的检测样本前期的处理方法是不一样的。正确的处理样本是保证ELISA检测的正确性和准确性的第一步，下面简单介绍一下不同类型的样本的处理方法。
1、 血清
血清是最常用于ELISA检测的一类样本，处理也比较简单。
用无热原、无内毒素的试管或离心管采集血液标本，将试管或离心管室温放置2小时或4℃过夜，使血清析出。（最好将试管或离心管倾斜放置，使液面横截面增大，能使血清更大程度的析出。）4℃ 1000×g离心20分钟，仔细收集上清。建议将血清分装多份，-20℃或-80℃保存，避免反复冻融。
采血过程中应避免溶血，因为红细胞裂解时会释放具有过氧化酶活性的物质，在以HRP为标记的ELISA检测中会有非特异性的显色，而导致检测的不准确性。同时也应避免细菌污染，因为菌体内可能含有内源性的HRP从而导致检测的假阳性。
2、 血浆
用含抗凝剂的采血管或离心管采集血液标本，标本采集后30min内4℃ 1000×g离心15min，取上清即血浆。将上清分装多份，-20℃或-80℃保存，避免反复冻融。避免使用溶血或高血脂的标本。
常用的抗凝剂为EDTA、肝素钠和枸橼酸钠等，检测时还应仔细阅读试剂盒说明书，检查试剂盒是否对抗凝剂有特殊要求。
3、 细胞培养上清
elisa检测本身是不需要无菌操作的，但是细胞培养上清样本一般是需要无菌采集的.
细胞培养上清 ： 检测分泌性的成份时,用无菌管收集 .离心 20 分钟左右 （ 2000-3000 转 / 分 ）去掉其中可能有的细胞碎片等,仔细收集上清。如果当天做可以放4度直至加样，否则在-20度冻存一个月以内使用，更长时间则冻存至-80度。收集好的样本避免反复冻融。以上是大致常见的做法，具体保存的期限还是要跟待测物本身的稳定性有关。 检测细胞内的成份时，用 PBS （ PH7.2-7.4 ）稀释细胞悬液，细胞浓度达到 100 万 /ml 左右 。 通过反复冻融 ， 以使细胞破坏并放出细胞内成份 。 样本保存同上.
4、 细胞裂解液
1） 吸去培养板内的培养基，用胰酶消化细胞，加适量培养基将细胞从培养板上吹下来。悬浮细胞可省略。
2） 收集细胞悬液，1000×g离心10min，弃去培养基，用预冷的PBS润洗3次。
3） 加入适量的预冷PBS或细胞裂解液（临用前加入蛋白酶抑制剂）重悬细胞。通常6孔板一个孔的细胞量需要150~250μL PBS重悬。
4） 将样品放入-20℃或-80℃，使样品冷冻，再放室温解冻样品，反复冻融几次，使细胞充分裂解。也可将样品进行超声破碎，以达到裂解的目的。
5） 4℃ 10000×g 离心10min，除去细胞碎片，取上清，-20℃或-80℃保存，避免反复冻融。
5、组织匀浆液
组织匀浆的制备
1、取组织块（0.1g～0.5g）最少可到5～10mg在冰冷的PBS中漂洗，滤纸拭干，准确称重，放入5ml的匀浆管中。
2、按重量(g):体积(ml)=1:9的比例加入9倍体积的匀浆介质于匀浆管中，冰水浴条件下,用眼科小剪尽快剪碎组织块。
3、匀浆的方式：手工匀浆，机器匀浆。
① 手工匀浆：左手持匀浆管将下端插入盛有冰水混合物的器皿中，右手将捣杆垂直插入套管中，上下转动研磨数十次（6～8分钟），充分研碎，制成10%的匀浆液。
② 机器匀浆：用组织捣碎机10000～15000 转/分 上下研磨制成10%组织匀浆，也可用内切式组织匀浆机制备（匀浆时间10秒/次，间隙30秒，连续3～5次，在冰水中进行,可适当延长匀浆时间），镜检观察:
4、将制备好的10%匀浆液用普通离心机或低温低速离心机3000转/分左右,离心10～15分钟，取上清液进行测定.
二、匀浆介质 一般采用0.05 mol/L Tris-HCl, pH 7.4 磷酸盐缓冲液(PBS),客户可根据样本及测定指标的情况自行设定浓度,目的是保持样本的等渗环境。
三、样本保存:
组织样本暂时不测定，可立即低温冻存，温度越低越好，中间如不反复冻融，-20℃以下可保 存三个月,-70℃以下可保存六个月
6、 尿液、唾液等其他液体生物样本
1000×g离心20min，取上清即可检测。
7、植物样本怎么匀浆处理
1，样本在保持鲜重的同时，重量不能低于50mg
2，组织匀浆比例按照10%进行，相当于1g组织加9ml的匀浆液，匀浆液要求用PBS，浓度是0.01mol/L，PH控制在7.2-7.4我们并不要求没个样本的重量必须保持1g整数，只要大于50mg即可，相对应匀浆液按照1:9的关系随之改变即可
3，剪碎叶片组织放入碾钵，然后液氮碾磨成粉末，加入换算后的匀浆液的量
4，离心取上清
备注：换算结果是乘以加入匀浆液的量，除以称取的重量，这个最终换算比例相当于乘以9g除以1ml,如果检测细胞内部，需要加一个超声破碎过程！
8、粪便样本：
尽量采集干的粪便，粪便过稀会较难处理且降低检测的准确性。采集的重量应大于 50 mg，将粪便用 PBS 洗 3 次（最终粪便质量：PBS 体积=1:9），用超声粉碎（或捣碎）后于 5000×g 离心 10 分钟，取上清检测。
总的来说，因为ELISA只能检测可溶性蛋白的含量，所以应保证所有样本均为澄清的液体，沉淀或悬浮物都应离心去除。
为了保证检测的准确性，保存于-20℃或-80℃的样本最好在1~6个月内检测；4℃保存的样本应在1周内进行检测。
另外，还应保证样本不含NaN3，因为NaN3会抑制HRP的活性，从而导致假阴性的结果。
ELISA test sample processing method
Usually there are many types of samples available for ELISA testing, such as serum, plasma, urine, cell culture upper serum or tissue homogenate, and different types of test samples are different.Correct processing of samples is the first step in ensuring the correctness and accuracy of ELISA detection, and here is a brief introduction of the processing methods of different types of samples.
1, serum
Sera is the class of samples most commonly used for ELISA detection and is relatively simple to treat.
Blood specimens were collected with a heat-free, endotoxin-free test tube or centrifugal tube and placed at room temperature for 2 hours or 4℃ overnight to allow serum dissection.(It is better to tilt the test tube or centrifugal tube, so that the liquid surface cross-section increases, and can precipitate the serum to a greater extent.) 4℃ 1000 × g was centrifuged for 20 minutes and cleared carefully.It is recommended to save the serum in multiple parts, -20℃ or-80℃, to avoid repeated freezing and thawing.
Hemolysis should be avoided during blood collection because red blood cells release substances with peroxidase activity, with non-specific color rendering in ELISA detection marked with HRP, resulting in inaccuracy of detection.Bial contamination should also be avoided because bacteria may contain endogenous HRP resulting in false positive tests.
2, plasma
Blood specimens were collected with vascular or centrifugal tubes containing an anticoagulant, with 4℃ 1000 × g centrifugal 15 min, within 30min.Save multiple copies, -20℃ or-80℃ to avoid repeated freezing and thawing.Avoid the use of hemolytic or hyperlipidemia specimens.
The commonly used anticoagulant are EDTA, heparin sodium and sodium citrate, etc. During testing, we should read the kit instructions carefully to check whether the kit has special requirements for anticoagulant.
3, cell culture is qing
elisa testing itself does not require sterile operation, but cell culture clearance samples generally require sterile collection.
Cell culture clearance: collected with sterile tubes when detecting secretory components.For about 20 minutes (2000-3000 rpm), remove the possible cell fragments and collect them carefully.If done on that day, you can put 4 degrees until the sample, otherwise use frozen within-20 degrees for a month, and frozen to-80 degrees.Collect the samples to avoid repeated freezing and thawing.The above is a general common practice, the specific period of preservation or related to the stability of the object to be measured.When detecting components in the cell, use PBS (PH7.2-7.4 ) Dilute the cell suspension with a cell concentration of around 1 million / ml.Through repeated freezing and thawing to destroy the cell and release intracellular components.Sample save ditto.
4, cell lysis solution
1) sucks the medium in the board, digested the cells with trypsin, and adds an appropriate medium to blow the cells off the plate.Suspended cells can be omitted.
2) collected cell suspension, 1000 × g centrifugation 10min, abandoned medium and washed 3 times with pre-cooled PBS.
3) adds an appropriate amount of precooled PBS or cyrolysis (protease inhibitor before use) to resuspension cells.Usually the cell volume of a 6 hole plate requires 150~250 μL PBS resuspension.
4) places the sample into-20℃ or-80℃ to freeze and thaw it at room temperature and freeze it several times to fully crack the cells.Samples can also be broken Ultrasound for the purpose of cracking.
5) 4℃ 10000 × g centrifugation 10min, removes cell fragments, removes up, -20℃ or-80℃ to avoid repeated freezing and thawing.
5, tissue slurry
Preparation of tissue slurry 1. Taking tissue block (0.1g~0.5g) can be washed by at least 5~10mg in cold PBS, filter paper dried, accurately weighed, and put into 5ml slurry pipe.2, Add 9 times the volume of the slurry medium in the slurry tube by weight (g): volume (ml) =1:9. Under ice bath, cut the tissue pieces as soon as possible with the eye shear.3, uniform way: manual uniform slurry, machine uniform slurry.① Manual homogen: the left hand inserts the lower end into the vessel with ice water mixture, the right hand inserts the tamping rod vertical into the sleeve, and grind up and down dozens of times (6~8 minutes), fully ground to make 10% homogen.② machine slurry: 10000~15000 rpm by tissue mill (10 seconds / time, clearance 30 seconds continuously, 3 ~ 5 times, in ice water), microscopic observation: 4. Prepare 10% slurry with normal centrifuge or 3000 rpm centrifuge, 10 ~ 15 minutes, take clear liquid for determination.II. The homogenmedium generally adopts 0.05 mol/L Tris-HCl, pH 7.4 Phosphate Buffer (PBS), customers can set their own concentrations according on the sample and measurement indicators to maintain the seepage environment of the sample.III. Sample preservation: If the tissue samples are not temporarily determined, they can be frozen immediately at low temperature, the lower the better. Without repeated freezing and thawing, they can be saved for three months below-20℃ and-70℃ for 60 months
6, urine and other liquid biological samples, saliva
1000 × g centrifuge 20min, for detection.
How 7, plant samples homogenize 1, the sample cannot weigh less than 50mg while maintaining fresh weight
2. The tissue slurry ratio is 10%, equivalent to 1g tissue plus 9ml homogenate, PBS, concentration is 0.01mol/L, PH controlled at 7.2-7.4 We do not require that the weight of no sample must be 1g integer, as long as it is greater than 50mg, the relationship is 1:9
3. Cut the blade tissue into the bowl, then grind the liquid nitrogen into a powder, and add the amount of the converted slurry
4. Remove the centrifugation
Note: The conversion result is multiplied by the amount of slurry added, divided by the claimed weight, and the final conversion ratio is equivalent to that multiplied by 9g divided by 1ml, If the cell is detected, an ultrasonic crushing process is required!
8, stool samples:
Try to collect dry feces, too sparse feces will be more difficult to handle and reduce the accuracy of detection.The collected weight shall be greater than 50 mg, and wash feces 3 times with PBS (final fecal mass: PBS volume =1:9), ultrasonic crushing (or pounded) for 10 minutes at 5000 × g and removed for test.
Overall, since ELISA can only detect the content of soluble proteins, it should be ensured that all samples are clarified liquids and that precipitation or suspended objects should be centrifuged removed.
To ensure the accuracy of testing, samples stored at-20℃ or-80℃ shall preferably be tested within 1~6 months; 4℃ saved samples shall be tested within 1 week.
In addition, it should be ensured that the sample does not contain NaN3 because NaN3 suppresses HRP activity leading in false negative results.
