免疫类

氨苄青霉素（AMP）酶联免疫分析（ELISA）

试剂盒使用说明书

本试剂盒仅供研究使用。

1 使用目的：

本试剂盒用于微生物，植物，饲料、鱼、虾和肉类组织（如鸡、牛肉和猪肉），鸡蛋、蜂蜜、牛奶、羊奶，血清和尿样中氨苄青霉素（AMP）残留的定量检测。

2 实验原理

本试剂盒采用竞争ELISA方法，在微孔板包被有氨苄青霉素（AMP）偶联抗原，加入氨苄青霉素（AMP）标准品或样品，游离氨苄青霉素（AMP）与微孔条上预包被的氨苄青霉素（AMP）偶联抗原互相竞争抗氨苄青霉素（AMP）抗体酶标记物，用TMB底物显色，加入终止液后颜色由蓝色变为黄色，用酶标仪在450nm波长下进行检测，吸光值与样品中氨苄青霉素（AMP）含量成反比，通过标准曲线计算样品中氨苄青霉素（AMP）的含量。  

3 试剂盒组成

3.1 预包被的氨苄青霉素（AMP）偶联抗原的可拆酶标板：1块（12孔×8条）。
3.2 氨苄青霉素（AMP）标准品：6瓶（1ml/瓶），含量分别是： 0 ppb，0.1 ppb,0.3 ppb,0.9 ppb,2.7 ppb,8.1 ppb。
3.3抗氨苄青霉素（AMP）抗体酶结合物：1瓶（6ml）。
3.4显色液A：1瓶（6ml）。
3.5显色液B：1瓶（6ml）。
3.6终止液：1瓶（6ml），2M硫酸。
3.7样本稀释液：1瓶（10×,  6ml），用于样品稀释用。
3.8浓缩洗涤液：1瓶（20×，20ml），用于洗板。
3.9说明书一份。

4 需要而未提供的材料
4.1 设备
4.1.1波长450nm酶标仪。
4.1.2粉碎机。
4.1.3量筒。
4.1.4振荡器。
4.1.5漏斗。
4.1.6Whatman No 1或相当的滤纸。
4.1.7微量移液器。
4.2 试剂
4.2.1去离子水或蒸馏水。
4.2.2 甲醇。
5 贮存
5.1 试剂盒贮存于2~8℃，切勿冷冻
5.2 未用完的微孔板应该密封干燥保存
6 注意事项
6.1 使用试剂盒前请仔细阅读说明书。
6.2 不要使用过期试剂盒。
6.3 试剂盒使用前，将试剂恢复至室温（25±2℃），建议至少回温2小时。
6.4 标准品中含有氨苄青霉素（AMP），使用时应特别注意，操作时应带手套。
6.5 终止液中含有硫酸，使用时防止灼伤皮肤及腐蚀衣物。
6.6 不同标准品、样品所用吸头不能混用，否则会影响试验结果。
6.7 不同批号试剂盒中的试剂不得混用；不同标准品、样品所用吸头不得混用，否则会影响实验结果。
6.8 稀释样本时必须用本试剂盒中的样本稀释液，否则会影响实验结果
6.9 混合试剂时应避免起泡。
7 工作液准备
7.1 氨苄青霉素（AMP）标准品溶液：0ppb,0.1ppb,0.3 ppb,0.9ppb,2.7 ppb,8.1ppb
7.2 浓缩洗涤液：用蒸馏水按1:20(1+19)稀释备用

7.3 样本稀释液：用蒸馏水按1:10(1+9)稀释备用
7.3 显色剂：已备用，避免光线直照
7.4 反应终止液：已备用
8 样品处理程序（样品在提取过程中，要严格按说明书操作，提取过程中应准确稀释，否则会出现结果不准确，样品应当保存在阴凉避光之处及冷藏保存）
8.1取10g粉碎的样品，加 20ml 70%甲醇溶液
8.2强力振荡3分钟
8.3用Whatman No 1滤纸过滤
8.4取25µl处理后的样品，加入25µl样本稀释液于反应孔中（样本稀释倍数为2）
9 分析步骤
9.1 实验须知
9.1.1 实验开始前请将所有试剂于盒外充分恢复至室温（25±2℃），时间约2小时。回温至室温（25±2℃）后再取出微孔条，多余的微孔条重新密封立即于2~8℃干燥保存
注：一定保证回温充分，否则影响检测的精确度和准确度。
9.1.2 使用后请立即将试剂放回2~8℃保存
9.1.3 请不要改变分析程序
9.1.4 请使用精确的微量移液器
9.1.5 操作一旦开始，请不要中断任何程序
9.1.6 ELISA结果的可重复性极大程度的取决于操作程序，请严格按照要求操作
9.1.7 为避免交叉污染，每个标准品和样品均应使用不同的吸头加样
9.1.8 加样时请勿让吸头接触微孔中的溶液或内表面
9.2 分析步骤
9.2.1 预先进行编号，标记B0、标准品和样品的位置，推荐进行双孔检测
9.2.2 取所需数量的微孔（微孔条可拆），将多余板条重新密封并立即放回2~8℃保存
9.2.3 样品稀释液（10×）、浓缩洗涤液（20×）稀释成工作液(蒸馏水或去离子水稀释)
9.2.4 在B0孔中加入50µl  0.0ppb标准品溶液
9.2.5 在各标准孔中加入50μl的标准品溶液
9.2.6 在各样品孔中加入50µl样品溶液
9.2.7 在所有孔中加入50µl的抗氨苄青霉素（AMP）抗体酶结合物
9.2.8 轻轻晃动反应板几秒钟。
9.3 37℃温浴30min （温浴过程中不时轻拍反应板，可以减少双孔误差）
9.3.1 甩掉孔中液体，用洗液洗涤微孔板5次，最后一次应在吸水纸上拍打以完全除去孔中液体。
9.4 反应
9.4.1 洗涤程序完成后，立即用微量移液器在每个微孔中先加入50µl显色液A，再加 50µl显色液B；轻微晃动反应板使之彻底混匀
9.4.2 37℃温浴10min
9.4.3 每孔中加入50µl终止液，混匀
9.4.4 在450nm下检测吸光度，结果在5min内读取。
10 结果计算
10.1定量分析
10.1.1所获得的每个浓度标准溶液和样本吸光度值的平均值（B）除以第一个标准（0标准）的吸光度值（B0）再乘以100%，即百分吸光度值。
B—标准溶液或样本溶液的平均吸光度值
B0—0ppb标准溶液的平均吸光度值
10.1.2以氨苄青霉素（AMP）浓度的对数值为X轴，百分吸光度值为Y轴，绘制标准曲线图。根据样品百分吸光度值，可从曲线上得到对应点的横坐标，即为氨苄青霉素（AMP）浓度的对数值，求得反对数即为测定液中氨苄青霉素（AMP）浓度C（ppb）
10.1.3由于样品经过了预先稀释，因此根据标准曲线所得出的样品浓度一定要再乘以其稀释倍数。
10.2 半定量测定
10.1.1目测半定量测定：首先选择一个适当的标准液与样品同运行，根据样品与标准品吸光度值的高低比较，判断样品浓度值是小于还是大于标准值。
10.1.2仪器半定量测定：首先选择一个适当的标准液与样品同运行，根据样品与标准品颜色深浅比较，判断样品浓度值是小于还是大于标准值。

11 特异性
物质 交叉反应
氨苄青霉素（AMP） 100%

 

 

12 试剂盒参数
本试剂盒检测下限为0.1ppb
B0吸光度最佳值应大于1.0
试剂盒吸光度板内误差小于8％，板间误差小于15％。
用本说明书提供的组织样本提取方法回收率大于80％。
13
试剂盒提供的标准曲线范围为0.1ppb~8.1ppb。

14 分析限制
本试剂盒检测为阳性的样品应该用另一种方法如HPLC或GC/MS加以确证。

 

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用户评论(共6条评论)

• 办事效率蛮高，几天，就帮我搞定了这个试剂盒，谢谢！
• 盒子蛮不错，包含所需试剂，并提供了中英文双版说明书及增值税发票，很正规，销售人员的态度也谦和。
• 灵敏度不错，重复性也行，试剂盒在我做的这些实验中，与国内同行的盒子相比来说，性价比挺高的，尤其是价格占有不错的优势，赞一个！
• 操作挺方便的，也简单，当然，这也多亏了公司的技术人员详细的实验指导，很好，谢谢，实验也相当成功。
• 实验出来的标准曲线很不错（很优美），而且价格相对合理，技术指导很到位......
• 我购买了两盒，批次最新，保质期内，实验结果做出来了，货到的挺快，那么远，几天就到了，加上我做实验，一共五天就搞定了。
• 经朋友介绍，购买了此公司的ELISA试剂盒，用过，还不错，实验效果很好，基本达到实验参数要求，如果，下次还做实验的话，我还买这家的，朋友这几年都在用这家的（长期订货）。

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酶联生物经过不断的实验优化和改进，积累了大量的经验，拥有专业的酶联研发团队。利用专业的酶联免疫技术自主研发的elisa试剂盒，能对血清及其它样本定量检测抗原，定性检测特异性抗体。优质的试剂，先进的仪器和正确的操作是保证ELISA检测结果准确可靠的必要条件。ELISA检测的方便性、稳定性、重复性和可靠性方面都具有很大的优势。

ELISA检测技术服务内容：
1、双抗体夹心法检测抗原 2、间接法检测抗体 3、为客户提供各种ELISA技术进行样本检测。

以上代测费，凡购买本公司试剂盒，我们免费代测！
凡购买本公司目录任何一种酶联免疫检测试剂盒，您只需将需要检测的动物（Human, Rat, Mouse, Rabbit, Monkey, Pig……）种类和检测指标（白介素类、激素类）及标本数量（48T/96T）通知公司业务员即可。在接到客户标本当日起，现货产品一周内将检测报告交到客户手中！
欢迎各科研单位在各种项目上与我们公司开展不同层次的密切合作，以双赢求发展，共同进步，为中国检测事业的发展积累经验。

二、样本要求
在收集标本前都必须有一个完整的计划，必须清楚要检测的成份是否足够稳定。我们提倡新鲜标本尽早检测，对收集后当天就进行检测的标本，及时储存在4℃备用，如有特殊原因需要周期收集标本，请造模取材后，将标本及时分装后放在-20℃或-70℃条件下保存。因冰室与室温存在一定温差，蛋白极易降解，直接影响实验质量，所以避免反复冻融。代测放免标本的客户取材前须向我司销售人员索要说明书，具体操作注意事项请与我司技术人员沟通。

液体类标本：标本必须为液体，不含沉淀。包括血清、血浆、尿液、胸腹水、脑脊液、细胞培养上清、组织匀浆等。

血清：室温血液自然凝固10-20分钟后，离心20分钟左右（2000-3000转/分）。收集上清。如有沉淀形成，应再次离心。

血浆：应根据试剂盒的要求选择EDTA、柠檬酸钠或肝素作为抗凝剂，加入10%（v/v）抗凝剂(0.1M柠檬酸钠或1%heparin 或2.0%EDTA.Na2)混合10-20分钟后，离心20分钟左右（2000-3000转/分）。仔细收集上清。如有沉淀形成，应再次离心。

尿液、胸腹水、脑脊液：用无菌管收集。离心20分钟左右（2000-3000转/分）。仔细收集上清。如有沉淀形成，应再次离心。

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）。仔细收集上清。检测细胞内的成份时，用PBS（PH7.0-7.4）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。离心20分钟左右（2000-3000转/分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。

组织标本：切割标本后，称取重量。加入一定量的PBS，缓冲液中可加入1μg/L蛋白酶抑制剂或50U/ml的Aprotinin（抑肽酶）。用手工或匀浆器将标本匀浆充分。离心20分钟左右（2000-3000转/分）。仔细收集上清置于-20度或-70度保存，如有必要，可以将样品浓缩干燥。分装后一份待检测，其余冷冻备用。

三、寄标本时需注明以下情况：
1、标本编号；2、所测项目；3、是否做复孔；3、联系方式；4、实验后标本是否寄回。

客户须知：
客户应对所提供的材料及信息负责，如因客户提供的材料及信息不准确而引起的实验延误或经济损失由客户承担。

Q:1. how to collect samples and preparation of ELISA?
Performed by ELISA test is generally common clinical samples including blood (finger blood, blood), urine, feces, cerebrospinal fluid, pleural effusion, prostatic fluid, semen, vaginal secretions, which
Some time of sample collection, preservation methods and has certain requirements.
Collection (a) clinical specimens
A, blood samples:Some physiological factors, such as smoking, eating, exercise, mood swings, pregnancy, postural changes in blood can affect certain ingredients, even some of diurnal variation. Therefore, blood samples
Acquisition should avoid interference physiological factors, consistent with appropriate conditions, such as can not be avoided, should indicate the factors on the specimen.
1. Peripheral:Usually select the inside of blood left ring finger, the portion should be no frostbite, inflammation, edema, damage. If the site does not meet the requirements to other parts of the fingers instead. For burn patients, optional leather
Intact skin at the blood. As part of routine blood tests (eg, white blood cell count, sort, etc.) affected by physiological factors fluctuation is too large, when compared to the conditional should be consistent. It relates to the body, blood clotting function
Can test items (such as platelet count, bleeding time or clotting time) testing, we must pay attention to understand whether the patient used anticoagulant, procoagulant drugs in order to reduce or avoid interfering factors
influences.
2. Blood:In addition to involving a variety of projects such as hemostasis and thrombosis detector requires the use of anticoagulated blood plasma, the current analysis to detect the vast majority of projects can be directly detected using blood serum. In the serum test items
, Some (such as blood sugar, blood fat) diet and circadian factors influenced, fasting blood samples were generally appropriate; some decay rapidly in the blood (serum enzyme activity assay such as ACP activity, etc.),
0 ~ 4 ℃ storage is not an activity decreased, the detection of these projects must be timely and fast; some (such as creatine kinase) influenced by exercise and other factors. Avoid hemolysis occurs when blood is also important
And, more particularly potassium, LDH and other measurement.
B, urine samples:With the same blood samples, urine samples affect diet, exercise, medication and other factors that are also large, especially on the diet, so the morning urine generally superior to random urine. Means getting up early morning urine
After the first urine specimens, representing concentrated and acidified visible components (such as blood cells, epithelial cells, tubular) easy to observe the relative concentration. Random urine that is a random urine specimens convenient, but by diet,
Sports, and even more the influence of drugs, prone to false positive and false negative results, such as diet proteinuria, glucosuria diet, vitamin C interference occult blood results and the like. Postprandial urine (patient 2 hours after lunch, collected
Human Urine) suitable for urine, urine protein and urobilinogen check urine samples at this time to increase the sensitivity of the test, the detection of minor lesions. 12 hours in urine cell count is Addis count (last night 8:00
After emptying the bladder to all specimens of urine 8 o'clock the next morning), because a long time, easy to breed bacteria shall be added preservative formaldehyde. 24-hour urine (the first day of the morning after emptying the bladder specimens from 8:00 to 8:00 the next morning
All urine) quantification of chemical substances, including proteins, sugars, urinary 17-one, 17-hydroxy steroids, catecholamines, Ca2 +, etc., to detect different substances, choose a different preservative preservative. clean
Urine used for urine bacterial culture requires sterile specimens were taken after washing the vulva. Urine specimens should be enough to collect all, at least 12 ml, preferably 50 ml, the timing must collect all the urine of women
Patients should avoid vaginal secretions, blood contamination of urine specimens.
C, stool samples:Stool samples for the detection judgment digestive diseases has important reference value. Collection requirements with a clean bamboo select faecal mucus, pus and blood components and other abnormality, no abnormal appearance
Droppings shall be drawn from multiple surface and deep manure end. Get parasitemia and for egg counts should be collected 24 hours feces. Dysentery amoeba trophozoites check should immediately check in after a bowel movement, and from there sepsis
Softer at the drawn, insulation inspection. Charles S. japonicum eggs should take mucus, pus and blood portion 30g stool specimens from at least miracidia hatching, and to be treated as soon as possible. Check pinworm eggs must use transparent film swab
Night before 12:00 or early in the morning from defecation wrinkled folds around the anus and immediately swabbing at microscopic examination. Occult blood test (chemistry), fasting before the test on the 3rd of meat and foods containing animal blood and ban clothing iron, vitamin C and so on.
Should be checked in all 1 hour stool specimen collection is completed, in order to prevent damage to physical components of digestive enzymes and pH by. For clinical samples above the detection indicators.
D, CSF samples:CSF samples collected immediately after submission, place too long will affect the test results: such as cell degeneration, destruction, leading to counting and classification are not allowed; some chemicals such as glucose content will decompose Save
Less; bacteria occur autolysis affect bacteria detection rate. Cerebrospinal fluid extracted three general dispensing a sterile tube, the first tube for bacterial culture, a second tube for chemical analysis and immunological tests, the third tube for general
Characters and microscopic examination, three of the order should be reversed. Specimen collection is difficult because all inspection and testing process should pay attention to safety.
E, ascites and pleural effusion samples:CSF samples with the same attention to safety after the specimen collection, and timely submission. Generally separated into three tubes, one for routine cytology, a biochemical examination, a bacterial culture, in order
CSF same is appropriate.
F, prostatic fluid sample:Prostatic fluid specimen after prostate massage by the acquisition, directly drop when less liquid on a glass slide and timely submission shall be taken to prevent sample evaporation to dryness, the amount collected for a long time in a clean, dry test tube. If massage
No prostatic fluid, urine sediment can be checked after the massage.
G, semen samples:Abstinence before semen collection should be 3 to 7 days, drain the urine after masturbation or other available methods of semen directly into clean containers, insulation and timely submission. Due to changes in sperm production during the day and
Large, generally should be checked 2 to 3 times (each time interval of 1 to 2 weeks) in order to make a diagnosis.
H, samples of vaginal secretions:Vaginal samples were collected 24 hours before intercourse should be prohibited, bath, vaginal examination, vaginal lavage and local on the drug, etc., drawing instruments used need to be cleaned. Usually with brine-soaked cotton swab from the vagina deep
Or rear vaginal fornix, cervical canal mouth drawn, etc., made after saline smear vaginal secretion samples observation, women with menstrual vaginal secretions were not checking.
2, do before each sample by ELISA experiment how to prepare?
Before collecting the sample must have a comprehensive plan must clearly be detected component is stable enough. To be collected on the same day Sample testing, and timely backup stored at 4 ℃. For the next day re-testing samples frozen in a timely manner after dispensing -20 ℃ spare, conditional, preferably -70 ℃ cryopreservation standby. Avoid repeated freezing and thawing specimens
. Liquid samples: including serum, plasma, urine, pleural effusion, cerebrospinal fluid, cell culture supernatant and the like.
1. serum:
Coagulation at room temperature 10-20 mins, centrifugation 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again.
2. Plasma:
EDTA should be selected according to the requirements of the specimen, sodium citrate or heparin as an anticoagulant, mix 10-20 mins, centrifugation 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. Save process
If precipitation appeared, Centrifugal again.
3. Urine:
Sterile collection tube. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again. Pleural and peritoneal effusions, and cerebrospinal fluid Reference to this practice. 4. The cell culture supernatant:
The detection of secretory component with a sterile collection tube. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant.
5. cultured cells
????When the detection of intracellular components, diluted with PBS (PH7.2-7.4) cell suspension, the cell concentration reached 1 million / ml or so. By repeated freezing and thawing or tissue protein extraction reagent was added to the cells
Damage and release of intracellular components. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again.
6. tissues
????After cutting samples, check the weight. Adding a certain amount of PBS, PH7.4. Rapidly frozen with liquid nitrogen. After thawing samples remained at 2-8 ℃. Adding a certain amount of PBS
(PH7.4), or tissue protein extraction reagent, or by hand homogenizer homogenized sample. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. A new package to be detected, which
I alternate freezing.
Q:Do I have to run all of my standards and samples in duplicate?
A:Yes, the duplicates are run in order to monitor assay precision and increase confidence in the assay results obtained.
Q:Do I have to run all of my samples at one time?
A:No, each kit uses stripwell microplate. This allows the user to analyse different numbers of samples at different times.
Q:What types of reproducible results are obtained with the assays?
A:Each kit comes with a manual containing a graph of typical data obtained. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
Q:Is it possible to store the reagents other than indicated?
A:Storage of the kit components under conditions other than indicated is not recommended in order to assure proper performance of the test.
Q:How should I store my samples?
A:Samples should be stored at -20oC or lower temperature. For long-term storage, it is recommended to freeze them at -70oC -80oC.
Q:Can I modify the protocol?
A:BG ELISA kits have been optimized to provide the best possible results. Modifying the format or protocol may give inaccurate and wrong results.
Q:Can I use a sample type that is not recommended in the kit insert?
A:The kit has been validated for the sample types listed in the kit insert. Sample types other than those validated have not been tested. Contact Technical Service for further information.
Q:My samples generated values that were outside the dynamic range of the assay. Can I use these values?
A:It is recommended that only sample values that fall within the range of the standard curve be used. Values outside the range of the standard curve are generally non-linear, which can lead to incorrectly extrapolated values. Samples that generate values higher than the highest standard should be (further) diluted and the assay repeated. If samples fall below the range of the assay, the sample is considered to be non-detectable.
Q:Do I have to run a Blank or Zero Standards every time?
A:Yes, these are required for the calculations, and reflect any subtle but significant performance changes from day to day and assay to assay. They are also extremely helpful when troubleshooting the source of a particular assay problem.
Q:Can I alter the volume of sample I use in the assay?
A:It is not recommended that you alter the volumes since all BG kits are designed for optimal performance at the given volumes
Q:Can components from different kits be used?
A:Each kit contains components which have specific lot numbers to ensure that all of the components are performing optimally alone, as well as with all of the other components in the kit. QC testing is performed on these specific lots. It is never recommended to use your own components or components from other kits or vendors.
Q:My standard curve looked fine, but I didn’t get a signal in my sample when I expected to, why?
A:The sample may not contain the analyte. A matrix effect may be masking the detection. Ensure that the recommended dilution was followed as stated in the kit insert. If dilution was recommended, check to be sure that the dilution was performed properly. Over-dilution may cause the sample to fall below the range of the standard curve.
Q:How do you recommend I wash my plate?
A:If you are using an automated plate washer we recommend that the calibration be checked on a regular basis, and that the system is flushed with the Plate Washing Buffer prior to washing. The same is true for a manual washer. A repeater or a wash bottle can also be used. The user should be careful to ensure that all of the contents are aspirated and the plate tapped dry on lint-free paper.
Q:Do I need to use a plate shaker?
A:Reliable results can be obtained without a plate shaker, but the O.D.'s will generally be lower than those obtained using a plate shaker.
Q:Why do I have to use wavelength correction between 450-570nm?
A:For the ELISA assay, reading at dual wavelengths is done to correct for the optical density contributed by the plastic well, the lamp and optical fluctuations.
Q:If I extract my sample, do I still need to follow the recommended dilutions given in the kit insert?
A:The amount of sample dilution needed after an extraction procedure will be affected by the effects of purification and concentration in the protocol used. The amount of dilution or concentration will have to be determined by the end-user.
Q:What is the expected concentration of analyte that I should expect to find?
A:The amount of a given analyte may vary not only from species-to-species, but also between tissue and cellular sources. The best source of this information is the current literature that is easily accessed through the Internet at multiple scientific databases.
Q:My optical densities were a little higher (or lower) than those in the manual that came with my kit. Why?
A:The optical density is affected by a number of physical conditions such as time and temperature. We suggest that you shorten or lengthen the final incubation with substrate solution to compensate.
Q:What are the reasons for High Background?
A:1) Improper Washing: Check volume of washing buffer reservoir and make sure all recommended washing steps are performed. 2) Contaminated Substrate: Make sure there is no contamination of the substrate with metal ions or oxidizing reagents, before use. Keep the extra substrate solution separately during the ELISA substrate development time. 3) Substrate exposed to light: Exposure to light may result in a blue color of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate. 4) Wrong Incubation Times/Temperatures: Generally follow the test protocol regarding incubation times and temperatures. However, if all wells are intensely and equally colored with no intensity gradient observed in the standard dilution series, then it may be necessary to observe the substrate reaction as the color is developing, in order to stop the reaction sooner.