斑馬魚(yú)5羥色胺(5-HT)ELISA試劑盒試驗(yàn)原理：
斑馬魚(yú)5羥色胺(5-HT)ELISA試劑盒是固相夾心法酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）.已知待測(cè)物質(zhì)濃度的標(biāo)準(zhǔn)品、未知濃度的樣品加入微孔酶標(biāo)板內(nèi)進(jìn)行檢測(cè)。先將待測(cè)物質(zhì)和生物素標(biāo)記的抗體同時(shí)溫育。洗滌后，加入親和素標(biāo)記過(guò)的HRP。再經(jīng)過(guò)溫育和洗滌，去除未結(jié)合的酶結(jié)合物，然后加入底物A、B，和酶結(jié)合物同時(shí)作用。產(chǎn)生顏色。顏色的深淺和樣品中待測(cè)物質(zhì)的濃度呈比例關(guān)系。
試劑盒內(nèi)容及其配制
自備材料
1）蒸餾水。
2）加樣器：5ul、10ul、50ul、100ul、200ul、500ul、1000ul。
3）振蕩器及磁力攪拌器等。
安全性
1）避免直接接觸終止液和底物A、B。一旦接觸到這些液體，請(qǐng)盡快用水沖洗。
2）實(shí)驗(yàn)中不要吃喝、抽煙或使用化妝品。
3）不要用嘴吸取試劑盒里的任何成份。
操作注意事項(xiàng)
1）試劑應(yīng)按標(biāo)簽說(shuō)明書(shū)儲(chǔ)存，使用前恢復(fù)到室溫。稀稀過(guò)后的標(biāo)準(zhǔn)品應(yīng)丟棄，不可保存。
2）實(shí)驗(yàn)中不用的板條應(yīng)立即放回包裝袋中，密封保存，以免變質(zhì)。
3）不用的其它試劑應(yīng)包裝好或蓋好。不同批號(hào)的試劑不要混用。保質(zhì)前使用。
4）使用一次性的吸頭以免交叉污染，吸取終止液和底物A、B液時(shí)，避免使用帶金屬部分的加樣器。
5）使用干凈的塑料容器配置洗滌液。使用前充分混勻試劑盒里的各種成份及樣品。
6）洗滌酶標(biāo)板時(shí)應(yīng)充分拍干，不要將吸水紙直接放入酶標(biāo)反應(yīng)孔中吸水。
7）底物A應(yīng)揮發(fā)，避免長(zhǎng)時(shí)間打開(kāi)蓋子。底物B對(duì)光敏感，避免長(zhǎng)時(shí)間暴露于光下。避免用手接觸，有毒。實(shí)驗(yàn)完成后應(yīng)立即讀取OD值。
8）加入試劑的順序應(yīng)一致，以保證所有反應(yīng)板孔溫育的時(shí)間一樣。
9）按照說(shuō)明書(shū)中標(biāo)明的時(shí)間、加液的量及順序進(jìn)行溫育操作。
樣品收集、處理及保存方法
1）血清-----操作過(guò)程中避免任何細(xì)胞刺激。使用不含熱原和內(nèi)毒素的試管。收集血液后，1000×g離心10分鐘將血清和紅細(xì)胞迅速小心地分離。
2）血漿-----EDTA、檸檬酸鹽、肝素血漿可用于檢測(cè)。1000×g離心30分鐘去除顆粒。
3）細(xì)胞上清液---1000×g離心10分鐘去除顆粒和聚合物。
4）組織勻漿-----將組織加入適量生理鹽水搗碎。1000×g離心10分鐘，取上清液
5）保存------如果樣品不立即使用，應(yīng)將其分成小部分-70
℃保存，避免反復(fù)冷凍。盡可能的不要使用溶血或高血脂血。如果血清中大量顆粒，檢測(cè)前先離心或過(guò)濾。不要在37℃或更高的溫度加熱解凍。應(yīng)在室溫下解凍并確保樣品均勻地充分解凍。
試劑的準(zhǔn)備
1）標(biāo)準(zhǔn)品：標(biāo)準(zhǔn)品的系列稀釋?xiě)?yīng)在實(shí)驗(yàn)時(shí)準(zhǔn)備，不能儲(chǔ)存。稀釋前將標(biāo)準(zhǔn)品振蕩混勻。
2）洗滌緩沖液（50×）的稀釋?zhuān)赫麴s水50倍稀釋。
操作步驟
1）使用前，將所有試劑充分混勻。不要使液體產(chǎn)生大量的泡沫，以免加樣時(shí)加入大量的氣泡，產(chǎn)生加樣上的誤差。
2）根據(jù)待測(cè)樣品數(shù)量加上標(biāo)準(zhǔn)品的數(shù)量決定所需的板條數(shù)。每個(gè)標(biāo)準(zhǔn)品和空白孔建議做復(fù)孔。每個(gè)樣品根據(jù)自己的數(shù)量來(lái)定，能使用復(fù)孔的盡量做復(fù)孔。標(biāo)本用標(biāo)本稀釋液1：1稀釋后加入50ul于反應(yīng)孔內(nèi)。
3）加入稀釋好后的標(biāo)準(zhǔn)品50ul于反應(yīng)孔、加入待測(cè)樣品50ul于反應(yīng)孔內(nèi)。立即加入50ul的生物素標(biāo)記的抗體。蓋上膜板，輕輕振蕩混勻，37℃溫育1小時(shí)。
4）甩去孔內(nèi)液體，每孔加滿洗滌液，振蕩30秒，甩去洗滌液，用吸水紙拍干。重復(fù)此操作3次。如果用洗板機(jī)洗滌，洗滌次數(shù)增加一次。
5）每孔加入80ul的親和鏈酶素-HRP，輕輕振蕩混勻，37℃溫育30分鐘。
6）甩去孔內(nèi)液體，每孔加滿洗滌液，振蕩30秒，甩去洗滌液，用吸水紙拍干。重復(fù)此操作3次。如果用洗板機(jī)洗滌，洗滌次數(shù)增加一次。
7）每孔加入底物A、B各50ul，輕輕振蕩混勻，37℃溫育10分鐘。避免光照。
8）取出酶標(biāo)板，迅速加入50ul終止液，加入終止液后應(yīng)立即測(cè)定結(jié)果。
9）在450nm波長(zhǎng)處測(cè)定各孔的OD值。
局限
6號(hào)標(biāo)準(zhǔn)品以上的結(jié)果為非線性的，根據(jù)此標(biāo)準(zhǔn)曲線無(wú)法得到精確的結(jié)果。
試劑盒性能
1. 靈敏度：最小的檢測(cè)濃度小于1號(hào)標(biāo)準(zhǔn)品。稀釋度的線性。樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值為0.990。
2. 特異性：不與其它細(xì)胞因子反應(yīng)。
3. 重復(fù)性：板內(nèi)、板間變異系數(shù)均小于10%。
結(jié)果判斷與分析
1、儀器值：于波長(zhǎng)450nm的酶標(biāo)儀上讀取各孔的OD值
2、以吸光度OD值為縱坐標(biāo)（Y），相應(yīng)的待測(cè)物質(zhì)標(biāo)準(zhǔn)品濃度為橫坐標(biāo)（X），做得相應(yīng)的曲線，樣品的待測(cè)物質(zhì)含量可根據(jù)其OD值由標(biāo)準(zhǔn)曲線換算出相應(yīng)的濃度。
酶聯(lián)生物經(jīng)過(guò)不斷的實(shí)驗(yàn)優(yōu)化和改進(jìn)，積累了大量的經(jīng)驗(yàn)，擁有專(zhuān)業(yè)的酶聯(lián)研發(fā)團(tuán)隊(duì)。利用專(zhuān)業(yè)的酶聯(lián)免疫技術(shù)自主研發(fā)的elisa試劑盒，能對(duì)血清及其它樣本定量檢測(cè)抗原，定性檢測(cè)特異性抗體。優(yōu)質(zhì)的試劑，先進(jìn)的儀器和正確的操作是保證ELISA檢測(cè)結(jié)果準(zhǔn)確可靠的必要條件。ELISA檢測(cè)的方便性、穩(wěn)定性、重復(fù)性和可靠性方面都具有很大的優(yōu)勢(shì)。
ELISA檢測(cè)技術(shù)服務(wù)內(nèi)容：
1、雙抗體夾心法檢測(cè)抗原 2、間接法檢測(cè)抗體 3、為客戶提供各種ELISA技術(shù)進(jìn)行樣本檢測(cè)。
以上代測(cè)費(fèi)，凡購(gòu)買(mǎi)本公司試劑盒，我們免費(fèi)代測(cè)！
凡購(gòu)買(mǎi)本公司目錄任何一種酶聯(lián)免疫檢測(cè)試劑盒，您只需將需要檢測(cè)的動(dòng)物（Human, Rat, Mouse, Rabbit, Monkey,
Pig……）種類(lèi)和檢測(cè)指標(biāo)（白介素類(lèi)、激素類(lèi)）及標(biāo)本數(shù)量（48T/96T）通知公司業(yè)務(wù)員即可。在接到客戶標(biāo)本當(dāng)日起，現(xiàn)貨產(chǎn)品一周內(nèi)將檢測(cè)報(bào)告交到客戶手中！
歡迎各科研單位在各種項(xiàng)目上與我們公司開(kāi)展不同層次的密切合作，以雙贏求發(fā)展，共同進(jìn)步，為中國(guó)檢測(cè)事業(yè)的發(fā)展積累經(jīng)驗(yàn)。
二、樣本要求
在收集標(biāo)本前都必須有一個(gè)完整的計(jì)劃，必須清楚要檢測(cè)的成份是否足夠穩(wěn)定。我們提倡新鮮標(biāo)本盡早檢測(cè)，對(duì)收集后當(dāng)天就進(jìn)行檢測(cè)的標(biāo)本，及時(shí)儲(chǔ)存在4℃?zhèn)溆茫缬刑厥庠蛐枰芷谑占瘶?biāo)本，請(qǐng)?jiān)炷Ｈ〔暮螅瑢?biāo)本及時(shí)分裝后放在-20℃或-70℃條件下保存。因冰室與室溫存在一定溫差，蛋白極易降解，直接影響實(shí)驗(yàn)質(zhì)量，所以避免反復(fù)凍融。代測(cè)放免標(biāo)本的客戶取材前須向我司銷(xiāo)售人員索要說(shuō)明書(shū)，具體操作注意事項(xiàng)請(qǐng)與我司技術(shù)人員溝通。
液體類(lèi)標(biāo)本：標(biāo)本必須為液體，不含沉淀。包括血清、血漿、尿液、胸腹水、腦脊液、細(xì)胞培養(yǎng)上清、組織勻漿等。
血清：室溫血液自然凝固10-20分鐘后，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。收集上清。如有沉淀形成，應(yīng)再次離心。
血漿：應(yīng)根據(jù)試劑盒的要求選擇EDTA、檸檬酸鈉或肝素作為抗凝劑，加入10%（v/v）抗凝劑(0.1M檸檬酸鈉或1%heparin
或2.0%EDTA.Na2)混合10-20分鐘后，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。如有沉淀形成，應(yīng)再次離心。
尿液、胸腹水、腦脊液：用無(wú)菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。如有沉淀形成，應(yīng)再次離心。
細(xì)胞培養(yǎng)上清：檢測(cè)分泌性的成份時(shí)，用無(wú)菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。檢測(cè)細(xì)胞內(nèi)的成份時(shí)，用PBS（PH7.0-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬(wàn)/ml左右。通過(guò)反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過(guò)程中如有沉淀形成，應(yīng)再次離心。
組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的PBS，緩沖液中可加入1μg/L蛋白酶抑制劑或50U/ml的Aprotinin（抑肽酶）。用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清置于-20度或-70度保存，如有必要，可以將樣品濃縮干燥。分裝后一份待檢測(cè)，其余冷凍備用。
三、寄標(biāo)本時(shí)需注明以下情況：
1、標(biāo)本編號(hào)；2、所測(cè)項(xiàng)目；3、是否做復(fù)孔；3、聯(lián)系方式；4、實(shí)驗(yàn)后標(biāo)本是否寄回。
客戶須知：
客戶應(yīng)對(duì)所提供的材料及信息負(fù)責(zé)，如因客戶提供的材料及信息不準(zhǔn)確而引起的實(shí)驗(yàn)延誤或經(jīng)濟(jì)損失由客戶承擔(dān)。
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.
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