RayBio?Human Ferritin ELISA Kit
User Manual
RayBio? Human Ferritin
ELISA Kit Protocol
(Cat#: ELH-Ferritin-001)
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RayBiotech, Inc.
RayBio? Human Ferritin
ELISA Kit Protocol
TABLE OF CONTENTS
I.Introduction (2)
II.Reagents (2)
III.Storage (3)
IV.Additional Materials Required (3)
V.Reagent Preparation (4)
VI.Assay Procedure (6)
VII.Assay Procedure Summary (7)
VIII.Calculation of Results
A.Typical Data (7)
B.Sensitivity (8)
C.Recovery (8)
D.Linearity (8)
E.Reproducibility (9)
IX.Specificity (9)
X.Troubleshooting Guide (10)
I. INTRODUCTION
The RayBio? Human Ferritin ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Ferritin in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human Ferritin coated on a 96-well plate. Standards and samples are pipetted into the wells and Ferritin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human Ferritin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Ferritin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
II. REAGENTS
1.Ferritin Microplate (Item A): 96 wells (12 strips x 8 wells) coated with
anti-human Ferritin.
2.Wash Buffer Concentrate (20x) (Item B): 25 ml of 20x concentrated
solution.
3.Standards (Item C): 2 vials of recombinant human Ferritin.
4.Assay Diluent A (Item D): 30 ml diluent buffer, 0.09% sodium azide as
preservative. For Standard/Sample (serum/plasma) diluent.
5.Assay Diluent B (Item E): 15 ml of 5x concentrated buffer. For
Standard/Sample (cell culture medium/urine) diluent.
6.Detection Antibody Ferritin (Item F): 2 vial of biotinylated anti-human
Ferritin (each vial is enough to assay half microplate).
7.HRP-Streptavidin Concentrate (Item G): 8 μl 20,000x concentrated
HRP-conjugated streptavidin.
8.TMB One-Step Substrate Reagent (Item H): 12 ml of 3,3’,5,5’-
tetramethylbenzidine (TMB) in buffer solution.
9.Stop Solution (Item I): 8 ml of 2 M sulfuric acid.
III. STORAGE
May be stored for up to 6 months at 2o to 8o C from the date of shipment. Standard (recombinant protein) should be stored at -20 o C or -80
o C (recommended at –80 o C) after reconstitution. Opened Microplate Wells or reagents may be stored for up to 1 month at 2o to 8o C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: the kit can be used within one year if the whole kit is stored at -20 o C. Avoid repeated freeze-thaw cycles.
IV. ADDITIONAL MATERIALS REQUIRED
1Microplate reader capable of measuring absorbance at 450 nm.
2Precision pipettes to deliver 2 μl to 1 ml volumes.
3Adjustable 1-25 ml pipettes for reagent preparation.
4100 ml and 1 liter graduated cylinders.
5Absorbent paper.
6Distilled or deionized water.
7Log-log graph paper or computer and software for ELISA data analysis.
8Tubes to prepare standard or sample dilutions.
V. REAGENT PREPARATION
1.Bring all reagents and samples to room temperature (18 - 25°C) before
use.
2.Sample dilution: Assay Diluent A (Item D) is used for dilution of
serum/plasma samples. Recommend 2 - 20 fold dilution for
serum/plasma samples; 1x Assay Diluent B (Item E) can be used for
dilution of cell culture supernates/urine.
3.Assay Diluent B should be diluted 5-fold with deionized or distilled
water before use.
4. Preparation of standard: Briefly spin the vial of Item C and then add 400 μl Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent
B (for cell culture supernates/urine) into Item
C vial to prepare a 200 ng/ml standard. Dissolve the powder thoroughly by a gentle mix. Pipette 270 μl Assay Diluent A or 1x Assay Diluent B into each tube. Use the 200 ng/ml standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Gently vortex to mix. Assay Diluent A or 1x Assay Diluent B serves as the zero
standard (0 ng/ml).
5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 μl of 1x Assay Diluent B into the vial to prepare a detection antibody
concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4°C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
180 μl 200 80 32 12 5.12 2.04 0.81 0 ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml Standard, Item C + 400 μl
180μl 180 μl 180 μl 180 μl 180 μl
7.Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette
up and down to mix gently before use. HRP-Streptavidin concentrate
should be diluted 20,000-fold with 1x Assay Diluent B.
For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . A dd 2 μl of HRP-Streptavidin concentrate into a tube with 198 μl 1x Assay Diluent B to prepare a 100-fold diluted HRP-
Streptavidin solution (don’t store the diluted solution for next day use). Mix through and then pipette 50 μl of prepared 100-fold diluted
solution into a tube with 10 ml 1x Assay Diluent B to prepare a final
10,000 fold diluted HRP-Streptavidin solution.
VI. ASSAY PROCEDURE:
1.Bring all reagents and samples to room temperature (18 - 25°C) before
use. It is recommended that all standards and samples be run at least in
duplicate.
2.Add 100 μl of each standard (see Reagent Preparation step 2)and
sample into appropriate wells. Cover well and incubate for 2.5 hours at
room temperature or over night at 4°C with gentle shaking.
3.Discard the solution and wash 4 times with 1x Wash Solution. Wash by
filling each well with Wash Buffer (300 μl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is
essential to good performance. After the last wash, remove any
remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4.Add 100 μl of 1x prepared biotinylated antibody (Reagent Preparation
step 6) to each well. Incubate for 1 hour at room temperature with
gentle shaking.
5.Discard the solution. Repeat the wash as in step 3.
6.Add 100 μl of prepared Streptavidin solution (see Reagent Preparation
step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
7.Discard the solution. Repeat the wash as in step 3.
8.Add 100 μl of TMB One-Step Substrate Reagent (Item H) to each well.
Incubate for 30 minutes at room temperature in the dark with gentle
shaking.
9.Add 50 μl of Stop Solution (Item I) to each well. Read at 450 nm
immediately.
VII. ASSAY PROCEDURE SUMMARY
1. Prepare all reagents, samples and standards as instructed.
2. Add 100 μl standard or sample to each well.
Incubate 2.5 hours at room temperature or over night at 4o C.
3. Add 100 μl prepared biotin antibody to each well.
Incubate 1 hour at room temperature.
4. Add 100 μ
Incubate 45 minutes at room temperature.
5. Add 100 μl TMB One-Step Substrate Reagent to each well.
Incubate 30 minutes at room temperature.
6. Add 50 μl Stop Solution to each well.
Read at 450 nm immediately.
VIII. CALCULATION OF RESULTS
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
A. TYPICAL DATA
These standard curves are for demonstration only. A standard curve must be run with each assay.
B. SENSITIVITY
The minimum detectable dose of Ferritin is typically less than 0.6 ng/ml. C. RECOVERY
Recovery was determined by spiking various levels human Ferritin into human serum, plasma and cell culture media. Mean recoveries are as follows:
Sample Type Average % Recovery Range (%)
Serum 97.32 86-105
Plasma 103.1 93-112
Cell culture media 96.46 85-93
D. LINEARITY
Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 97.78 102.8 105.4
Range (%) 86-106 91-110 96-113
1:4 Average % of Expected 101.1 75.54 86.67
Range (%) 91-110 67-81 75-94
E. REPRODUCIBILITY
Intra-Assay: CV<10%
Inter-Assay: CV<12%
IX.SPECIFICITY
Cross Reactivity: This ELISA kit shows no cross-reactivity with the following cytokines tested: human Angiogenin, BDNF, BLC, CNTF, ENA-78, FGF-4, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, IL-309, IP-10, FGF-4, FGF-6, FGF-7, G-CSF, GDNF, GM-CSF, IFN-γ, IGFBP-2, IGF-BP-3, IGF-BP-4, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIF, MIG, MIP-1α, MIP-1 β, MIP-1δ, PARC, PDGF, RANTES, SCF,SDF-1alpha, TARC, TGF-β, TIMP-1, TIMP-2, TNF-α, TNF-β, TPO, VEGF.
X. TROUBLESHOOTING GUIDE
Problem Cause Solution
1. Poor standard
curve
1. Inaccurate pipetting 1. Check pipettes
2. Improper standard dilution 2. Ensure a brief spin of Item C
and dissolve the powder thoroughly by a gentle mix.
2. Low signal 1.Too brief incubation
times 1. Ensure sufficient incubation time; assay procedure step 2 may change to over night
2. Inadequate reagent volumes or improper dilution 2. Check pipettes and ensure correct
preparation
3. Large CV 1. Inaccurate pipetting 1. Check pipettes
4. High background 1. Plate is insufficiently
washed 1. Review the manual for proper wash. If using a plate
washer, check that all ports are
unobstructed.
2. Contaminated wash buffer 2. Make fresh wash buffer
5. Low sensitivity 1. Improper storage of the
ELISA kit 1. Store your standard at<-20o C after
reconstitution,
others at 4 o C. Keep substrate solution protected from light
2. Stop solution 2. Stop solution should
be added to each
well before measure
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胃蛋白酶原（PGⅠ＆PGⅡ） 定量检测试剂盒（化学发光法） 项目推荐书 胃蛋白酶原简介 一、胃蛋白酶原 胃蛋白酶原（pepsinogen，PG）是胃分泌的一种消化酶前体[1]，是胃液中胃蛋白酶的无活性前体，为一个由375个氨基酸组成的蛋白多肽链，平均相对分子质量为42,000，人胃粘膜中有7组胃蛋白同工酶原。PG在核糖体上合成，由高尔基体分泌出细胞，被盐酸激活后变成胃蛋白酶。根据生化性质、免疫原性、细胞来源及组织内分布可分成PGⅠ、PGⅡ两个亚群，1～5组分免疫原性近似，称为PGⅠ(PGA)，主要由胃腺的主细胞的粘液颈细胞分泌；6～7组分免疫原性近似，称为PGⅡ，除由胃体和胃底黏膜的泌酸腺的主细胞分泌外，泌酸腺的黏液颈细胞、贲门腺和胃窦的幽门腺的黏液细胞及十二指肠上段的Brunner腺也能产生PGⅡ。 PG含量与良、恶性胃溃疡的鉴别有关，血清PGⅠ水平与萎缩性胃炎、PGⅠ/PGⅡ水平与胃癌和胃癌前期病变呈负相关。血清PGⅠ与胃泌酸腺细胞功能相关，PGⅡ与胃底粘膜病变的相关性较大，血清PG 反映胃总体分泌PG水平。消化性溃疡的发生与胃分泌酸过多有密切关系。萎缩性胃炎、胃癌前期病变或胃癌发生时，尤其是幽门螺杆菌感染等因素所干扰，胃酸分泌过多的浅表性胃炎和幽门螺杆菌感染的胃
炎，PGⅠ和PGⅡ的分泌会增加；而在慢性严重萎缩性胃炎当主细胞减少时PGⅠ含量下降；当萎缩性胃炎伴有肠化、胃窦宪假幽门腺化生，PGⅡ含量会随之增高。血清PG可作为检测胃癌的一个可靠标志物。 约有1%的PG透过胃黏膜毛细血管进入血液循环，进入血液循环的PG在血液中非常稳定。血清PG I和PG II反映胃黏膜腺体和细胞的数量，也间接反映胃黏膜不同部位的分泌功能。当胃黏膜发生病理变化时，血清胃蛋白酶原含量也随之改变。因此，监测血清中胃蛋白酶原的浓度可以作为监测胃黏膜状态的手段。 二、专家共识 2010年2月25日，由中国医师协会主办“全民胃部重大疾病普查行动”中，将胃蛋白酶原检测作为重要普查方法：进一步以胃镜确诊。 已被卫生部疾控中心列为： 《中国癌症筛查及早诊早治技术方案》标准筛查手段。 胃癌预防亚太地区共识与指南,本次共识会议由亚太胃肠病学会发起。于2006年11月11～ 12日在泰国曼谷召开。共有38条公示条文被提出评估。 第15条：低血清PG I水平和低PG I／II比例反映了胃萎缩， 低血清PG可作为萎缩性胃炎的一个替代标志物。 第16条：低血清PG I水平和低PG I／II比例可作为鉴别胃癌高危人群的标志物。 上世纪90年代，日本在临床试验的基础上，血清PG作为慢性萎缩
胃蛋白酶（英文名称：Pepsin）是一种消化性蛋白酶，由胃部中的胃粘膜主细胞（gastricchiefcell）所分泌，功能是将食物中的蛋白质分解为小的肽片段。胃蛋白酶的前体被称为胃蛋白酶原。胃腺主细胞分泌的蛋白酶。初分泌时为无活性的胃蛋白酶原，在胃酸或已激活的胃蛋白酶的作用下转变为具活性的胃蛋白酶。在适宜环境下(pH约为2)可将蛋白质分解为和胨，很少产生小分子肽或氨基酸。自猪、牛、羊等胃粘膜提取的胃蛋白酶用作助消化药。常与稀盐酸同时用于幼畜消化不良性腹泻和慢性萎缩性胃炎。 蛋白酶原由胃底主细胞分泌，在pH1.5～5.0条件下，被活化成胃蛋白酶，将蛋白质分解为胨，而且一部分被分解为酪氨酸、苯丙氨酸等氨基酸。 胃液胃蛋白酶测定可用于鉴别神经性低酸症和胃性低酸症，当胃酸过少或 缺乏时，前者胃蛋白酶的含量有时正常而后者盐酸与胃蛋白酶同时缺乏。一般认为胃性低酸症是由于胃粘膜的重症器质性变化所致，特别是对于恶性贫血、无酸症、无胃蛋白酶分泌是诊断上的重要所见。慢性胃炎、慢性胃扩张、慢性十二脂肠炎等胃蛋白酶的分泌常减少。一般胃酸基础分泌高的疾患，如十二指肠溃疡等，胃蛋白酶活性增高。1836年，索多·施旺（TheodorSchwann）在对消化过程进行的研究中，发现了一种能够参与消化作用的物质，并将其命名为胃蛋白酶。胃蛋白酶也是第一个从动物身上获得的酶。胃中惟一的一种蛋白水解酶。其最适pH值为1～2。胃蛋白酶作用的主要部位是芳香族氨基酸或酸性氨基酸的氨基所组成的肽键。此酶由胃腺的主细胞合成，以酶原颗粒形式分泌，经胃液中盐酸激活后，具有消化蛋白质的能力。药用胃蛋白酶，可以从猪胃中提取，用于消化不良。消化性溃疡禁用此药。 性状本品为白色或淡黄色的粉末；无霉败臭；有引湿性；水溶液显酸性反应。 鉴别取本品的水溶液，加鞣酸、没食子酸或多数重金属盐的溶液，即发生沉淀。 检查干燥失重 取本品，在100℃干燥4小时，减失重量不得过5.0％（附录ⅧL）。 效价测定 对照品溶液的制备 精密称取经105℃干燥至恒重的酪氨酸适量，加盐酸溶液〔取1mol/L盐酸溶液65ml，加水至1000ml〕制成每1ml中含0.5mg的溶液。 胃蛋白酶在对蛋白或多肽进行剪切时，具有一定的氨基酸序列特异性。例如，它倾向于剪切氨基端或羧基端为芳香族氨基酸（如苯丙氨酸、色氨酸和酪氨酸）或亮氨酸的肽键；而如果往某一肽键氨基端数第三个氨基酸为碱性氨基酸（如赖氨酸、精氨酸和组氨酸）或者该肽键的氨基端为精氨酸
蛋白酶的论述 摘要：蛋白酶（英语：Protease）是生物体内的一类酵素（酶），它们能够分解蛋白质。分解方法是打断那些将氨基酸连结成多肽链的肽键。抑制蛋白酶活性的小分子化合物被称蛋白酶抑制剂。许多病毒蛋白酶的抑制剂是很有效的抗病毒药。 1．木瓜蛋白酶 1.1木瓜蛋白酶简介 木瓜蛋白酶，是一种蛋白水解酶，可将抗体分子水解为3个片段。是番木瓜中含有的一种低特异性蛋白水解酶，活性中心含半胱氨酸，属巯基蛋白酶，应用于啤酒及食品工业。 1.2木瓜蛋白酶的特点 木瓜蛋白酶（Papain）简称木瓜酶，又称为木瓜酵素。是利用未成熟的番木瓜（Carica papaya）果实中的乳汁，采用现代生物工程技术提炼而成的纯天然生物酶制品。它是一种含巯基(-SH)肽链内切酶，具有蛋白酶和酯酶的活性，有较广泛的特异性，对动植物蛋白、多肽、酯、酰胺等有较强的水解能力，同时，还具有合成功能，能把蛋白水解物合成为类蛋白质。溶于水和甘油，水溶液无色或淡黄色，有时呈乳白色；几乎不溶于乙醇、氯仿和乙醚等有机溶剂。最适合PH值6～7(一般3～9.5皆可)，在中性或偏酸性时亦有作用，等电点（pI)为8.75；最适合温度55～65℃(一般10～85℃皆可)，耐热性强，在90℃时也不会完全失活；受氧化剂抑制，还原性物质激活。木瓜蛋白酶由212个氨基酸残基组成，当用氨基肽酶从N末端水解掉分子中的2/3肽链后，剩下的1/3肽链仍保持99%的活性，说明木瓜蛋白酶的生物活性集中表现在C末端的少数氨基酸残基及其所构成的空间结构区域。 木瓜蛋白酶papain属巯基蛋白酶，具有较宽的底物特异性，作用于蛋白质中L-精氨酸、L-赖氨酸、甘氨酸和L-瓜氨酸残基羧基参与形成的肽键。此酶属内肽酶，能切开全蛋蛋白质分子内部肽链—CO—NH—生成分子量较小的多肽类。存在于木瓜胚乳中的蛋白酶。EC3．4．22．2。作为植物来源的蛋白酶来说，此酶研究进展的最快。此酶主要是以内肽酶的形态起作用。活性的产生，而半胱氨酸残基是不可缺少的，所以是硫基蛋白酶的一种，底物的特异性不太严格，分子量为23400，氨基酸残基数212。 木瓜蛋白酶是一种在酸性、中性、碱性环境下均能分解蛋白质的蛋白酶。它的外观为白色至浅黄色的粉末，微有吸湿性。 酪蛋白被木瓜蛋白酶降解生成的酪氨酸在紫外光区 275nm 处有吸收峰。1.3木瓜蛋白酶物理化学性质 本品为乳白色至微黄色粉末，具有木瓜特有的气味,稍具有吸湿性。水解蛋白质能力强,但几乎不能分解蛋白胨，易溶于水,甘油,不溶于一般的有机溶剂,耐热性强。由木瓜制得的商品酶制剂中，含有如下三种酶：(1)木瓜蛋白酶，分
胃蛋白酶原Ⅰ/Ⅱ（PGⅠ/PGⅡ）定量检测 试剂盒（化学发光法） 简介： 胃蛋白酶原（pepsinogen，PG）是胃分泌的一种消化酶前体，是胃液中胃蛋白酶的无活性前体，为一由375个氨基酸组成的蛋白多肽链，平均相对分子质量为42,000，人胃粘膜中有7组胃蛋白同工酶原。PG 在核糖体上合成，由高尔基体分泌出细胞，被盐酸激活后变成胃蛋白酶。根据生化性质、免疫原性、细胞来源及组织内分布可分成PGⅠ、PGⅡ两个亚群，1～5组分免疫原性近似，称为PGⅠ(PGA)，主要由胃腺的主细胞的粘液颈细胞分泌。利用化学发光分析法得出结果。【包装规格】 48人份/盒、96人份/盒 【预期用途】 用于血清中胃蛋白酶原Ⅰ的定量检测。 【样本要求】 1.采用正确医用技术收集血清样本。 2.样本中的沉淀物可能会影响试验结果，应离心除去，并确定样本未变质方可使用。 3.严重溶血或脂血的样本不能用于测定。 4.样本在24h内测定，则应存放于2℃～8℃冰箱中，如果长期使用，则应冻存于-20℃以下，避免反复冻融。使用前恢复到室温，轻轻摇动混匀。
【检测步骤】 1.取出一定量的包被孔，每孔分别加入20μl校准品或血清样品。 2.每孔分别加入酶结合物50μl。 3.在微型振荡器上振荡30秒使孔内液体混合均匀，置37℃温育60分钟。 4.洗板5次（推荐使用洗板机），最后在吸水纸上拍干。 5.每孔分别加入发光底物A和B各50μl，室温（18℃～25℃）避光反应5分钟。 6.化学发光检测仪检测发光强度。 【结果计算】 选择适当的曲线拟合方式，本试剂盒推荐使用线性回归拟和方程建立标准曲线，但也可根据不同情况采用其他拟和方式。以系列校准品浓度的对数值为横坐标（X轴），以系列校准品发光强度值的对数值为纵坐标（Y轴）建立标准曲线（log-log），进行计算。 【胃蛋白酶原试剂盒检测优势】 优点：1.血清检测无创伤、更安全。 2.费用低廉，适用于普查体检。 3.操作简单，时间短，不会造成受检人员的长时间滞留。 4.胃癌检出率高，特异性强 5.操作简便 6.无创，病人耐受好 7.费用低