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Human Interferon γ (IFN-γ)
ELISA Kit
Catalog No. CSB-E04577h
(96T)
This immunoassay kit allows for the in vitro quantitative determination of human IFN-γ
concentrations in cell culture supernates, serum, plasma and other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
CUSABIO BIOTECH CO., Ltd.
http://www.cusabio.com/ http://www.cusabio.cn/
E-mail: cusabio@cusabio.com cusabio@cusabio.cn
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INTRODUCTION
Interferon-gamma (IFN-γ) is a dimerized soluble cytokine that is the only member
of the type II class of interferons. This interferon was originally called
macrophage-activating factor. The IFN-γ monomer consists of a core of six
α-helices and an extended unfolded sequence in the C-terminal region. This is
shown in the structural models below. The α-helices in the core of the structure are
numbered 1 to 6. Full length IFN-γ is 143 amino acids in length, the models are
126 amino acids in length. Affinity for the glycosaminoglycan heparan sulfate
resides solely within the deleted sequence of 17 amino acids. In contrast to
interferon-α and interferon-β which can be expressed by all cells, IFN-γ is secreted
by Th1 cells, Tc cells, dendritic cells and NK cells. Also known as immune
interferon, IFN-γ is the only Type II interferon. It is serologically distinct from
Type I interferons and it is acid-labile, while the type I variants are acid-stable.
IFN-γ has antiviral, immunoregulatory, and anti-tumour properties. It alters
transcription in up to 30 genes producing a variety of physiological and cellular
responses. Amongst the effects are: Increase antigen presentation of macrophages.
Activate and increase lysosome activity in macrophages. Suppress Th2 cell
activity. Cause normal cells to increase expression of class I MHC molecules.
Promotes adhesion and binding required for leukocyte migration Promotes NK cell
activity. Activation by IFN-γ is achieved by its interaction with a heterodimeric
receptor consisting of IFNGR1 & IFNGR2 (interferon gamma receptors). IFN-γ
binding to the receptor activates the JAK-STAT pathway. In addition, IFN-γ
activates APCs and promotes Th1 differentiation by upregulating the transcription
factor T-bet. IFN-γ is the hallmark cytokine of Th1 cells (whereas Th2 cells
produce IL-4 and Th17 cells produce IL-17). NK cells and CD8+ cytotoxic T cells
also produce IFN-γ. IFN-γ suppresses osteoclast formation by rapidly degrading
the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway,
which otherwise stimulates the production of NFκB.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an antibody
specific to IFN-γ. Standards or samples are then added to the appropriate
microtiter plate wells with a biotin-conjugated polyclonal antibody preparation
specific for IFN-γ and Avidin conjugated to Horseradish Peroxidase (HRP) is
added to each microplate well and incubated. Then a TMB (3,3’5, 5’
tetramethyl-benzidine) substrate solution is added to each well. Only those wells
that contain IFN-γ, biotin-conjugated antibody and enzyme-conjugated Avidin will
exhibit a change in color. The enzyme-substrate reaction is terminated by the
addition of a sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of
IFN-γ in the samples is then determined by comparing the O.D. of the samples to
the standard curve.
DETECTION RANGE
0.78 ng/ml-50 ng/ml. The standard curve concentrations used for the ELISA’s were
50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml, 1.56 ng/ml, 0.78 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural human IFN-γ. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human IFN-γ is typically less than 0.195 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as
the lowest protein concentration that could be differentiated from zero.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120l
HRP-avidin 1 x 120l
1 x 20 ml
Wash Buffer
(25×concentrate)
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
STORAGE
1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter
plate should be kept in a sealed bag with desiccants to minimize exposure to
damp air. The test kit may be used throughout the expiration date of the kit.
Refer to the package label for the expiration date.
2. Opened test kits will remain stable until the expiring date shown, provided it is
stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical
density range of 0-3 OD or greater at 450nm wavelength is acceptable for use
in absorbance measurement.
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REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm to room
temperature and mix gently until the crystals have completely dissolved.
Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to
prepare 500 ml of Wash Buffer.
2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This
reconstitution produces a stock solution of 50 ng/ml. Allow the standard to sit
for a minimum of 15 minutes with gentle agitation prior to making serial
dilutions. The undiluted standard serves as the high standard (50 ng/ml). The
Sample Diluent serves as the zero standard (0 ng/ml).
3. Biotin-antibody Dilute to the working concentration specified on the vial
label using Biotin-antibody Diluent(1:100), respectively.
4. HRP-avidin Dilute to the working concentration specified on the vial label
using HRP-avidin Diluent(1:100), respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450 nm, with the
correction wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate washer.
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SAMPLE COLLECTION AND STORAGE
Cell Culture Supernates Remove particulates by centrifugation and assay
immediately or aliquot and store samples at -20° C. Avoid repeated
freeze-thaw cycles.
Serum Use a serum separator tube (SST) and allow samples to clot for 30
minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and
assay immediately or aliquot and store samples at -20° C. Avoid repeated
freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant.
Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay
immediately or aliquot and store samples at -20° C. Avoid repeated
freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
1. Add 100l of Standard, Blank, or Sample per well. Cover with the adhesive
strip. Incubate for 2 hours at 37° C.
2. Remove the liquid of each well, don’t wash.
3. Add 100l of Biotin-antibody working solution to each well. Incubate for 1
hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm to
room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of
three washes. Wash by filling each well with Wash Buffer (200l) using a
squirt bottle, multi-channel pipette, manifold dispenser 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.
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5. Add 100l of HRP-avidin working solution to each well. Cover the microtiter
plate with a new adhesive strip. Incubate for 1 hour at 37° C.
6. Repeat the aspiration and wash three times as step 4.
7. Add 90l of TMB Substrate to each well. Incubate for 30 minutes at 37°C.
Keeping the plate away from drafts and other temperature fluctuations in the
dark.
8. Add 50l of Stop Solution to each well. If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm.
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and subtract
the average zero standard optical density. Create a standard curve by reducing the
data using computer software capable of generating a four parameter logistic (4-PL)
curve-fit. As an alternative, construct a standard curve by plotting the mean
absorbance for each standard on the y-axis against the concentration on the x-axis
and draw a best fit curve through the points on the graph. The data may be
linearized by plotting the log of the IFN-γ concentrations versus the log of the O.D.
and the best fit line can be determined by regression analysis. This procedure will
produce an adequate but less precise fit of the data. If samples have been diluted,
the concentration read from the standard curve must be multiplied by the dilution
factor.
LIMITATIONS OF THE PROCEDURE
The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
It is important that the Calibrator Diluent selected for the standard curve be
consistent with the samples being assayed.
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If samples generate values higher than the highest standard, dilute the samples
with the appropriate Calibrator Diluent and repeat the assay.
Any variation in Standard Diluent, operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can cause variation in
binding.
This assay is designed to eliminate interference by soluble receptors, binding
proteins, and other factors present in biological samples. Until all factors have
been tested in the Quantikine Immunoassay, the possibility of interference
cannot be excluded.
TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoid foaming.
To avoid cross-contamination, change pipette tips between additions of each
standard level, between sample additions, and between reagent additions. Also,
use separate reservoirs for each reagent.
When using an automated plate washer, adding a 30 second soak period
following the addition of wash buffer, and/or rotating the plate 180 degrees
between wash steps may improve assay precision.
To ensure accurate results, proper adhesion of plate sealers during incubation
steps is necessary.
Substrate Solution should remain colorless until added to the plate. Keep
Substrate Solution protected from light. Substrate Solution should change
from colorless to gradations of blue.
Stop Solution should be added to the plate in the same order as the Substrate
Solution. The color developed in the wells will turn from blue to yellow upon
addition of the Stop Solution. Wells that are green in color indicate that the
Stop Solution has not mixed thoroughly with the Substrate Solution.
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人人γ干扰素干扰素(IFN-γ)酶联免疫分析酶联免疫分析
人人 干扰素干扰素 酶联免疫分析酶联免疫分析
试剂盒说明书试剂盒说明书
试剂盒说明书试剂盒说明书
本试剂盒仅供研究使用本试剂盒仅供研究使用
本试剂盒仅供研究使用本试剂盒仅供研究使用
产品编号产品编号：：CSB-E04577h
产品编号产品编号：：
检测范围检测范围：：0.78 ng/ml - 50 ng/ml
检测范围检测范围：：
最低检测限最低检测限：：0.195 ng/ml
最低检测限最低检测限：：
特异性特异性：：本试剂盒可同时检测天然或重组的人IFN-γ，且与其他相关蛋白无
特异性特异性：：
交叉反应。
有效期有效期：：6 个月
有效期有效期：：
预期应用预期应用：：ELISA 法定量测定人血清、血浆、细胞培养上清或其它相关生
预期应用预期应用：：
物液体中IFN-γ含量。
说明说明：：
说说明明：：
1. 试剂盒保存：-20℃（较长时间不用时）；2-8℃（频繁使用时）。
2. 浓洗涤液低温保存会有盐析出，稀释时可在水浴中加温助溶。
3. 中、英文说明书可能会有不一致之处，请以英文说明书为准。
4. 刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实
验结果造成任何影响。
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概述概述：：
概述概述：：
干扰素 （IFN）是1957 年被发现的。它是一类分泌性蛋白，具有广谱抗
病毒、抗肿瘤和免疫调节功能。根据产生干扰素细胞来源不同、理化性质和
生物学活性的差异，可分为α-1b 型干扰素、β-干扰素和γ干扰素。γ干扰素
（IFN-γ）也叫Ⅱ型IFN，主要由活化T 细胞产生，活化NK 细胞也可产生IFN-γ。
人IFN-γ基因定位于12 号染色体，在DNA 水平上IFN-γ基因与IFN-α/β基因
无同源性。人IFN-γ 成熟分子由143 个氨基酸组成，糖蛋白，以同源双体形
式存在，分子量为40kDa 。IFN-γ 的生物学作用有较严格的种属特异性，人
IFN-γ只作用于人或灵长类动物的细胞。其生物学作用有：(1)诱导单核细胞、
巨噬细胞、树突状细胞、皮肤成纤维细胞、血管内皮细胞、星状细胞等MHC Ⅱ
类抗原的表达，使其参与抗原提呈和特异性免疫的识别过程。此外，IFN-γ可
上调内皮细胞ICAM-1(CD54)表达，促进巨噬细胞FcγR 表达，协同诱导TNF
并促进巨噬细胞杀伤病原微生物。(2)促进LPS 体外刺激小鼠B 细胞分泌
IgG2a，降低IgG1、IgG2b、IgG3 和IgE 的产生；抑制由IL-4 诱导的小鼠B
细胞增殖，IgG1 和IgE 产生以及FcεR Ⅱ表达；促进SAC 诱导的人B 细胞的
增殖。(3)协同IL-2 诱导LAK 活性，促进T 细胞IL-2R 表达。(4)诱导急性期
蛋白合成，诱导髓样细胞分化。
实验原理实验原理：：
实验原理实验原理：：
用纯化的抗体包被微孔板，制成固相载体，往包被抗IFN-γ 抗体的微孔
中依次加入标本或标准品、生物素化的抗IFN-γ抗体、HRP 标记的亲和素，
经过彻底洗涤后用底物TMB 显色。TMB 在过氧化物酶的催化下转化成蓝色，
并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的IFN-γ呈正相关。
用酶标仪在450nm 波长下测定吸光度 （OD 值），计算样品浓度。
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试剂盒组成及试剂配制试剂盒组成及试剂配制：：
试剂盒组成及试剂配制试剂盒组成及试剂配制：：
1. 酶联板酶联板(Assay plate )： 一块（96 孔）。
酶联板酶联板
2. 标准品标准品（Standard）： 2 瓶(冻干品)。
标准品标准品
3. 样品稀释液样品稀释液(Sample Diluent)： 1×20ml/瓶。
样品稀释液样品稀释液
4. 生物素标记抗体稀释液生物素标记抗体稀释液（（Biotin-antibody Diluent）：） 1×10ml/瓶。
生物素标记抗体稀释液生物素标记抗体稀释液（（ ））
5. 辣根过氧化物酶标记亲和素稀释液辣根过氧化物酶标记亲和素稀释液（（HRP-avidin Diluent）：） 1×10ml/瓶。
辣根过氧化物酶标记亲和素稀释液辣根过氧化物酶标记亲和素稀释液（（ ））
6. 生物素标记抗体生物素标记抗体（（Biotin-antibody）：） 1×120l/瓶（1：100）。
生物素标记抗体生物素标记抗体（（ ））
7. 辣根过氧化物酶标记亲和素辣根过氧化物酶标记亲和素（（HRP-avidin ）：） 1×120l/瓶（1：100）。
辣根过氧化物酶标记亲和素辣根过氧化物酶标记亲和素（（ ））
8. 底物溶液底物溶液（（TMB Substrate）：） 1×10ml/瓶。
底物溶液底物溶液（（ ））
9. 浓洗涤液浓洗涤液（（Wash Buffer ）：） 1×20ml/瓶，使用时每瓶用蒸馏水稀释25 倍。
浓洗涤液浓洗涤液（（ ））
10. 终止液终止液（（Stop Solution）：） 1×10ml/瓶（2N H SO4）。
终止液终止液（（ ）） 2
需要而未提供的试剂和器材需要而未提供的试剂和器材：：
需要而未提供的试剂和器材需要而未提供的试剂和器材：：
1. 标准规格酶标仪
2. 高速离心机
3. 电热恒温培养箱
4. 干净的试管和Eppendof 管
5. 系列可调节移液器及吸头，一次检测样品较多时，最好用多通道移液器
6. 蒸馏水，容量瓶等
标本的采集及保存标本的采集及保存：：
标本的采集及保存标本的采集及保存：：
1. 血清：全血标本请于室温放置2 小时或4℃过夜后于1000 x g 离心20 分
钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻
融。
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2. 血浆：可用EDTA 或肝素作为抗凝剂，标本采集后30 分钟内于2 - 8° C
1000 x g 离心15 分钟，或将标本放于-20℃或-80℃保存，但应避免反复冻
融。
3. 细胞培养物上清或其它生物标本：1000 x g 离心20 分钟，取上清即可检
测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。
注：注：标本溶血会影响最后检测结果标本溶血会影响最后检测结果，因此溶血标本不宜进行此项检测，因此溶血标本不宜进行此项检测。。
注注：：标本溶血会影响最后检测结果标本溶血会影响最后检测结果，，因此溶血标本不宜进行此项检测因此溶血标本不宜进行此项检测。。
标本的稀释原则标本的稀释原则：：
标本的稀释原则标本的稀释原则：：
首先通过文献检索的方式了解待测样本的大致含量，确定适当的稀释倍数。
只有稀释至标准曲线的范围内，检测的结果才是准确的。稀释的过程中，应
做好详细的记录。最后计算浓度时，稀释了“N”倍，标本的浓度应再乘以“N”。
标准品的稀释原则标准品的稀释原则：：
标准品的稀释原则标准品的稀释原则：：
2 瓶，每瓶临用前以样品稀释液稀释至1ml，盖好后静置10 分钟以上，然后
反复颠倒/搓动以助溶解，其浓度为50 ng/ml，做系列倍比稀释后，分别稀释
50 ng/ml，25 ng/ml，12.5 ng/ml，6.25 ng/ml，3.12 ng/ml，1.56 ng/ml，0.78 ng/ml，
样品稀释液直接作为标准浓度0 ng/ml，临用前15 分钟内配制。
如配制25 ng/ml 标准品：取0.5ml （不要少于0.5ml）50 ng/ml 的上述标准品
加入含0.5ml 样品稀释液的Eppendorf 管中，混匀即可，其余浓度以此类推。
生物素标记抗体的稀释原则生物素标记抗体的稀释原则：：
生物素标记抗体的稀释原则生物素标记抗体的稀释原则：：
临用前以生物素标记抗体稀释液稀释，稀释前根据预先计算好的每次实验所
需的总量配制（每孔100l），实际配制时应多配制0.1-0.2ml。如10l 生物素
标记抗体加990l 生物素标记抗体稀释液的比例配制，轻轻混匀，在使用前
一小时内配制。
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辣根过氧化物酶标记亲和素的稀释原则辣根过氧化物酶标记亲和素的稀释原则：：
辣根过氧化物酶标记亲和素的稀释原则辣根过氧化物酶标记亲和素的稀释原则：：
临用前以辣根过氧化物酶标记亲和素稀释液稀释，稀释前根据预先计算好的
每次实验所需的总量配制 （每孔100l），实际配制时应多配制0.1-0.2ml。如
10l 辣根过氧化物酶标记亲和素加 990l 辣根过氧化物酶标记亲和素稀释
液的比例配制，轻轻混匀，在使用前一小时内配制。
操作步骤操作步骤：：
操作步骤操作步骤：：
实验开始前，请提前配置好所有试剂，试剂或样品稀释时，均需混匀，混匀
时尽量避免起泡。每次检测都应该做标准曲线。如样品浓度过高时，用样品
稀释液进行稀释，以使样品符合试剂盒的检测范围。
1. 加样：分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100l，
余孔分别加标准品或待测样品100l，注意不要有气泡，加样将样品加于
酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀，酶标板加上盖或覆膜，
37℃反应120 分钟。
为保证实验结果有效性，每次实验请使用新的标准品溶液。
2. 弃去液体，甩干，不用洗涤。每孔加生物素标记抗体工作液 100l （取1l
生物素标记抗体加99l 生物素标记抗体稀释液的比例配制，轻轻混匀，
在使用前一小时内配制），37℃,60 分钟。
3. 温育60 分钟后，弃去孔内液体，甩干，洗板3 次，每次浸泡1-2 分钟，
200l/每孔，甩干。
4. 每孔加辣根过氧化物酶标记亲和素工作液 （同生物素标记抗体工作液）
100l，37℃，60 分钟。
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5. 温育60 分钟后，弃去孔内液体，甩干，洗板5 次，每次浸泡1-2 分钟，
200l/每孔，甩干。
6. 依序每孔加底物溶液90l，37℃避光显色 （（30 分钟内，此时肉眼可见标
（（
准品的前3-4 孔有明显的梯度蓝色，后3-4 孔梯度不明显，即可终止）。
7. 依序每孔加终止溶液50l，终止反应（此时蓝色立转黄色）。终止液的加
入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性，底
物反应时间到后应尽快加入终止液。
8. 用酶联仪在450nm 波长依序测量各孔的光密度（OD 值）。在加终止液后
15 分钟以内进行检测。
实验备注实验备注
实验备注实验备注
1. 用户在初次使用试剂盒时，应将各种试剂管离心数分钟，以便试剂集中到
管底。
2. 每次实验留一孔作为空白调零孔，该孔不加任何试剂，只是最后加底物溶
液及2N H SO4。测量时先用此孔调OD 值至零。
2
3. 为防止样品蒸发，试验时将反应板放于铺有湿布的密闭盒内，酶标板加上
盖或覆膜。
4. 未使用完的酶标板或者试剂，请于2-8℃保存。标准品、生物素标记抗体
工作液、辣根过氧化物酶标记亲和素工作液请依据所需的量配置使用。请
勿重复使用已稀释过的标准品、生物素标记抗体工作液、或辣根过氧化物
酶标记亲和素工作液。
5. 建议检测样品时均设双孔测定，以保证检测结果的准确性。
14
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洗板方法洗板方法：：
洗板方法洗板方法：：
手工洗板方法：吸去（不可触及板壁）或甩掉酶标板内的液体；在实验台上
铺垫几层吸水纸，酶标板朝下用力拍几次；将推荐的洗涤缓冲液至少0.3ml
注入孔内，浸泡1-2 分钟。根据需要，重复此过程数次。
自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中。
计算计算：：
计算计算：：
以标准物的浓度为横坐标（对数坐标），OD 值为纵坐标（普通坐标），在半对
数坐标纸上绘出标准曲线，根据样品的OD 值由标准曲线查出相应的浓度；
再乘以稀释倍数；或用标准物的浓度与OD 值计算出标准曲线的直线回归方
程式，将样品的OD 值代入方程式，计算出样品浓度，再乘以稀释倍数，即
为样品的实际浓度。
注意事项注意事项：：
注意事项注意事项：：
1. 当混合蛋白溶液时应尽量轻缓，避免起泡。
2. 洗涤过程非常重要，不充分的洗涤易造成假阳性。
3. 一次加样时间最好控制在5 分钟内，如标本数量多，推荐使用排枪加样。
4. 请每次测定的同时做标准曲线，最好做复孔。
5. 如标本中待测物质含量过高，请先稀释后再测定，计算时请最后乘以稀释
倍数。
6. 在配制标准品、检测溶液工作液时，请以相应的稀释液配制，不能混淆。
7. 底物请避光保存。
8. 不要用其它生产厂家的试剂替换试剂盒中的试剂。
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