Elisa实验报告6p
ELISA生化实验报告
ELISAObjective:1.To know about the basic operation and thoery of themeasurement of ELISA.2.To learn how to use microplate reader, how to dilute thesolution in certain ratio quickly.3.Understand the advantage of ELISA to measure theconcentration of protein.Theory:In the general procedure, the antigen was absorbed by the solid support, then the antibody we want to test was added in. Then the conjugated enzyme was added in to label the antibody. Then the substrate of the enzyme was added in and caused the formation of colored chemicals. Then the amount of antibody cloud be calculated by light absorption of spectrophotometer.In this experiment, we use Rabbit-anti-human IgG antiserum as first antibody. It can combine with Goat-antirabbit IgG-HRP, which can facilitate the oxidation of OPD. The colored product absorbs light at 492 nm. So the concentration of products can be measured by spectrophotometry. We can get a linear relation and cauculate the concentration.Reagens:1.Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) :2.Phosphate buffered saline (PBS)3.Washing buffer (PBS-T): PBS + 0.05% Tween 20.4.Blocking solution: 0.5% BSA in PBS.5.First antibody: Rabbit-anti-human IgG antiserum (1/400~1/12800).6.Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:40000).7.Substrate: substrate buffer(fresh):8.Stopping reagent: 2mol/L H2SO4.Equioment:1.microplate reader2.microtiter plate3.PipetteProcedure:Antigen Coating1. Prepare human IgG solution at 0.025mg/ml in carbonate-bicarbonate buffer (coating buffer).2. Pipette 0.2 ml of the above solution to each well of the 96-well microtiter plate from 2B-D to 10 B-D. Incubate at 37 ℃for 30-45 min.3. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well).4. Blocking step: pipette 0.02% (w/v) non-fat dry milk in-PBST (0.2ml/well) to each well of the 96-well microtiter plate from 2B-D to 11 B-D except 10 B-D. Keep the plate at Room Temperature for 5-10 min. Wash as mentioned above.B. Primary Antibody Reaction5. Dilute the primary (first) antibodies, Rabbit-anti-human IgG, in PBS-T. (different dilutions of 1st Ab., 1:400~1:12800).6. Add 0.2 ml of the diluted first antibody to each well according to Figure 1.Note: Add sample A (1st Ab. of unknown concentration) to well 8B-D. Negative control should be included (9B-D, No first Ab.).7. Incubate at 37 ℃for 1 hour.8. Wash three times with PBS-T (0.2 ml/well).C. Application of Secondary Antibody9. Dilute (1:40,000) the peroxidase conjugated Goat-anti-rabbit IgG-HRP (secondary antibody) in PBS-T. Add 0.2 ml of this solution to each well.10. Incubate at 37 ℃for 30 min.11. Wash three times with PBS-T (0.2 ml/well).D. Substrate Preparation12. During the last incubation and immediately before use, dissolve o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer; add 30% H 2 O 2 before use.E. Development13. Add 0.2 ml of the freshly prepared substrate to each well.14. Orange-yellow color should develop in positive wells after 3-5 minutes.15. Stop reaction with 50 μl per well of preferred stopping reagent and read at 490 nm in a microplate reader.The element of each well was shown in the table 1.Table 1Data:Table 2. A490 of the sampleNo. dilution of the sample 1 sample 2 sample 3 average first antibody1 1/400 1.252 1.307 1.433 1.3312 1/800 1.079 1.294 1.295 1.2233 1/1600 1.186 0.910 1.070 1.0554 1/3200 0.939 0.954 0.818 0.9045 1/6400 0.698 0.722 0.783 0.7346 1/12800 0.646 0.577 0.526 0.5837. 1/4000 0.801 0.730 0.788 0.7738 - 0.023 0.021 0.020 0.0219 1/400 1.123 1.350 1.177 1.21710 1/400 0.133 0.140 0.242 0.172 Then Drawing the curves of absorbency and the concentration of the series of diluted first antibody.The dilution of sample7 as calculate is 5560.DiscussionAs we can see in the result, when the concentration of the first antibody decrease, A will decrease, too. And the –lg(ratio) and A follow a linear relation approximately. The result can be explainedthat with equal amount of antigen and 2nd antibody in every well, (in exception of Group 9and10) the concentration of 1st antibody dominates the product yield in restricted time. The higher first antibody added into the well, the more second-antibody-peroxidase complex attached to the well. So we can get the higher concentration of colored product. And the aborsance changes as lambert-beer law.The result of No..9 is similar No.1 but lower than it, blocking will decrease the influence of the other irrelevant protein’s adsorption.The result of No.8 is so low,,nearly zero,because there is no first antibody in No.8, and No.10 is low because without the antigen, the adsoption of first antibody will be much lower.Easy to see, our curve is not a perfect linear relation. And the calculation of the No.7 is different from the theoric so much. There may be several reasons.1.When we pipette the stopping reagent, I spilled alittle of the first sample, the height of the liquid isnot absolutely equal with the others.2.The stopping method mayt be too late so thecolored product was generated too much, whichinfluences the linearity.3.The move of the microtiter plate leads to thebubble in the sulotion, which may influence theresult.Literature references:《ELISA》 《The practical approach of biochemistry》Bingbin Yu。
elisa实验报告结果分析
Elisa实验报告结果分析1. 引言Elisa（酶联免疫吸附实验）是一种常用于检测和定量分析生物样本中特定分子的方法。
本文旨在对实验结果进行分析，并根据分析结果得出结论。
2. 实验设计在本次实验中，我们选择了特定的生物分子作为目标，通过Elisa方法来检测样本中的该分子含量。
实验过程中，我们遵循了以下步骤： 1. 准备样本和试剂：收集样本并处理，准备所需的试剂。
2. 涂覆酶标板：将待测样本加入酶标板孔中，使样本中的目标分子与酶标板表面发生特异性结合。
3. 洗涤：去除未结合的物质。
4. 加入检测抗体：加入特异性抗体，与已结合的目标分子发生反应。
5. 加入标记物：加入标记物，用于检测目标分子的存在。
6. 洗涤：去除未结合的物质。
7. 反应底物：加入底物，观察颜色变化。
8. 停止反应：加入停止液停止反应。
9. 测量：使用酶标仪或光谱仪测量吸光度。
3. 结果分析根据实验结果，我们得到了一系列吸光度值。
下面我们将对吸光度值进行分析，并根据实验设计和已有知识得出结论。
3.1. 标准曲线分析实验中通常会使用一系列已知浓度的标准样品来构建标准曲线。
通过测量标准样品的吸光度值，我们可以绘制出标准曲线。
在实验中，我们可以使用标准曲线来确定未知样本中目标分子的浓度。
3.2. 样本浓度分析根据实验设计，我们在酶标板中加入了待测样本，并测量了其吸光度值。
通过标准曲线，我们可以将吸光度值转化为目标分子的浓度。
根据样本中目标分子的浓度，我们可以进行进一步的分析。
3.3. 结果验证为了验证实验结果的准确性和可靠性，我们可以进行重复实验或与其他方法进行比较。
此外，还可以进行质控实验来评估实验的可重复性和准确性。
4. 结论通过对Elisa实验结果的分析，我们得出以下结论： 1. 根据标准曲线分析，我们可以确定未知样本中目标分子的浓度。
2. 样本浓度分析结果显示，样本中目标分子的含量为X单位。
3. 实验结果经过验证，具有较高的准确性和可靠性。
elisa检测实验报告
elisa检测实验报告ELISA 检测实验报告一、实验目的本次 ELISA 检测实验的目的是定量检测样本中特定抗原或抗体的浓度，以评估实验对象的生理或病理状态。
二、实验原理ELISA（酶联免疫吸附测定）是一种基于抗原抗体特异性结合反应的免疫测定技术。
其基本原理是将已知的抗原或抗体固定在固相载体（如聚苯乙烯微孔板）表面，然后加入待检样本，样本中的待测抗原或抗体与固相载体上的抗原或抗体发生特异性结合。
接着，加入酶标记的第二抗体（或抗原），形成抗原抗体酶标记抗体复合物。
最后，加入底物，酶催化底物反应生成有色产物，通过测定有色产物的吸光度值，即可定量分析样本中待测抗原或抗体的浓度。
三、实验材料与设备1、试剂包被缓冲液（碳酸盐缓冲液，pH 96）封闭液（含 1% 5% 牛血清白蛋白的 PBS 缓冲液）洗涤液（含 005% Tween-20 的 PBS 缓冲液）样本稀释液（PBS 缓冲液）标准品（已知浓度的抗原或抗体）酶标记的二抗（辣根过氧化物酶或碱性磷酸酶标记）底物溶液（如 TMB 或 pNPP）终止液（如 2M 硫酸或 1M 氢氧化钠）2、仪器与设备酶标仪（能够读取 450nm 或 492nm 波长的吸光度值）恒温培养箱移液器（量程分别为20μL、200μL、1000μL）聚苯乙烯微孔板离心机四、实验步骤1、包被将已知浓度的抗原或抗体用包被缓冲液稀释至适当浓度。
向聚苯乙烯微孔板的每孔中加入100μL 稀释后的包被液，4℃过夜或 37℃孵育 2 3 小时。
2、封闭倒掉包被液，用洗涤液洗涤微孔板 3 次，每次浸泡 3 分钟，然后拍干。
每孔加入200μL 封闭液，37℃孵育 1 2 小时。
3、加样倒掉封闭液，用洗涤液洗涤微孔板 3 次，然后拍干。
将待检样本和标准品用样本稀释液进行梯度稀释。
向微孔板的每孔中分别加入100μL 稀释后的样本和标准品，空白对照孔加入100μL 样本稀释液。
37℃孵育 1 2 小时。
ELISA(酶联免疫吸附测定)实验报告
ELISALin Chengyu Bio 04 2010030007Experiment Date: 2012-03-12 Submitting Date: 2012-03-211Introduction1.1Background informationELISA (Enzyme-linked Immunosorbent Assay) is a solid-phase assay for antibodiesemploying ligands labeled with enzymes which is widely used for immunological assays.This technique can be applied to detect antigens or antibodies for qualitative orquantitative purpose. Since enzyme reactions are very well known amplificationprocesses, the signal is generated by enzymes which are linked to the detection reagentsin fixed proportions to allow accurate quantification.11.2Major principlesFigure 1 Schematic diagram of ELISA2Figure 2 Procedure of indirect ELISA3As shown in Figure 1 & 2, the general procedure of indirect ELSIA is to: incubate theplate well with antigen, wash off unbounded antigen, incubate with 1st antibody, washoff unbounded 1st antibody, incubate with labeled 2nd antibody, wash off unbounded 2ndantibody, incubate with enzyme substrate solution, and detect optical density or otherindex showing enzyme activity.2Experiment Operation2.1Antigen coating(1)Prepare an antigen solution in coating buffer (human IgG at 0.025mg/ml);(2)Pipette 200 μl antigen solution to each well (Row: B~G; Column: 2~10; Column 11is negative control without antigen) of the microtiter plate;(3)Incubate the plate at 37 ℃for 30 min;(4)Remove the antigen solution;(5)Wash each well with 200 μl with PBS-T for 3 times;(6)Block each well (Row: B~G; Column: 2~11) with 200 μl 0.5% BSA-PBS, andincubate the plate at 37 ℃for 30 min;(7)Remove the blocking solution;(8)Wash each well with 200 μl with PBS-T for 3 times.2.2Primary antibody reaction(1)Dilute the primary antibody (rabbit-anti-human IgG antiserum) in PBS-T fordifferent dilution (from 1:400 to 1:51,200 in 2-folds dilution);(2)Add 200 μl diluted antibody solution to each well following Table 1;Table 1 Scheme to add primary antibody(3)Incubate the plate at 37 ℃for 1 hour;(4)Remove the primary antibody solution;(5)Wash each well with 200 μl PBS-T for 3 times.2.3Application of secondary antibody(1)Dilute the peroxidase conjugated secondary antibody (Goat-anti-rabbit IgG-HRP) inPBS-T at the dilution of 1:20,000 and 1:40,000;(2)Add 200 μl secondary antibody solution to each well following Table 2;(3)Incubate the plate at 37 ℃for 1 hour;(4)Remove the secondary antibody solution;(5)Wash each well with 200 μl PBS-T for 3 times.2.4Substrate development(1)Add 200 μl substrate solution to each well (Row: B~G ,Column: 2~11);(2)Incubate for approximately 3 min;(3)Add 50 μl 2 M H2SO4 to each well to terminate the reaction;(4)Measure optical density at 490 nm.3Raw data and its processing3.1Raw data3.2Data processingSet Row B, C, and D as Group I, and Row E, F, and G as Group II. The processed datais shown in Table 4Table 4 Processed data: optical density of each groupSet different dilutions of primary antibody as x axis, optical density as y axis, drawFigure 3 to illustrate their relation.Figure 3 Relationship between optical density and dilutions of primary antibodyFor the reason that the curve cannot illustrate the relationship enough, change the x axis to nature logarithm of different dilutions of primary antibody. See Figure 4:Figure 4 Relationship between optical density and natural logarithm of dilutions of primary antibodyUsing linear fit for each group, we can figure out that two lines are approximately parallel.In the black curve in Figure 3, there is an oblivious point of inflection which corresponds with the dilution of 1:800. The curve after this point becomes flat, which indicates that the binding between antigens and primary antibodies is saturated in the dilution of 1:800 and higher. This data can suggest that in other immunoenzymatic experiment, the proper dilution of primary antibody will be around, and no higher than 1:800.What’s more, from the red line in Figure 4 we can figure out that the optical density hasa linear relation with natural logarithm of dilutions of the primary antibody.As for comparison between Group I and Group II, from Figure 3 we can figure out thatthe point of infection of blue curve, which corresponds with the dilution of 1:40,000, ison the left, about 1:1600.In Figure 4, the green line (1:40,000) is positioned lower than the red line (1:20,000),which is easy to understand. Lower concentration of secondary antibody means lessbinding with primary antibody during application of secondary antibody.4Results and discussion4.1Results(1)The optical density has an approximately linear relation with the natural logarithmof the dilutions of the primary antibody;(2)For secondary antibody in the dilution of 1:20,000, the proper dilution of primaryantibody is 1:800; for secondary antibody in the dilution of 1:40,000, primaryantibody is recommended to be 1:1600;(3)With the same dilution of antigen and primary antibody, higher concentration ofsecondary antibody will get a higher optical density;4.2Discussion(1)What is the significance of the negative control groups?I.The no primary antibody groups proved that there is no specific bindingbetween antigen and secondary antibody, and provided a background ofnon-specific binding between secondary antibody and antigen;II.The no antigen groups can provide a background of non-specific binding between primary antibody and BSA.(2)Why washing step is essential?Washing each well with PBS-T, which contains tween-20 as detergent, can wash offunbounded antigens and antibodies, including those non-specifically binding. Ifwashing step is omitted, the background index will be higher, and might causeinterference to the result.(3)Why blocking step is essential?After the antigen coating step, the surface of the well is not covered by antigenentirely, i.e. there is still some site leaving blank, which allows other proteins bindto them. Blocking step is to block those blank sites with non-specific bindingmaterial that will not cause interference to the experiment. Thus, the primaryantibody will only bind to the antigen coated in the first step, rather than coat on thesurface as well.(4)What’s the advantage of indirect ELISA comparing with direct ELISA?I.Indirect ELISA can amplify the optical density which we measure.Compared to direct ELISA, the number of secondary antibody binding tothe primary antibody is way larger than the number of primary antibodybinding to the antigen. Thus, optical density will be higher and easier tomeasure, which means a lower error;II.The secondary antibody contains HRP, which is essential for substrate development. Compared to direct ELISA, indirect ELISA need only onekind of antibody contains HRP to perform many kinds of experiment, ratherthan one antibody linked to enzyme for one experiment, which isinconvenient.5Reference【1】/wiki/ELISA【2】/post/9314400054【3】/indirect_elisa。
elisa技术实验报告
elisa技术实验报告实验目的：本实验旨在通过酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）技术，检测特定抗原或抗体的存在，以评估ELISA技术在生物医学研究和临床诊断中的应用。
实验原理：ELISA技术是一种基于抗原-抗体特异性结合的免疫学检测方法。
通过将抗原或抗体固定在固相载体上，利用酶标记的二抗体与待测物结合，通过酶催化底物产生可检测的信号，从而定量或定性分析目标分子。
实验材料：1. 96孔ELISA板2. 待测样本3. 特异性一抗4. 酶标记的二抗5. 底物溶液6. 终止液7. 洗涤缓冲液8. 标准品或阳性对照9. 酶标仪实验步骤：1. 准备96孔ELISA板，将标准品或阳性对照按照不同浓度稀释后加入板中，同时加入待测样本。
2. 将特异性一抗加入每个孔中，室温下孵育1小时。
3. 用洗涤缓冲液洗涤ELISA板，去除未结合的一抗。
4. 加入酶标记的二抗，室温下孵育1小时。
5. 再次用洗涤缓冲液洗涤板子。
6. 加入底物溶液，根据实验需要设定孵育时间。
7. 加入终止液，终止酶反应。
8. 使用酶标仪在特定波长下测定吸光度（OD值）。
实验结果：实验结果显示，随着标准品浓度的增加，OD值呈线性增加，表明ELISA实验具有较好的灵敏度和特异性。
待测样本的OD值与标准曲线进行比较，可以计算出待测物的浓度。
实验讨论：本次实验中，ELISA技术成功地检测了目标分子的存在，验证了其在生物医学研究中的实用性。
然而，实验中也存在一些可能影响结果的因素，如样本的稀释倍数、孵育时间和洗涤次数等。
在未来的实验中，需要进一步优化实验条件，以提高检测的准确性和重复性。
实验结论：通过本次ELISA实验，我们成功地检测了特定抗原或抗体的存在，证明了ELISA技术在生物医学研究和临床诊断中的有效性。
未来，我们将继续探索和优化ELISA技术，以满足更广泛的应用需求。
参考文献：[1] Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871-874.[2] Voller A, Bartlett A, Bidwell D. Enzyme immunoassays with special reference to ELISA techniques. Journal of General Virology. 1976;33(2):165-169.请注意，本实验报告是一个示例，实际实验应根据具体的实验设计和结果进行编写。
ELISA实验报告
ELISA实验报告实验目的：本实验旨在通过ELISA（酶联免疫吸附试验）技术来检测细胞培养上清液中的蛋白质含量，从而了解该细胞株的蛋白质表达水平，进一步研究相关的细胞分子机制。
实验原理：酶联免疫吸附试验是一种常用的免疫学实验方法，通过特异性抗体与待测物结合来实现对特定蛋白质的检测。
该实验主要分为三个步骤：包被抗原、特异性抗体结合和酶底物显色。
实验材料：1.待测细胞上清液2.包被抗原3.特异性抗体4.酶标记的二抗5.酶底物6.缓冲液7.清洗缓冲液8.吸光度测定仪实验步骤：1.包被抗原的处理：将抗原溶液加入微孔板孔中，使其均匀附着在孔壁上。
然后将孔中的液体弃去，用清洗缓冲液进行孔的清洗。
2.探针的结合：将待测样品加入已经包被抗原的孔中，使其与抗原结合。
然后将孔中的液体弃去，用清洗缓冲液进行孔的清洗。
3.酶标记的二抗结合：将酶标记的二抗加入已经含有特异性抗体的孔中，使其与特异性抗体结合。
然后将孔中的液体弃去，用清洗缓冲液进行孔的清洗。
4.酶底物加入：将酶底物加入到孔中，使其在酶的作用下发生显色反应。
5.吸光度测定：使用吸光度测定仪读取吸光度值，根据吸光度值可以推断待测样品中蛋白质的含量。
实验结果：经过ELISA实验，我们得到了待测细胞上清液中蛋白质的含量。
根据吸光度值和标准曲线的对照，我们可以计算出待测样品中蛋白质的浓度。
实验结论：实验分析：本次实验利用ELISA技术成功检测了待测细胞上清液中的蛋白质含量。
该方法具有高灵敏度、高特异性、操作简单等优点，可以在生物医学、生物工程等领域广泛应用。
但需要注意的是，ELISA实验在操作过程中需要严格控制实验条件，避免交叉污染和误差的产生。
实验改进：为了进一步提高实验的准确性和可靠性，可以进行以下改进：1.增加重复次数，提高数据的可靠性和稳定性；2.使用更加准确的仪器和试剂；3.对实验流程进行优化，减少操作的差异性。
总结：本次实验通过ELISA技术成功检测了待测细胞上清液中的蛋白质含量，得出了蛋白质表达水平较高的结论。
elisa实验报告
竭诚为您提供优质文档/双击可除elisa实验报告篇一：eLIsA实验报告experiment:eLIsA(enzyme-linkedImmunosorbentAssay) Date:20XX.10.17objectives(1)LearneLIsAprinciple(2)mastereLIsAtechnology,quantitativelydetermineant ibodyandantigen.experimentalprincipleTheprincipleofeLIsAistomakeantigenorantibodycombine tothesurfaceofsomesolidphasecarrierandmaintainitsim munocompetence,andthenmaketheantibodyorantigentoconnectwithsomeenzymetobecomeenzymelabeledantigenorant ibodyinwhichtheimmunocompetenceofbothantigenorantibodyandenzymeisk ept.Inthedetermination,makethespecimentobetestedand enzymelinkedantigenreactwiththeantigenorantibodyont hesurfaceofsolidphasecarrierfollowingdifferenceproc edures.Throughwashing,antigen-antibodycomplexformed onsolidphasecarrierandothermaterialsareseparated.Fi nally,addingsubstrateintoenzymereaction,thesubstrat ewillbecatalyzedandbecomecoloredproduct.Theamountof producthasdirectrelationwiththeamountofenzymeonthes olidphasecarrier,inotherwords,theamountofthemateria ltobetestedinthespecimen.Quantitativeanalysiscanbec onductedbasedontheshadeofcolorreaction.Thisdetermin ationhasaveryhighsensitivity.eLIsAcanbedividedintomainlyfourtypes:thedirectmetho d,indirectmethod,doubleantibodysandwichmethodandcom petitiveinhibitionmethod.Also,differentkindsofenzym eandsubstratecanbeusedineLIsA.DoubleantibodymethodisakindofeLIsAtodetermineantige ninthespecimen.Inthismethod,antibodyiscoatedonthesu rfaceofsolidphasecarrier.Antigeninthesamplebindswit hantibodyonthesolidphase,whileothermaterialsarewash edaway.Thenaddenzymelinkedantibodytobindtheantigenw hichareboundonthesolidphase.Afterwashing,superfluou santibodyiswashedaway.Thesubstrateisthenaddedtoprod ucecolorandthequantitativetestsareconducted.Inourexperiment,weadopteddoubleantibodysandwichmeth odtodeterminemousetumornecrosisfactoralpha(TnFα)withanti-mouseTnFα.TheenzymeandsubstrateweusedareAvidin-hRpandTmbre spectively.Reagentsandequipment(1)equipments40-wellreactionplatecoatedwithanti-mouseTnF α;liquidtransfergun(1ml,200μl);thermotank(37℃).(2)Reagentswashingliquid(pbsT);sealingfluid;antigen(1000pg/ml);anti-mouseTnFα;Tmb;Avidin-hRp;1mh2so4.methodofoperation(1)emptyoutthesolutionintheplateandcleantheplatewit hwashingliquidforthreetimes.Topupeachholeandflapona bsorbentpapertocleanawaytheremainingliquidineachhol e.Add200μlsealingfluidineachholeandputtheplatein37℃for30mins.(2)washtheplatefor3times.Addreagentsin8holesaccordi ngtothetable:(numberrefertothe(3)washtheplatefor3times,add100μlantibodyineachhole,puttheplatein37℃for30mins.(4)washtheplatefor3times,add100μlenzymeineachhole,puttheplatein37℃for20mins.(5)washtheplatefor5times,add100μlsubstrateineachhole,puttheplatein37℃for5mins.observethecolorationandtakeapicture.(6)Add50μl1mh2so4ineachhole,observethecolorchangeandtakeapic ture.notes:(1)Allreagentsshallbekeptindarkplaceat2-8℃.(2)Liquidfeedingshallbelimitedatthebottomofholesins teadofwall,withoutspillage.(3)whencleaningtheplatemanually,donotspillouttheliq uidintheholesincasethattheadjacentholespolluteeachothertocausefalsepositive.(4)Theresultsareonlyvalidwithin30minsafterthetermin ationoftest.(5)experimentwastesshallbetreatedasinfectioussample s.Discussion(1)Fromtheresult,wecanseethegradientshadeofcolorati on,whichmeanstheshadeofcolorationisinproportiontoth econcentrateofantigen.Theexactshadeofcolorationcanb edetectedbyspectrophotometer,andbecomparedwiththest andardcurvetogettheconcentratevalue.(2)everytimewhencleaningtheplate,thewashingliquidmu stbecompletelyremoved.Questions(1)Analyzetheeffectofimpureenzymelabeledantibodytot hetestingresultofantigen.(DoubleAntibodysandwichmet hod)Iftheimpuritycannotbebondwithantigen,ithasnoaffectt otheresult;iftheimpuritycanbebondwithantigen,itwill causetheresultlowerthantherealconcentrate.(2)comparethemostsuitabletestingobjectsofIFAandeLIsA.IFAisusedinvivotolocateacertainantigen;whileeLIsAis usedinvitroconditiontodeterminethepresenceandconcen trateofacertainantigenorantibody.篇二：eLIsA英文版实验报告Test1.productionofantibody[principle]1.Inthespecialhumoralimmuneresponse,antigencanstimu lateimmunesystemtoproducespecificantibodies.eachant igenmoleculehasseveraldifferentantigenicdeterminant s,soitcanberecognizedbydifferentbcellsandthentheseb cellswillproducedifferentspecificantibodiesthatcanb indwithepitopesspecifically.Theimmunityserumproduce dbyusingantigentoimmuneanimalisamixtureofmanydiffer entantibodies,calledpolyclonalantibody.Themostcommo nmethodtoproducepolyclonalantibodyisusingpureantige ntoimmuneanimal.2.Thefactorsinfluencingproducingofantibodiesisrelat edtoantigenpurity,animal,amountofantigen,waysofinje ction,timesofimmunity,etc.primaryimmuneanimal,theantigenenteranimalforthefirs ttime,thebodywillproduceasmallamountantibodiesafter alatency,butwhensecondinjectionantigentotheanimal,t herewillbeafastresponsetotheantigenandalotantibodie swillbeproduced.sointhistest,useantigentoimmunemice 3times,thenwecandetectantibodiesintheserum.。
ELISA实验报告
ELISA实验报告一、实验目的ELISA（酶联免疫吸附测定）是一种广泛应用于生物医学研究和临床诊断的免疫学技术。
本次实验的目的是通过 ELISA 方法检测样本中特定抗原或抗体的含量，熟悉 ELISA 的实验原理、操作步骤，并对实验结果进行分析和解读。
二、实验原理ELISA 的基本原理是将已知的抗原或抗体固定在固相载体（如聚苯乙烯微量反应板）表面，然后加入待检样本，使其中的抗原或抗体与固相载体上的抗原或抗体发生特异性结合。
洗去未结合的物质后，再加入酶标记的第二抗体或抗原，形成抗原抗体酶标抗体复合物。
最后加入底物，通过酶催化底物显色，根据颜色的深浅来定量测定样本中抗原或抗体的含量。
三、实验材料1、试剂包被缓冲液（pH 96 的碳酸盐缓冲液）洗涤缓冲液（含 005% Tween-20 的 PBS 缓冲液）封闭液（含 1% BSA 的 PBS 缓冲液）样本稀释液（PBS 缓冲液）酶标抗体工作液底物溶液（TMB 溶液）终止液（2 M 硫酸溶液）2、仪器酶标仪移液器恒温箱微量反应板3、样本待检血清样本四、实验步骤1、包被用包被缓冲液将抗原稀释至适当浓度，每孔加入100 μL，4℃过夜包被。
2、洗涤次日，弃去孔内液体，用洗涤缓冲液洗涤 3 次，每次 3 分钟，拍干。
3、封闭每孔加入200 μL 封闭液，37℃孵育 1 小时，洗涤 3 次。
4、加样将待检血清样本用样本稀释液进行梯度稀释，每孔加入100 μL，同时设置阴性对照和阳性对照，37℃孵育 1 小时，洗涤 3 次。
5、加酶标抗体每孔加入100 μL 酶标抗体工作液，37℃孵育 1 小时，洗涤 5 次。
6、显色每孔加入100 μL 底物溶液，37℃避光显色 15 分钟。
7、终止每孔加入50 μL 终止液，终止反应。
8、测定使用酶标仪在 450 nm 波长处测定各孔的吸光度值（OD 值）。
五、实验结果1、原始数据记录各孔的 OD 值，如下表所示：｜样本|OD 值|｜｜｜｜阴性对照|0085|｜阳性对照|1258|｜待检样本 1|0325|｜待检样本 2|0158|｜待检样本 3|0685|2、结果判断计算阴性对照和阳性对照的平均 OD 值，分别记为 OD 阴平和 OD阳平。
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1.333
1.317
1.131
1.186
1.084
0.854
0.682
0.419
0.285
0.175
P.S:紫色数据表示异常
实验讨论：
1无一抗（10）数据值存在异常。理论上应趋近于零。可能是由于受污染所致
实验现象比较清晰，随浓度由浓到稀，颜色由深黄渐变为浅黄。
2线性关系比较明显。但斜率随浓度减小而有所增大。这可能是在使用取液器时有气泡混入，导致实际浓度低于理论上的浓度。
B
1.408
1.240
1.211
1.228
1.114
0.810
0.698
0.519
0.374
0.094
C
1.098
1.357
0.943
0.996
1.019
0.895
0.682
0.415
0.201
0.067
D
1.494
1.354
1.240
1.334
1.118
0.859
0.667
0.472
0.281
0.083
３．加封闭液200μl，37℃温育30min。
４．洗涤同2。
５．加被检血清：用稀释液将被检血清作几种稀释，每孔200μl。同时作稀释液对照。37℃温育30min。
６．洗涤同2。
７．加辣根过氧化物酶羊抗兔IgG,每孔200μl, 37℃温育30min。
８．洗涤同2。
９．加底物：邻苯二胺溶液加200μl，室温暗处5min。
加蒸馏水12.5ml,
取邻苯二胺8mg（溶解）；
临用前加30%（体积分数）H2O240μl。
9．终止液：2mol/LH2SO4。
实验步骤：
１．包被抗原：用包被液将抗原作适当稀释,一般为1~10μg/孔，每孔加200μl, 37℃温育30min。
２．洗涤：倒尽板孔中液体，加200μl洗涤液，反复三次，最后将反应板倒置在吸水纸上，使孔中洗涤液流尽。
KH2PO40.2g,
Na2HPO4·12H2O2.9g,
Tween-20，0.5ml，
蒸馏水加至1000ml。
6．洗涤液：同稀释液。
7．封闭液：0.5%（质量分数）BSA（用PBS配制）。
8．邻苯二胺溶液(底物)：
配制：
0.1mol/L柠檬酸(2.1g/100ml),取6.1ml，
0.2mol/LNa2HPO4·12H2O(7.163g/100ml),取6.4ml,
实验材料与试剂：
1．聚苯乙烯微量细胞培养板(平板, 96孔)。
2．酶联免疫检测仪。
3．辣根过氧化物酶羊抗兔IgG。
4．包被液：0.05mol/L碳酸缓冲液（pH9.6）：
Na2CO30.15g,
NaHCO30.293g，
蒸馏水稀释至100ml。
5．稀释液（PBS-Tween）：
NaCl8g,
KCl 0.2g，
另外由于操作不熟练、不仔细，也带来了一些粗大误差，如无一抗（10）。这是我们实验中应该避免的。吸附剂测定
实验原理：
酶联免疫吸附测定是一种免疫测定技术。测定中，先使抗原吸附在固相载体上，然后加待测的抗体，再用某种酶标记抗体，形成抗原-抗体-酶标记抗体的“双抗体夹心”，此时酶仍保有活性，同时标记抗体亦有免疫活性。之后再加入酶的底物，在酶的催化下产生反应并产生有色物，颜色深浅与待测物质的量直接相关。至此，酶的催化放大作用与免疫反应的特异性相完美结合，提高了测定的准确性与灵敏度。
3组号（2）（4）（5）中3个样本数据差别较大，可能是因为配血清时混合不够均匀所致。
这个实验采用了间接法测定，利用酶的催化放大作用提高了检测的灵敏度。作为一名精仪系的学生，这给予我的启示就是，在直接测量精度如果遇到瓶颈时，没有合适的方法提高精度，那么可以考虑寻找一个“中介”，而这个“中介”应该具有“在变量产生微小变化时能放大这种变化，或者用另一种形式体现这种变化”的特性，从而测量变得可行。而该实验中，最终效果就是我们可以直接通过颜色深浅大致看出浓度的不同。这一点很值得思考。
１０．加终止液：每孔50μl。
１１．观察结果：用酶联免疫检测仪记录OD490nm读数。
实验结果
数据如下：
血清稀释倍数
样本号(组号)
1/400
(2)
1/800
(3)
1/1600
(4)
1/3200
(5)
1/6400
(6)
1/12800
(7)
1/25600
(8)
1/51200
(9)
无1抗
(10)
无抗原
(11)