ELISA 분석: 간접, 샌드위치 및 길항
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출처: 휘트니 스완슨1,2,프랜시스 V. 자스타드2,3,토마스 에스 그리피스1,2,3,4
1 미네소타 대학교 비뇨기과, 미니애폴리스, MN 55455
2 면역학 센터, 미네소타 대학, 미니애폴리스, MN 55455
3 미생물학, 면역학 및 암 생물학 대학원 프로그램, 미네소타 대학, 미니애폴리스, MN 55455
4 Masonic 암 센터, 미네소타 대학, 미니애폴리스, MN 55455
효소-연결된 면역흡소생식분석(ELISA)은 생물학적 샘플에서 항원, 항체, 펩타이드, 단백질, 호르몬 또는 기타 생체 분자의 존재 및/또는 농도를 측정하는 데 자주 사용된다. 그것은 매우 민감한, 낮은 항 원 농도 감지 할 수. ELISA의 감도는 단일 항원 항체 복합체(1)간의 상호 작용을 검출하는 능력에 기인한다. 더욱이, 효소-공주 항원 특이적 항체의 포함은 무색 기판의 변환을 플레이트 판독기에 의해 검출되고 쉽게 양수할 수 있는 염색체 또는 형광 제품으로 변환할 수 있게 한다. 알려진 항원의 적정량에 의해 생성된 값과 비교하면, 실험 샘플에서 동일한 항원의 농도를 결정할 수 있다. 다른 ELISA 프로토콜은 다양한 실험 샘플에서 항원 농도를 측정하도록 조정되었지만 모두 동일한 기본 개념(2)을 가지고 있습니다. 수행, 간접, 샌드위치, 또는 경쟁력을 수행하기 위해 ELISA의 유형을 선택하는 것은 테스트 할 샘플의 복잡성과 사용할 수있는 항원 특정 항체를 포함하여 여러 가지 요인에 달려 있습니다. 간접 ELISA는 시료에서 항체의 농도를 측정하는 것과 같은 면역 학적 반응의 결과를 결정하는 데 자주 사용됩니다. 샌드위치 ELISA는 분석물 또는 관심있는 항원이 혼합 샘플의 일부인 조직 배양 초월제 또는 조직 용액과 같은 복잡한 샘플을 분석하는 데 가장 적합합니다. 마지막으로, 경쟁적인 ELISA는 관심 있는 항원 검출을 위해 사용할 수 있는 항체가 하나일 때 가장 자주 사용된다. 경쟁적 ELISA는 또한 스테릭 방해로 인해 두 개의 다른 항체를 수용할 수 없는 단일 항체 에피토프만으로 작은 항원검출에 유용하다. 이 프로토콜은 간접, 샌드위치 및 경쟁력있는 ELISA 분석의 기본 절차를 설명합니다.
간접 ELISA 분석체는 일반적으로 혈청 또는 혼종 배양의 상부에서 항체의 양을 측정하는 데 사용됩니다. 간접 ELISA 분석법에 대한 일반적인 절차는 다음과 같이 합니다.
- 항원으로 우물을 코팅
- 항체를 포함하는 혈청 또는 hybridoma 배양 상피제 추가 (1 차 또는 1° 항체)
- 인큐베이션 및 세척
- 이차(또는 2°) 효소 컨쥬게이드 항체 추가
- 인큐베이션 및 세척
- 기판 추가
샌드위치 ELISA 분석법은 정제된 항원으로 플레이트를 코팅하는 것을 포함하지 않는다는 점에서 간접 ELISA 분석과 다릅니다. 대신, “캡처”항체는 플레이트의 우물을 코팅하는 데 사용됩니다. 항원은 포획 항체와 두 번째 “검출” 효소-컨쥬게이트 항체 사이에 “끼어들”이다- 두 항체가 동일한 항원에 특이적이지만, 상이한 에피토프(3)에서. 포획 항체/항원 복합체에 결합함으로써 검출 항체는 플레이트 내에 남아 있다. 단일 클론 항체 또는 폴리클론 항세라는 포획 및 검출 항체로서 사용될 수 있다. 샌드위치 ELISA의 주요 장점은 샘플이 분석하기 전에 정제 될 필요가 없다는 것입니다. 또한, 분석은 매우 민감 할 수있다 (4). 많은 시판되는 ELISA 키트는 샌드위치 품종이며 테스트된 테스트된 항체 쌍을 사용합니다. 샌드위치 ELISA 분석의 일반적인 절차는 다음과 같이 합니다.
- 포획 항체가 있는 코트 웰
- 항원 함유 테스트 샘플 추가
- 인큐베이션 및 세척
- 효소-컨쥬게이드 검출 항체를 추가합니다.
- 인큐베이션 및 세척
- 기판 추가
대부분의 시판 가능한 샌드위치 ELISA 키트는 효소-컨쥬게이트 검출 항체와 함께 제공됩니다. 효소-컨쥬게이트 검출 항체가 제공되지 않는 경우, 검출 항체에 특이적인 이차 효소-컨쥬게이트 항체를 사용할 수 있다. 이차 항체의 효소는 무색 기판을 염색체 또는 형광 제품으로 변환하는 동일한 역할을 수행한다. 상기 언급된 이차 효소-컨쥬게이드 항체는 예를 들어, 자신의 단일클론 항체를 생성한 조사관에 의해 개발된 “수제” 샌드위치 ELISA에 더 많이 사용되고 싶습니다. 이차 효소-컨쥬게이드 항체를 사용하는 한 가지 단점은 검출 항체에만 결합하고, 플레이트에 결합된 항체를 포획하지 않도록 하는 것이다. 이것은 항원 또는 검출 항체의 존재 또는 부재에 관계없이 모든 우물에서 측정 가능한 제품을 초래할 것입니다.
마지막으로, 경쟁적인 ELISA 분석은 수용성 항원검출을 위해 이용됩니다. 수행이 간단하지만 정제 된 항원이 비교적 많은 양으로 사용할 때만 적합합니다. 경쟁력 있는 ELISA 분석법에 대한 일반적인 절차는 다음과 같이 합니다.
- 항원으로 코트 우물
- 인큐베이션 및 세척
- 효소 접합 1차 항체를 갖춘 프리인큐베이트 테스트 샘플
- 잘 혼합물을 추가
- 무한한 효소를 공주한 1차 항체를 배양하고 씻어내다
- 기판 추가
이 분석에서 “경쟁”은 3단계에서 사용되는 시험 샘플에서 더 많은 항원들이 항원을 코팅하여 결합할 수 있는 항체를 적게 초래할 것이라는 사실에서 비롯된다. 따라서, 분석의 끝에 잘 있는 염색체/불소생성 생성물의 강도는 시험 샘플에 존재하는 항원의 양과 반비례한다.
ELISA의 모든 유형의 핵심 성분은 사용자가 시험 샘플에 존재하는 항원 농도를 결정할 수 있도록 알려진 농도의 적정 기준이다. 전형적으로, 일련의 우물은 표준 곡선을 만들기 위해 지정되며, 정제된 재조합 단백질의 알려진 양이 양을 감소시키는 우물에 첨가됩니다. 이러한 우물이 시험 샘플과 동시에 처리될 때, 사용자는 시험 샘플에 대한 흡광도 값과 함께 이동하는 알려진 단백질 농도에 대한 마이크로 플레이트 판독기로부터 얻어진 흡광도 값의 기준 세트를 가질 수 있다. 그런 다음 사용자는 현재 관심 있는 단백질의 양을 결정하기 위해 테스트 샘플을 비교할 수 있는 표준 곡선을 계산할 수 있습니다. 표준 곡선은 또한 사용자의 희석 제조의 정밀도 정도를 결정할 수 있습니다.
마지막으로 위에 나열된 각 ELISA 유형의 마지막 단계는 기판의 추가를 요구합니다. 제품에 기판의 변환 정도는 우물에 존재하는 효소의 양과 직접적으로 관련이 있다. 고추성 과산화구체 (HRP) 및 알칼리인포스파아제 (AP)는 항체에 공주된 가장 흔한 효소이다. 예상대로, 염색체 또는 형광 제품을 생성하는 효소에 대해 특정 한 많은 기판이 있습니다. 더욱이, 기판은 분석의 전반적인 감도를 증가시킬 수 있는 감도의 범위에서 유효하다. 사용자는 또한 해당 효소-컨쥬게이트 항체와 함께 사용할 기판 유형을 선택할 때 실험이 끝날 때 플레이트를 읽을 수 있는 계측의 유형을 고려해야 합니다.
HRP에 일반적으로 사용되는 염색체 기판에는 2,2′-Azinobis [3-ethylbenzothiazoline-6-설포닉 산]-디아MM모늄 염(ABTS) 및 3,3′,5′-테트라메틸벤지딘(TMB), p-니트로펜일 포산염(PMB)이 포함됩니다. ABTS와 TMB는 각각 수용성 녹색 및 파란색 색 반응 제품을 생산합니다. 녹색 ABTS 제품에는 두 개의 주요 흡광도 봉우리, 410 및 650 nm가 있으며 파란색 TMB 제품은 370 및 652 nm에서 가장 잘 감지됩니다. 450 nm에서 가장 잘 읽는 산성 정지 용액을 추가하면 ABTS 및 TMB의 색상이 노란색으로 변경됩니다. ABTS의 색상 개발은 느리지만 TMB의 경우 빠릅니다. TMB는 ABTS보다 더 민감하며 효소 반응이 너무 오래 진행되면 더 높은 배경 신호를 생성할 수 있습니다. PNPP는 405 nm에서 빛을 흡수하는 AP 변환 후 노란색 수용성 제품을 생산합니다.
Procedure
Results
In the following example of an indirect ELISA, the presence of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice was determined. C57Bl/6 mice were infected with influenza A virus (A/PR/8; 105 PFU in 100 µL PBS i.p.) and serum was collected 28 days later. To quantitate the amount of IAV-specific IgG in the serum, 96-well ELISA plates were coated with purified A/PR/8 Influenza A virus (50 µL/well of 2 mg/ml PBS virus) overnight at 4°C. Coated plates were blocked for 1 hour at room temperature with 5% normal donkey serum in PBS, followed by incubation with diluted serum samples from IAV-challenged mice overnight at 4°C. The serum was initially diluted 1:12.5, followed by 1:4 dilutions (dilution range – 1:12.5 to 1:204,800). After washing, plates were incubated with an alkaline phosphatase (AP)-conjugated donkey anti-mouse IgG for 1 h. The plates were washed, and then p-Nitrophenyl Phosphate (PNPP; 1 mg/mL, 100 µL/well) was added. The colorless PNPP solution turns to a yellow color when AP is present. After 5-10 min, the enzymatic reaction was stopped by adding 100 µL/well 2N H2SO4. The plate was read on a microplate reader at 405 nm. The results obtained are shown in Table 1 and Figure 1.
| Sample | Wells | OD405 | Mean |
| Serum 1:12.5 | A1 | 2.163 | 2.194 |
| B1 | 2.214 | ||
| C1 | 2.204 | ||
| Serum 1:50 | A1 | 1.712 | 1.894 |
| B1 | 2.345 | ||
| C1 | 1.624 | ||
| Serum 1:200 | A1 | 1.437 | 1.541 |
| B1 | 1.73 | ||
| C1 | 1.456 | ||
| Serum 1:800 | A1 | 1.036 | 0.957 |
| B1 | 0.912 | ||
| C1 | 0.923 | ||
| Serum 1:3200 | A1 | 0.579 | 0.48 |
| B1 | 0.431 | ||
| C1 | 0.429 | ||
| Serum 1:12800 | A1 | 0.296 | 0.281 |
| B1 | 0.312 | ||
| C1 | 0.236 | ||
| Serum 1:51200 | A1 | 0.308 | 0.283 |
| B1 | 0.299 | ||
| C1 | 0.243 | ||
| Serum 1:204800 | A1 | 0.315 | 0.303 |
| B1 | 0.298 | ||
| C1 | 0.297 |
Table 1: Indirect ELISA assay data. Serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-infected mice containing IAV-specific IgG, optical density (OD) (405 nm) values and mean OD405 values.
Figure 1: Indirect ELISA assay scatter plot of mean OD405 values(+ S. D.) and serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice. The OD405 values can be inversely correlated to the serum dilutions.
In the following example of a sandwich ELISA, a 1:2.5 dilution of recombinant human TNFα standards (starting at a concentration of 75 pg/mL) was added to the indicated wells of a 96-well flat-bottom plate. These standards led to a corresponding 2.5-fold change in the absorbance readings.
| Sample | Concentration (pg/mL) | Wells | Values | Mean Value | Back Concentration Calculation | Average |
| Standard 1 | 75 | A1 | 1.187 | 1.169 | 76.376 | 75.01 |
| A2 | 1.152 | 73.644 | ||||
| Standard 2 | 30 | B1 | 0.534 | 0.52 | 30.827 | 29.962 |
| B2 | 0.506 | 29.098 | ||||
| Standard 3 | 12 | C1 | 0.23 | 0.217 | 12.838 | 12.105 |
| C2 | 0.204 | 11.372 | ||||
| Standard 4 | 4.8 | D1 | 0.09 | 0.084 | 5.055 | 4.726 |
| D2 | 0.078 | 4.398 | ||||
| Standard 5 | 1.92 | E1 | 0.033 | 0.031 | 1.941 | 1.86 |
| E2 | 0.03 | 1.778 | ||||
| Standard 6 | 0.768 | F1 | 0.009 | 0.011 | 0.626 | 0.764 |
| F2 | 0.014 | 0.901 | ||||
| Standard 7 | 0.307 | G1 | 0.002 | 0.004 | 0.238 | 0.377 |
| G2 | 0.007 | 0.516 |
Table 2: TNFα Sandwich ELISA standard curve data. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL), OD (450 nm) values, mean OD450 values, back concentration calculations and their averages.
Figure 2: Standard Curve for TNFα sandwich ELISA. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL) was analyzed using sandwich ELISA.The OD450 values can be directly correlated to the standard dilution concentrations. The amount of TNFα protein in the test sample was determined using the standard curve, which corresponds to a concentration of 38.72 pg/mL.
Once the standard curve was generated, the amount of TNFα protein in the test sample was determined. In this sandwich ELISA example, the test samples gave OD450 readings of 0.636 and 0.681, which give an average of 0.6585. When plotting this OD450 reading on the above chart, this corresponds to a TNFα concentration of 38.72 pg/ml.
Applications and Summary
As demonstrated, a range of immunoassays (with slight variation in protocols) fall within the ELISA technique family. Determining which version of ELISA to use depends on a number of factors, including what antigen is being detected, the monoclonal antibody available for a particular antigen, and the desired sensitivity of the assay (5). Some strengths and weaknesses of the different ELISAs described herein are:
| ELISA | Strengths | Weaknesses |
| Indirect | 1) High sensitivity due to the fact that multiple enzyme-conjugated secondary antibodies can bind to the primary antibody | 1) High background signal may occur because the coating of the antigen of interest to the plate is not specific (i.e., all proteins in the sample will coat the plate) |
| 2) Many different primary antibodies can be recognized by a single enzyme-conjugated secondary antibody giving the user the flexibility of using the same enzyme-conjugated secondary antibody in many different ELISA (regardless of the antigen being detected) | ||
| 3) Best choice when only a single antibody for the antigen of interest is available | ||
| Sandwich | 1) The use of antigen-specific capture and detection monoclonal antibody increases the sensitivity and specificity of the assay (compared to the indirect ELISA) | 1) Optimizing the concentrations of the capture and detection monoclonal antibodies can be difficult (especially for non-commercial kits) |
| 2) Best choice for detecting a large protein with multiple epitopes (such as a cytokine) | ||
| Competitive | 1) Impure samples can be used | 1) Requires a large amount of highly pure antigen to be used to coat plate |
| 2) Less sensitivity to reagent dilution effects | ||
| 3) Ideal for detecting small molecules (such as a hapten) |
Table 3: Summary. A summary of the strengths and weaknesses of the different ELISA techniques.
While a simple and useful technique, there are also some drawbacks to any ELISA. One is the uncertainty of the amount of the protein of interest in the test samples. If the amount is too high or too low, the absorbance values obtained by the microplate reader may fall above or below the limits of the standard curve, respectively. This will make it difficult to accurately determine the amount of protein present in the test samples. If the values are too high, the test sample can be diluted prior to adding to the wells of the plate. The final values would then need to be adjusted according to the dilution factor. As mentioned, homemade kits often require careful optimization of the antibody concentrations used to yield a high signal-to-noise ratio.
References
- Porstmann, T. and Kiessig S.T. Enzyme immunoassay techniques. An overview. Journal of Immunological Methods. 150 (1-2), 5-21 (1992).
- Suleyman Aydin. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4-15 (2015).
- Gan. S. D. and Patel K. R. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. Journal of Investigative Dermatology, 133 (9), 1-3 (2013).
- Kohl, T. O. and Ascoli C.A. Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harbor Protocols, 1 (6), (2017).
- Sakamoto, S., Putalun, W., Vimolmangkang, S., Phoolcharoen, W., Shoyama, Y., Tanaka, H., and Morimoto S. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of natural medicines, 72 (1), 32-42 (2018).
Transcript
Enzyme-linked Immunosorbent Assay, or ELISA is a highly sensitive quantitative assay commonly used to measure the concentration of an analyte like cytokines and antibodies in a biological sample. The general principle of this assay involves three steps: starting with capture, or immobilization, of the target analyte on a micro plate, followed by the detection of the analyte by target-specific detection proteins, and lastly, enzyme reaction, where a conjugated enzyme converts its substrate to a colored product. Based on different methods of capture and detection, ELISA can be of four types: direct, indirect, sandwich, and competitive.
For direct ELISA, the target antigen is first bound to the plate, and is then detected by a specific detection antibody. This method is commonly used for screening antibodies for a specific antigen. Indirect ELISA is used for detecting antibodies in a sample in order to quantify immune responses. The plate is first coated with a specific capture antigen, which immobilizes the target antibody, and this antigen-antibody complex is then detected using a second antibody.
In the case of sandwich ELISA, the target analyte is an antigen, which is captured on the plate using a capture antibody and then detected by the detection antibody, hence forming an antibody-antigen-antibody sandwich. This method is useful for measuring the concentration of an antigen in a mixed sample.
Competitive ELISA is used when only one antibody is available for a target antigen of interest. The plate is first coated with the purified antigen. Meanwhile, the sample containing the antigen is pre-incubated with the antibody and then added to the plate, to allow any free antibody molecules to bind to the immobilized antigen. The higher the signal from the plate, the lower the antigen concentration in the sample. In all of the four types of ELISA, direct, indirect, sandwich, and competitive, the detection antibody is either directly conjugated to the enzyme or can be indirectly linked to it through another antibody or protein.
The enzymes commonly used for the reaction are horseradish peroxidase or alkaline phosphatase with their respective substrates, both producing a soluble, colored product that can be measured and quantified using a plate reader. In this video, you will observe how to perform indirect ELISA, sandwich ELISA, and competitive ELISA, followed by examples of quantification of the target analyte from the indirect and sandwich ELISA methods.
The first experiment will demonstrate how to use indirect ELISA to determine the presence of anti-influenza virus antibodies in serum obtained from influenza-infected mice.
To begin, add 50 microliters of purified antigen – in this case, 2 milligrams per milliliter of purified A/PR/8 Influenza A virus- to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive cover and incubate it overnight at 4 degrees celsius to allow the antigen to bind to the plate. The following day, remove the coating solution by flicking the plate over a sink. Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of a blocking buffer- here, 5% donkey serum in 1X PBS- to each well. Leave the plate to incubate for at least 2 hours at room temperature. Following the incubation, remove the blocking buffer and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink once more to remove the wash.
Then, prepare the test samples by adding 460 microliters of PBS to a fresh tube, and then adding 40 microliters of serum to make a 1 to 12.5 dilution. Then, add 300 microliters of PBS to a second tube, and then add 100 microliters of the first dilution. Continue this serial dilution range until obtaining a final sample with a dilution of 1 to 204,800. Add the serially diluted serum samples in triplicate to the wells. Cover the plate with an adhesive cover and incubate at room temperature for an hour. Next, remove the samples by flicking the plate into the sink and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Once again, flick the plate to remove the wash.
Now, add 100 microliters of an enzyme-conjugated secondary antibody, which in this experiment is a horseradish peroxidase, or HRP, conjugated donkey anti-mouse secondary, to each well. Incubate the plate for one hour at room temperature, and flick the plate to remove any excess liquid. Wash the plate with 1X PBS containing 1% Tween-20 and then apply 100 microliters of the indicator substrate at a concentration of one milligram per milliliter to each well. Incubate the plate with the substrate for 5 to 10 minutes at room temperature. In this example, the colorless 3,3′, 5,5′ – tetramethylbenzidine, or TMB, substrate turns a blue color when HRP is present. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid. The samples will turn a yellow color.
Within 30 minutes of adding the stop solution, insert the plate into a microplate reader and read the plate at the appropriate wavelength for the substrate to determine the absorbance of the wells.
To begin the sandwich ELISA, the plate must be coated with purified capture antibody. To do this, add 100 microliters of the capture antibody at a concentration within the 1-10 microgram per milliliter range, to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive plate cover and then incubate the plate overnight at 4 degrees celsius. After the incubation, remove the coating solution by flicking the plate over a sink.
Now, block the remaining protein- binding sites in the coated wells by adding 200 microliters of 5% nonfat dry milk to the wells. Incubate the plate at room temperature for at least 2 hours. Next, remove the blocking buffer, and then wash the wells with 1X PBS containing 1% Tween-20. Remove the wash by flicking the plate over the sink. Now, add 100 microliters of the test sample to the wells, seal the plate with an adhesive cover, and then incubate it at room temperature for 2 hours. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink to remove the wash and then add 100 microliters of enzyme-conjugated detection antibody to the wells.
Seal the plate with an adhesive cover. Leave the plate to incubate at room temperature for 2 hours. After the incubation, remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Next, add 100 microliters of the indicator substrate at a concentration of 1 milligram per milliliter, and incubate the plate for 5 to 10 minutes at room temperature. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid to the wells and then read the plate within 30 minutes of adding the stop solution in a microplate reader.
To perform a competitive ELISA, first coat the wells of a 96-well ELISA plate with 100 microliters of purified antigen at a concentration of 1-10 micrograms per milliliter. Cover the plate with an adhesive plate cover and then incubate overnight at 4 degrees celsius. Following this, remove the unbound antigen solution from the wells by flicking the plate over a sink.
Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of blocking buffer to each well- here, 5% nonfat dry milk in PBS. Incubate the plate for at least 2 hours at room temperature. While blocking the wells, prepare the antigen-antibody mixture in a 1. 5 milliliter tube by adding 150 microliters of sample antigen to 150 microliters of primary antibody for each well in the assay. Incubate this mixture for 1 hour at 37 degrees celsius. Now, remove the blocking buffer from the wells by flicking the plate over a sink. Then, wash the wells with 1X PBS containing Tween 20 and then add 100 microliters of the sample antigen- primary antibody mixture.
Leave the plate to incubate at 37 degrees celsius for one hour. Next, remove the sample mixture by flicking the plate over a sink and then wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody. Add 100 microliters of an enzyme-conjugated secondary antibody to each well and incubate the plate for one hour at 37 degrees celsius. Following this, wash the plate with 1X PBS containing 1% Tween-20 and then add 100 microliters of the substrate solution to each well. Wait for 5-10 minutes. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid and then measure the absorbance in a microplate reader within 30 minutes of adding the stop solution.
For the semi-quantitative indirect ELISA assay, the presence of influenza A virus antibodies in serially diluted samples of serum from influenza A- infected mice was determined by reading the absorbance of each well at 405 nanometers in a plate reader. This raw data is exported to a spread sheet for calculation purposes. In this experiment, the serially diluted serum samples, which range from 1 – 12.5, to 1 – 204,800, were repeated in triplicate.
To analyze the data, the mean absorbance value is therefore calculated for each set of triplicates by adding all the values for each dilution and dividing the sum by 3. Once the mean for each set of triplicates is determined, the mean OD450 readings are plotted against the serial dilutions. The OD readings decrease as the serum is diluted, indicating that less antibodies are found in the more diluted samples. In the quantitative sandwich ELISA, dilutions of known standard, in this case recombinate Human TNFalpha, were added to a 96-well plate and read along with the unknown samples.
To create the standard curve, the mean absorbance value for each set of readings of the known concentrations was calculated. Then, the mean absorbance value was plotted on the y-axis, against the known protein concentrations on the x-axis. A best fit curve is added through the points in the graph.
Once the standard curve is generated, the amount of TNFalpha protein in the test sample can be determined by first calculating the mean absorbance value for the test sample. In this example, the test samples gave OD450 readings of 0.636 and 0. 681. Adding these values and dividing the sum by 2 gives an average of 0.659. From the y-axis on the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration which, in this test sample, corresponds to a TNFalpha concentration of 38.72 picograms per milliliter.