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- 抗原
- Occult Blood
- 适用
- 人
- 检测方法
- Colorimetric
- 实验类型
- Sandwich ELISA
- 检测范围
- 0.5-745 ng/mL
- 最低检测浓度
- 0.5 ng/mL
- 应用范围
- ELISA
- 原理
- This q-FOB? test kit is intended for detection of fecal occult blood by the quantitative determination of human hemoglobin levels in stool samples. This assay exclusively measures human without cross-reaction to animal blood. The test is useful for detecting the severity of gastrointestinal bleeding and in the aid of screening for colorectal adenoma/polyps and cancer, as well as other inflammatory bowel diseases (Crohn's disease, Ulcerative Colitis, etc.).
- 品牌
- q-FOB™
- 样品类型
- Fecal
- Analytical Method
- Quantitative
- 组件
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1. q-FOB Antibody Coated Microplate
Two vials each contain human hemoglobin in a lyophilized bovine serum based matrix with a non-azide, non-mercury preservative. Refer to vials for exact concentration range for each control.
Both controls should be stored at 2-8 °C and are stable until the expiration date on the kit box. - 试剂未包括
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1. Fecal sample collection tube
2. Precision single channel pipettes capable of delivering 100 µL, and 1000 µL etc.
3. Disposable pipette tips suitable for above volume dispensing.
4. Disposable plastic 100 mL and 1000 mL bottle with caps.
5. Aluminum foil.
6. Deionized or distilled water.
7. Plastic microtiter well cover or polyethylene film.
8. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
9. Spectrophotometric microplate reader capable of reading absorbance at 450 nm and 620 nm.
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- 实验时间
- 4 h
- 板类型
- Pre-coated
- 实验流程
- This ELISA is designed, developed and produced for the quantitative measurement of human hemoglobin in stool sample. The assay utilizes the two-site sandwich technique with two selected antibodies that bind to different epitopes of human hemoglobin. Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with hemoglobin antibodies. Subsequently, a horseradish peroxidase (HRP)-conjugated human hemoglobin specific antibody is added to each well. After an incubation period, a sandwich of antibody human hemoglobin HRP-conjugated antibody is formed. The unbound antibody and buffer matrix is removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human hemoglobin in a test sample. A standard curve is generated by plotting the absorbance versus the respective human hemoglobin concentration for each standard on point-to-point or 4 parameter curve fitting. The concentration of fecal human hemoglobin in test samples is determined directly from this standard curve.
- 试剂准备
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(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details.
(3) Reconstitute q-FOB standards and controls with 1 mL of deionized or distilled water.
(4) Test Configuration
(5) Place a sufficient number of antibody coated microwell strips in a holder to run human hemoglobin standards, controls and unknown samples in duplicate.
(6) Prepare working Tracer Antibody by 1:21 fold dilution of the Hemoglobin Tracer Antibody by adding the tracer antibody to the tracer antibody diluent . Following is a table that outlines the relationship of strips used and antibody required. Note: this antibody mixture should be freshly prepared before running the assay.
(7) Load all reagents onto a DS2 or DSX system according to the computer program. - 样品收集
- Only one fecal sample is required. Fresh fecal sample must be collected by using Epitope Diagnostics Fecal Sample Collection Device. This tube is specially designed for easy collection of a substantially small amount of fecal sample into the tube pre-filled with sample extraction buffer. The collected fecal sample must be transported, kept at 2-8 °C and tested within 7 days. Otherwise, fecal sample must be stored below -20 °C for a longer storage period. Avoid more than three freeze- thaw cycles for each specimen. It is strongly recommended to use Epitope Diagnostics Fecal Sample Collection Device for sample collection. The clinical validation data of this test were generated by using this sampling tube!
- 样品制备
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If the Epitope Diagnostics Fecal Sample Collection Tube is use, there is no sample preparation required. Before assay bring all patient samples to room temperature. Unscrew the white cap of the sample collection tube and load the samples onto the DS2 or DSX system. It is important to make sure that there are no air bubbles on the surface of the collection tube.
- 实验流程
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(1) Add 100 μL of standards, controls and patient samples into the designated microwell.
(2) Incubate plate at 39 °C, for 20 minutes with initial shaking for two minutes at low speed.
(3) Wash each well 5 times by dispensing 350 μL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(4) Add 100 μL diluted Tracer Antibody to each of the wells.
(5) Incubate plate at 39 °C, for 12 minutes with initial shaking for two minutes at low speed.
(6) Wash each well 5 times by dispensing 350 μL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(7) Add 100 μL of ELISA HRP Substrate into each of the wells.
(8) Incubate plate at 39 °C for 8 minutes with initial shaking for 8 seconds at low speed.
(9) Add 100 μL of ELISA Stop Solution into each of the wells.
(10) Read the absorbance at 450 nm with a reference of 620 nm and initial shaking for 3 seconds at low speed. - 结果分析
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For the DS2 and DSX system, it is recommended to use a linear/linear, point-to-point or 4-parameter standard curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. Subtract the average absorbance of the Diluted Fecal Sample Extraction Buffer (0 ng/mL) from the average absorbance of all other readings to obtain corrected absorbance.
3. The standard curve is generated by the corrected absorbance of all standard levels on the ordinate against the standard concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The fecal human hemoglobin concentrations for the controls and the patient samples are read directly from the standard curve using their respective corrected absorbance. - 实验精密度
- The intra-assay precision is validated by measuring two controls samples in a single assay with 16-replicate determinations. The inter-assay precision is validated by measuring two control samples in duplicate in 16 individual assays.
- 限制
- 仅限研究用
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- 注意事项
- The reagents must be used in professional laboratory. The source material for reagents containing bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.
- 储存条件
- 4 °C
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- 抗原
- Occult Blood
- 背景
- Detection of abnormally high level of fecal hemoglobin is recommended by America Cancer Society (ACS), Center for Disease Control and Prevention (CDC) and Center for Medical Service (CMS) of the Department of Health of the United States. The Fecal Occult Blood (FOB) Rapid Test was used in the past 40 years. Screening for occult blood by means of guaiac tests has an unsatisfactory sensitivity for the detection of colorectal neoplasm with a dietary restriction drawback. Immunochemical FOB test dramatically increases the analytical sensitivity and specificity in the detection of human hemoglobin in feces. A few clinical trials showed that immunochemical FOB test is superior in clinical diagnostic sensitivity and specificity compared to guaiac FOB test. However, these rapid tests do not give an insight to the severity of the bleeding in the lower gastrointestinal system. This q-FOB™ assay using human hemoglobin-specific antibodies would bring significant advantages over the qualitative rapid FOB tests. The assay does not require dietary restrictions such as no raw meat, vitamin C rich food (salad, fruits, etc.). This q-FOB™ assay detects human hemoglobin level in 100-fold lower concentrations than the guaiac FOB test and avoids false-negative results. Because highly specific human hemoglobin antibodies are used, false positive results are practically excluded.
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