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|Application / Reactivity||Human|
|ELISA||5 ELISA Kits|
|Antigen||Luteinizing Hormone beta Polypeptide (LHB) ELISA Kits|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||0.156-10 ng/mL|
|Minimum Detection Limit||0.156 ng/mL|
|Supplier||Log in to see|
Product Details LHB ELISA KitTarget details Application Details Handling Images
|Purpose||This immunoassay kit allows for the specific measurement of human immunosuppressive acidic protein, IAP concentrations in cell culture supernates, serum and plasma.|
|Sample Type||Cell Culture Supernatant, Serum, Plasma|
|Specificity||This assay recognizes recombinant and natural human IAP.|
|Cross-Reactivity (Details)||No significant cross-reactivity or interference was observed.|
|Characteristics||Homo sapiens,Human,Lutropin subunit beta,Lutropin beta chain,Luteinizing hormone subunit beta,LH-B,LSH-B,LSH-beta,LHB|
|Material not included||Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.|
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|Alternative Name||LHB (LHB ELISA Kit Abstract)|
|Background||Immunosuppressive acidic protein (IAP) is an acute-phase reactant which has a close correlation with the impairment of the host's immunity. It has been found to suppress both phytohemagglutinin-induced lymphocyte blast formation and mixed lymphocyte reaction in vitro. The level of immunosuppressive acidic protein has revealed clear increases with the progression of cancer, and has been reported to be a useful diagnostic and follow-up marker for ovarian and renal cancer.|
|Pathways||TCR Signaling, Metabolism of Steroid Hormones and Vitamin D, Peptide Hormone Metabolism, Hormone Activity, C21-Steroid Hormone Metabolic Process|
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|Sample Volume||100 μL|
|Protocol||This assay employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for IAP has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IAP present is bound by the immobilized antibody. An enzyme-linked antibody specific for IAP is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IAP bound in the initial step. The color development is stopped and the intensity of the color is measured.|
|Reagent Preparation||Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard. The Sample Diluent serves as the zero standard (0 ng/ml).|
|Sample Collection||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 approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.|
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 °C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 °C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
1. Add 100 μL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 °C . Detection Reagent A 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 (approximately 400 μL) 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.
5. Add 100 μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 °C .
6. Repeat the aspiration/wash as in step 4.
7. Add 90 μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 °C . Protect from light.
8. Add 50 μL 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 at once, using a microplate reader set to 450 nm.
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the 5 strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. 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.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.
|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 x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SAA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. 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.|
|Restrictions||For Research Use only|
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1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay 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.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
|Storage||4 °C/-20 °C|
|Storage Comment||The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.|
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