Mouse Mannma binding protein/mannan binding lectin, MBP/MBL ELISA kit
Catalog No. HR-M0373
(96 tests)
FOR RESEARCH USE ONLY
Intended use
This immunoassay kit allows for the use in vitro quantitative determination of mouse Mannma binding protein/mannan binding lectin, MBP/MBL concentrati*** in cell culture supernates, serum, pla***a and other biological fluids.
Introduction
Mannose (or mannan)-binding lectin (MBL), also known as serum mannosebinding protein (MBP), initiates the lectin branch of the innate immune resp***e by binding to the surface of potentially pathogenic microorgani***s and initiating complement fixation through an N-terminal collagen-like domain. MBL is a key component in immune resp***e in that it can directly trigger neutralization of invading microorgani***s by an Ab-independent mechani***. It binds to sugars on the surface of bacterial, fungal and parasitic cells through C-terminal, Ca2+-dependent carbohydrate-recognition domains. Mutati*** of human MBL are associated with immunodeficiency resulting from a reduction in the ability of the mutant MBL to initiate complement fixation. In human, two types of MBL-associated serine proteases (MASP-1and MASP-2) and a truncated form of MASP-2, designated ***all MBLassociated protein (***AP) or MAp19, complex with MBL to activate the lectin pathway of the complement system. Activated MASPs subsequently cle***e and activate downstream components of the complement pathway.
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to MBP/MBL. Standa*** or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for MBP/MBL and ***idin 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 MBP/MBL, biotin-conjugated antibody and enzyme-conjugated ***idin 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 w***elength of 450 nm &plu***n; 2 nm. The concentration of MBP/MBL in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Materials and components
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20ml
Assay Diluent A 1 x 10ml
Assay DiluentB 1 x 10ml
Detection Reagent A 1 x 120ul
Detection Reagent B 1 x 120ul
Wash Buffer 1 x 30ml
(25 x concentrate)
Substrate 1 x 10ml
Stop Solution 1 x 10ml
Plate sealer for 96 wells 1 x 5
Sample collection and storage
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 or -80° C.
Pla***a- Collect pla***a 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 or -80° C. ***oid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids- Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C or -80° C. ***oid repeated freeze-thaw cycles.
Sample preparation - Serum/pla***a samples require a 5 fold dilution. A suggested 5-fold dilution is 20uL sample + 80uL Sample Diluent.
Note: Serum, pla***a, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 ° C, otherwise samples must stored at -20° C (≤ 3 months) or -80° C (≤ 6 months) to ***oid loss of bioactivity and contamination. ***oid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.
It is recommended that all samples be assayed in duplicate.
DO NOT USE HEAT-TREATED SPECIMENS.
Limitati*** of the procedure
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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 h***e been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.
Reagent preparation
Bring all reagents to room temperature before use.
Wash Buffer - If crystals h***e formed in the concentrate, warm to room temperature and mix gently until the crystals h***e completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Rec***titute the Standard with 1.0 mL of Sample Diluent. This rec***titution produces a stock solution of 80 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial diluti***. The undiluted standard serves as the high standard (80 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL).
Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100),respectively.
Assay procedure
Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. ***oid foaming. Arrange and label required number of strips. Prepare all reagents, working standa*** and samples as directed in the previous secti***.
1. Add 100 uL 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 uL ofDetection 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 (350 uL) 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 uL of Detection Reagent Bworking 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 uL 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 uL 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.
Specificity
This assay recognizes recombinant and natural mouse MBP/MBL. No significant cross-reactivity or interference was observed.
This assay recognizes recombinant and natural mouse MBP/MBL. No significant cross-reactivity or interference was observed.
Sensitivity
The minimum detectable dose of mouse MBP/MBL is typically less than 0.31 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.
Detection Range
1.25-80 ng/mL. The standard curve concentrati*** used for the ELISA’s were 80 ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL.
Important Note:
1. Please carefully rec***titute Standa*** or working Detection Reagent A and B according to the instruction, and ***oid foaming and mix gently until the crystals h***e completely dissolved. The rec***tituted Standa*** can be used only once.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
3. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standa***, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is ***ailable.
4. Duplication of all standa*** and specimens, although not required, is recommended.
5. When mixing or rec***tituting protein soluti***, always ***oid foaming.
6. To ***oid cross-contamination, change pipette tips between additi*** of each standard level, between sample additi***, and between reagent additi***. Also, use separate reservoirs for each reagent.
7. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
Calculation of results
***erage the duplicate readings for each standard, control, and sample and subtract the ***erage 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, c***truct 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 MBP/MBL concentrati*** 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 h***e been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Storage of test kits and instrumentation
1. Unopened test kits should be stored referring to the package label for frequent use, and stored at -20°C for long time storage. 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 (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
3. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
4. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm w***elength is acceptable for use in absorbance measurement.
5. Use fresh disposable pipette tips for each transfer to ***oid contamination.
6. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
Precaution
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
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