ለምርምር ጥቅም ብቻ
የመመሪያ መመሪያ

http://www.dldevelop.com

Mouse Huntingtin (HTT) ELISA Kit
ካታሎግ ቁጥር፡ DLR-HTT-Mu
96 ሙከራዎች

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE procedure BEFORE BEGINNING!

የታሰበ አጠቃቀም
ኪት በአይጥ ሴረም፣ ፕላዝማ፣ ቲሹ ሆሞጋኔቶች፣ ሴል ላይሳይቶች፣ የሕዋስ ባህል ሱፐርናቶች ወይም ሌሎች ባዮሎጂካል ፈሳሾች ውስጥ ያለውን የኤችቲቲ ኢን ቪትሮ መጠናዊ መለኪያ የሳንድዊች ኢንዛይም immunoassay ነው።

የቀረቡ REAGENTS እና ቁሳቁሶች

ሬጀንቶች	ብዛት	ሬጀንቶች	ብዛት
Pre-coated, ready to use 96-well strip plate	1	Plate sealer for 96 wells	2
መደበኛ	2	መደበኛ ማቅለጫ	1×20ml
Detection Solution A	1×12ml	TMB Substrate	1×9ml
Detection Solution B	1×12ml	መፍትሄ ማቆም	1×6ml
Wash Buffer (30 × concentrate)	1×20ml	መመሪያ መመሪያ	1

ቁሳቁሶች ያስፈልጋሉ ግን አልተሰጠም

1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting sampሌስ.
4. የተጣራ ወይም የተጣራ ውሃ.
5. የማይክሮቲተር ሰሃን ለማጥፋት የሚስብ ወረቀት።
6. Container for Wash Solution.

የኪትስ ማከማቻ

1. For unopened kits: All the reagents should be kept at -20°C upon receipt.
2. For opened kits: Once the kit is opened, the remaining reagents still need to be stored according to the above storage conditions. In addition, return the unused wells to the foil pouch containing the desiccant pack and reseal along entire edge of zip-seal.

ማስታወሻ፡-
For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this expiration date.
It is highly recommended to use the remaining reagents within 1 month of opening.

SAMPLE ስብስብ እና ማከማቻ

• ሴረም - የሴረም መለያያ ቱቦ ይጠቀሙ እና ፍቀድamples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
• Plasma – Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
• Tissue homogenates – The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues should be rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders also work). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates are centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20°C.
• Cell Lysates – Cells must be lysed before assaying according to the following directions.

1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4 times (or Freeze cells at ≤-20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.).
4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris.
    Cell culture supernatant or other biological fluids – Centrifuge samples ለ 20 ደቂቃዎች በ 1000×g. ቅንጣቶችን ያስወግዱ እና ወዲያውኑ ይመርምሩ ወይም s ያከማቹamples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

REAGENT ዝግጅት

1. ሁሉንም የኪት ክፍሎች እና ኤስamples to room temperature (18-25°C) before use.
2. Standard – Reconstitute the Standard with 1.0mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam).
   The concentration of the standard in the stock solution is 10ng/mL. Prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer.
   Prepare a dilution series with 7 points; for example: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/mL, 0.156ng/ml, እና የመጨረሻው የ EP ቱቦ ከመደበኛ ዲሊየንት ጋር በ0ng/mL ባዶ ነው።
3. Detection Solution A and Detection Solution B – Detection Solutions A and B are already at the correct concentrations and do not need to be diluted further.
4. Wash Solution – Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600mL of Wash Solution (1×).
5. TMB substrate – Aspirate the needed dosage of the solution with sterilized tips. Do not dump the residual solution back into the vial.

ማስታወሻ፡-

1. Do not perform a serial dilution directly in the wells.
2. Prepare standard within 15 minutes of performing the assay.
3. Carefully reconstitute Standards according to the instruction, avoid foaming and mix gently until the crystals are completely dissolved.
4. The reconstituted Standards can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals are completely dissolved.
6. Any contaminated water or container used during reagent preparation will influence the detection result.

SAMPLE ዝግጅት

1. DL Sci & Tech Development Co.,Ltd. is only responsible for the kit itself, not for the sampበምርመራው ወቅት ብዙ ጥቅም ላይ ይውላል ። ተጠቃሚው የሚቻለውን የ s መጠን ማስላት አለበት።ampበአጠቃላይ ፈተና ውስጥ ጥቅም ላይ የዋለ.
   እባክዎ በቂ samples በቅድሚያ.
2. እባክዎን ከመመርመርዎ በፊት ትኩረቱን ይተነብዩ. የእነዚህ ዋጋዎች በመደበኛ ከርቭ ክልል ውስጥ ከሌሉ ተጠቃሚዎች በጣም ጥሩውን s መወሰን አለባቸውampለተለየ ሙከራቸው ዳይሉሽን። ኤስamples should be diluted by standard diluent included in the kit. If included standard diluent is not enough, samples can also be diluted by 0.01 mol/L PBS (pH7.0-7.2)..
3. የኤስ.ኤስampበመመሪያው ውስጥ አልተገለፁም ፣ የኪቱን ትክክለኛነት ለመወሰን የመጀመሪያ ሙከራ አስፈላጊ ነው።
4. ቲሹ ወይም ሕዋስ ማውጣት samples prepared using a chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigens from other origin and antibodies used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be  recognized by our products.
6. Sampከሴሎች ባህል በላይ የሆነ ንጥረ ነገር እንደ ሴል አዋጭነት፣ የሕዋስ ቁጥር እና/ወይም ዎች ባሉ ተጽእኖዎች ምክንያት በኪቱ ላይገኝ ይችላል።ampረጅም ጊዜ.
7. ትኩስ ኤስampረዘም ላለ ጊዜ ያልተቀመጡት ለፈተናው ይመከራል. ያለበለዚያ በእነዚያ s ውስጥ የፕሮቲን መበስበስ እና የጥርስ መበላሸት ሊከሰት ይችላል።amples እና የተሳሳቱ ወይም የተሳሳቱ ውጤቶችን ይስጡ.

የ ASSAY ሂደት

1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for the standards, 1 well for blank. Add 100μL each of dilutions of standard (read Reagent Preparation), blank, and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 90 minutes at 37°C.
2. Remove the liquid from each well.
3. Add 100μL of Detection Solution A to each well. Incubate for 45 minutes at 37°C after covering it with the Plate sealer.
4. Aspirate the solution and wash with 300μL of 1× Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or auto-washer, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by tapping the plate onto absorbent paper.
   Wash thoroughly 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
5. Add 100μL of Detection Solution B to each well. Incubate for 45 minutes at 37°C after covering it with the Plate sealer.
6. Repeat the aspiration/wash process for a total of 5 times as conducted in step 4.
7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15-25 minutes at 37°C (Do not exceed 30 minutes). Protect from light. The liquid will turn blue with the addition of the Substrate Solution.
8. Add 50μL of Stop Solution to each well. The liquid will turn yellow with the addition of the Stop solution. Mix the liquid by tapping the side of the plate. If the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Remove any drops of water and fingerprints on the bottom of the plate and confirm there are no bubbles on the surface of the liquid. Run the microplate reader and take measurements at 450nm immediately.

ማስታወሻ፡-

1. Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from microplate. Remaining wells should be resealed and stored at -20°C.
2. Samples ወይም reagents መደመር፡ እባክዎን አዲስ የተዘጋጀውን መደበኛ ይጠቀሙ። በጥንቃቄ s ያክሉamples to wells and mix gently to avoid foaming. Do not touch the well walls. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change  pipette tips between additions of standards, samples, and reagents. In addition, use separated reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods in between incubation steps. Once reagents are added to the well strips, DO NOT let the strips dry at any time during the assay. Incubation time and temperature must be controlled.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting, and remove any drops of water or fingerprints on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in an inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Please protect it from light.
7. The environment humidity may have an effect on the results obtained from the kit. If the humidity in your facility is less than 60%, using a humidifier is recommended.

የፈተና መርህ
በዚህ ኪት ውስጥ የቀረበው የማይክሮቲተር ፕሌትስ ለኤችቲቲ በተለየ ፀረ እንግዳ አካል ቀድሞ ተሸፍኗል። ደረጃዎች ወይም ኤስamples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to HTT. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain HTT, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a
change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of HTT in the samples ከዚያም የ s OD በማወዳደር ይወሰናልamples ወደ መደበኛ ጥምዝ.

የውጤቶች ስሌት

ለእያንዳንዱ መደበኛ፣ ቁጥጥር እና ዎች የተባዙ ንባቦች አማካኝample፣ ከዚያ አማካዩን ዜሮ መደበኛ የኦፕቲካል እፍጋትን ይቀንሱ። ለእያንዳንዱ ስታንዳርድ አማካኝ ኦዲ እና ትኩረትን በማቀድ መደበኛ ኩርባ ይገንቡ እና በግራፉ ላይ ባሉት ነጥቦች ላይ በጣም ጥሩ የሆነ ኩርባ ይሳሉ ወይም በሎግ-ሎግ ግራፍ ወረቀት ላይ መደበኛ ኩርባ ይፍጠሩ በ y ዘንግ ላይ የኤችቲቲ ትኩረት እና በ x-ዘንግ ላይ። ሴራ ሶፍትዌርን መጠቀም (ለምሳሌ፣ ከርቭ ኤክስፐርት 1.30) እንዲሁም ይመከራል። ከሆነ samples been diluted, ከመደበኛው ከርቭ የተነበበው ትኩረት በ dilution ምክንያት ተባዝቶ አለበት.

የተለመደ ውሂብ

In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting the log of the data to establish a standard curve for each test is recommended.
The typical standard curve below is provided for reference only.

የማወቂያ ክልል
0.156-10ng/ml. ለ ELISA ጥቅም ላይ የዋሉት መደበኛ ኩርባዎች 10ng/ml, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/ml, 0.156ng/mL.

ስሜታዊነት
The minimum detectable dose of HTT is typically less than 0.055ng/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. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

ልዩነት
This assay has high sensitivity and excellent specificity for detection of HTT.
No significant cross-reactivity or interference between HTT and analogues was observed.
ማስታወሻ፡-
Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between HTT and all analogues, therefore, cross reactivity may still exist.

PRECISION
የውስጠ-ግምት ትክክለኛነት (በግምት ውስጥ ያለው ትክክለኛነት)፡ 3 ሴamples with low, middle and high level HTT were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level HTT were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

መረጋጋት
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
ማስታወሻ፡-
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

የ ASSAY ሂደት ማጠቃለያ

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 90 minutes at 37°C;
3. Aspirate and add 100µL Detection Solution A. Incubate 45 minutes at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL Detection Solution B. Incubate 45 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

ጠቃሚ ማስታወሻዎች

1. Limited by the current conditions and scientific technology, it is impossible to conduct comprehensive identification and analysis tests on the raw materials provided by suppliers. As a result, it is possible there are some qualitative and/or technical risks.
2. The final experimental results will be closely related to the validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available to obtain accurate results.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction manual included in your kit. Electronic ones on our website are for reference only.
4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
5. Protect all reagents from strong light during storage and incubation. All bottle caps of reagents should be closed tightly to prevent evaporation of liquids and contamination by microorganisms.
6. There may be a foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
7. Incorrect procedures during reagent preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
8. Even the same experimenter may get different results from two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before the general assay for each batch is recommended.
9. Each kit has undergone several rigorous quality control tests. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipment. Intra-assay variance among kits from different batches could arise from the above factors as well.
10. Kits from different manufacturers with the same item might produce different results, since we have not compared our products with other manufacturers.
11. The standard in this kit, as well as the antigens used in antibody preparation are typically recombinant proteins. Differently expressed sequences, expression systems, and/or purification methods can be used in the preparation of recombinant proteins. There is also the possibility of differences in the screening technique of antibodies and antibody pairs in our kits. As a result, we cannot guarantee that our kit will be able to detect recombinant proteins produced by other companies. We do NOT recommend using our ELISA kits for the detection of other recombinant proteins.
12. ተቀባይነት ያለው ጊዜ: 16 ወራት.
13. The instruction manual also works with the 48T kit, but all reagents in the 48T kit are reduced by half.

ጥንቃቄ
ከዚህ ኪት ጋር ለመጠቀም የተጠቆመው የማቆሚያ መፍትሄ የአሲድ መፍትሄ ነው። ይህን ሪጀንት ሲጠቀሙ የአይን፣ የእጅ፣ የፊት እና የልብስ መከላከያ ይልበሱ።

ሰነዶች / መርጃዎች

ዲኤልአር-ኤችቲቲ-ሙ አይጥ ሀንቲንግቲን ኤሊሳ ኪት አዳብሩ [pdf] መመሪያ መመሪያ AAA23957፣ DLR-HTT-Mu Mouse Huntingtin ELISA Kit፣ DLR-HTT-Mu፣ Mouse Huntingtin ELISA Kit፣ Huntingtin ELISA Kit፣ ELISA Kit፣ Kit

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