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Human Alpha-2-Macroglobulin (a2M) ELISA Kit
two product lines: Traditional ELISA Kit and Ready-to-Use ELISA Kit.
| Traditional ELISA Kit | Ready-to-Use ELISA KIT | |
| Product name: | Human Alpha-2-Macroglobulin (a2M) ELISA Kit | |
| Method: | Sandwich | |
| Synonyms: | A2MG; A2mg; CPAMD5; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5 |
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| Detection range: | 0.781-50ng/mL | |
| Target Protein: | a2M | |
| Size: | 96T/48T | |
| Quality guarantee period: | for 12 months, 16 months | |
| Catalog number: | DL-a2M-Hu (traditional) | (ready-to-use) |
| Assay length | 1-4.5Hours | 1-3.5Hours |
| Advantages: |
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| Instruction Manual |
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Other names：A2MG; A2mg; CPAMD5; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5
Function: Is able to inhibit all four classes of proteinases by a unique 'trapping' mechanism. This protein has a peptide stretch, called the 'bait region' which contains specific cleavage sites for different proteinases. When a proteinase cleaves the bait region, a conformational change is induced in the protein which traps the proteinase. The entrapped enzyme remains active against low molecular weight substrates (activity against high molecular weight substrates is greatly reduced). Following cleavage in the bait region a thioester bond is hydrolyzed and mediates the covalent binding of the protein to the proteinase.
Sequence：
MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL
LSYLNETVTV SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV
MFLTVQVKGP TQEFKKRTTV MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS
MDENFHPLNE LIPLVYIQDP KGNRIAQWQS FQLEGGLKQF SFPLSSEPFQ
GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT ILEEEMNVSV
CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF
YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL
SFVKVDSHFR QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD
EHGLVQFSIN TTNVMGTSLT VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL
VFSPSKSFVH LEPMSHELPC GHTQTVQAHY ILNGGTLLGL KKLSFYYLIM
AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL LIYAVLPTGD
VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV
DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDNE DCINRHNVYI
NGITYTPVSS TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLQQYEMHGP
EGLRVGFYES DVMGRGHARL VHVEEPHTET VRKYFPETWI WDLVVVNSAG
VAEVGVTVPD TITEWKAGAF CLSEDAGLGI SSTASLRAFQ PFFVELTMPY
SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE KEQAPHCICA
NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI
KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV
LGDILGSAMQ NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI
KSKAIGYLNT GYQRQLNYKH YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA
QARAYIFIDE AHITQALIWL SQRQKDNGCF RSSGSLLNNA IKGGVEDEVT
LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH GSHVYTKALL
AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ
APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS
TQDTVVALHA LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL
QQVSLPELPG EYSMKVTGEG CVYLQTSLKY NILPEKEEFP FALGVQTLPQ
TCDEPKAHTS FQISLSVSYT GSRSASNMAI VDVKMVSGFI PLKPTVKMLE
RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR DLKPAIVKVY
DYYETDEFAI AEYNAPCSKD LGNA
2. Features
INTENDED USE
The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of a2M in human serum, plasma, urine and other biological fluids.
DETECTION RANGE
0.781-50ng/mL. The standard curve concentrations used for the ELISA’s were 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.12ng/mL, 1.56ng/mL, 0.781ng/mL.
SENSITIVITY
The minimum detectable dose of a2M is typically less than 0.37ng/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.
SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of a2M.
No significant cross-reactivity or interference between a2M and analogues was observed.
Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between a2M and all analogues, therefore, cross reactivity may still exist.
IMPORTANT NOTES
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 of the kit and immunogen used for antibody preparation are commonly recombinant proteins. Different expressed sequence, expression systems, purification methods might be used in recombinant protein preparation. Besides, there might exist differences on the screening technique of antibody and antibody pairs in our kit. Thus we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to use the kit for the detection of recombinant protein.
12. Validity period: 12 months.
13. The instruction manual also works with the 48T kit, but all reagents in the 48T kit are reduced by half.
You can reference link of the kit as following
https://www.dldevelop.com/uploadfile/data/DL-a2M-Hu.pdf
Introduction
| Item | Standard | Test | |
| Description |
The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of a2M in human serum, plasma, urine and other biological fluids. |
Conform | |
| Identification | Colorimetric | Positive | |
| Composition | Traditional ELISA Kit | Ready-to-Use ELISA KIT | Conform |
| Pre-coated, ready to use 96-well strip plate 1 | Pre-coated, ready to use 96-well strip plate 1 | ||
| Plate sealer for 96 wells 2 | Plate sealer for 96 wells 2 | ||
| Standard 2 | Standard 2 | ||
| Diluents buffer 1×45mL | Standard Diluent 1×20mL | ||
| Detection Reagent A 1×120μL | Detection Solution A 1×12mL | ||
| Detection Reagent B 1×120μL | Detection Solution B 1×12mL | ||
| TMB Substrate 1×9mL | TMB Substrate 1×9mL | ||
| Stop Solution 1×6mL | Stop Solution 1×6mL | ||
| Wash Buffer (30 × concentrate) 1×20mL | Wash Buffer (30 × concentrate) 1×20mL | ||
| Instruction manual 1 | Instruction manual 1 | ||
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to the index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the index. 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 the index, 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 the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Recovery
Matrices listed below were spiked with certain level of recombinant a2M and the recovery rates were calculated by comparing the measured value to the expected amount of the index in samples.
| Matrix | Recovery range (%) | Average(%) |
| serum(n=5) | 81-93 | 86 |
| EDTA plasma(n=5) | 80-97 | 88 |
| heparin plasma(n=5) | 90-101 | 95 |
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the index and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
| Sample | 1：2 | 1：4 | 1：8 | 1：16 |
| serum(n=5) | 82-96% | 83-98% | 81-99% | 93-101% |
| EDTA plasma(n=5) | 88-101% | 86-95% | 90-102% | 80-93% |
| heparin plasma(n=5) | 80-91% | 82-90% | 95-104% | 79-95% |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%
Stability
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.
Note:
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.
Assay procedure summary
1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37℃;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
8. Add 50µL Stop Solution. Read at 450nm immediately.
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