人亮氨酰氨基肽酶（LAP）ELISA Kit

【資料簡(jiǎn)介】

產(chǎn)品特例：人亮氨酰氨基肽酶（LAP）試劑盒
英文名：HumanLAP ELISA KIT
檢測(cè)范圍：參見(jiàn)資料下載

本試劑盒用于測(cè)定人血清、血漿及相關(guān)液體樣本中亮氨酰氨基肽酶（LAP）含量。
實(shí)驗(yàn)原理
本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人亮氨酰氨基肽酶（LAP）水平。用純化的人亮氨酰氨基肽酶（LAP）抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入亮氨酰氨基肽酶（LAP）），再與HRP標(biāo)記的亮氨酰氨基肽酶（LAP）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的亮氨酰氨基肽酶（LAP）呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中人亮氨酰氨基肽酶（LAP）濃度。

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業(yè)務(wù):   何

INTRODUCTION

 

 LAP is a multifunctional protein that controls proliferation, differentiation and other functions in many cell types. Many cells synthesize TGFB1 and have specific receptors for it. It positively and negatively regulates many other growth factors. In addition, LAP plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation, and differentiation in committed osteoblasts. The precursor is cleaved into mature TGF-beta-1 and LAP, which remains non-covalently linked to mature TGF-beta-1 rendering it inactive.

 The inactive form consists of a TGFB1 homodimer non-covalently linked to a latency-associated peptide （LAP） homodimer. While the inactive complex can contain a latent TGFB1-binding protein, the active form is a disulfide-linked homodimer of mature TGFB1. Heterodimers of TGFB1/TGFB2 have been found in bone and interacts with CD109, DPT, and ASPN. LAP is highly expressed in bone, abundantly expressed in articular cartilage and chondrocytes and is increased in osteoarthritis （OA）. It also co-localizes with ASPN in chondrocytes within OA lesions of articular cartilage.

 Defects in TGFB1 are the cause of Camurati-Engelmann disease （CE）; also known as progressive diaphyseal dysplasia 1 （DPD1）. CE is an autosomal dominant disorder characterized by hyperostosis and sclerosis of the diaphyses of long bones. The disease typically presents in early childhood with pain, muscular weakness and waddling gait, and in some cases other features such as exophthalmos, facial paralysis, hearing difficulties and loss of vision.

 

Source: Entrez Gene; Swiss-Prot

  

 

ASSAY PRINCIPLES

 

 The OmniKine™ Human LAP ELISA Kit contains the components necessary for quantitative determination of natural or recombinant hLAP concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a “Sandwich” Enzyme-Linked Immunosorbent Assay （ELISA） where the target protein （antigen） is bound in a “sandwich” format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on the Human LAP cytokine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and “sandwiching” of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody （either covalently or via Avidin/Streptavidin-Biotin interactions）, allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB （3, 3’, 5, 5’-Tetramethylbenzidine） is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution （2 N Sulfuric Acid） where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration. 

 

 

ASSAY RESTRICTIONS

 

?This ELISA kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind.

?Materials included in this kit should NOT be used past the expiration date on the kit label.

?Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.

?Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided. 

?The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.

 

 

ADDITIONAL MATERIALS REQUIRED

 

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

 

?Microplate reader able to measure absorbance at 450 nm （with correction wavelength set to 540 nm or 570 nm）

?Micropipettes with capability of measuring volumes ranging from 1 μl to 1 ml

?Deionized or sterile water

?Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer 

?Graph paper or computer software capable of generating or displaying logarithmic functions

?Absorbent paper or vacuum aspirator

?Test tubes or microfuge tubes capable of storing ≥1 ml

?Bench-top centrifuge （optional）

?Bench-top vortex （optional）

?Orbital shaker （optional）

 

 

 

HEALTH AND SAFETY PRECAUTIONS

 

?Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.

?Stop Solution contains 2 N Sulfuric Acid （H2SO4） and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of