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血藍蛋白/KLH
產品名稱:血藍蛋白/KLH
品牌 :biosyn
貨號:KLH-NP
規格:10mg
價格:600元
詳細信息

血藍蛋白(KLH)來源于大鎖孔帽貝(透孔螺)的血淋巴,也被叫做匙孔血藍蛋白,是具有高度免疫原性的蛋白大分子,也是一種多功能蛋白,它不僅具有輸氧功能,而且還與能量的貯存、滲透壓的維持及蛻皮過程的調節有關,還具有酚氧化物酶活性和抗菌功能,被認為是節肢動物和軟體動物中的一種重要的免疫分子。
KLH廣泛作為載體蛋白應用,交聯于半抗原和其他抗原,使它們具有更強的免疫原性以用于抗體的制備。由于其質量大,復雜性高,與其他載體蛋白相比KLH會引出更強的免疫應答。因為此蛋白來源于軟體動物,它在系統發生上遠離哺乳動物物種,在測定過程中產生會與典型靶點樣品發生交叉反應的抗體的可能性較小。作為大的蛋白質,KLH含有上百個伯胺和羧基基團可以成為與戊二醛,N-羥基琥珀酰亞胺酯和EDC等交聯試劑交聯的靶點。
傳統凍干粉狀態血藍蛋白(KLH)會產生大量沉淀并可能導致在使用該產品過濾沉淀時損失高達30%-40%, 好的血藍蛋白(KLH)制劑溶液應在結合前極少沉淀,從而可產生高效的偶聯和增強抗體反應。

高分子量的天然KLH在近些年中得到了廣泛的應用。但是大多數可獲得的KLH配方并不適合在人體臨床試驗中使用。通常這些產品中的內毒素含量很高,因為血淋巴血清的生產是從冷凍動物中提取來完成的,在凍干KLH過程中可能導致沉淀產生。部分文獻表明,冷凍干燥天然KLH都會導致活性的喪失。
為了KLH產品的安全性、質量和一致性,Biosyn開發了非致命性血淋巴血清采集程序,并制定了必要的動物處理和檢疫技術,制定了這種關鍵原料規模生產的質量控制方法,在磷酸鹽緩沖液中生產低內毒素和高濃度天然KLH??蒲屑墑eNative KLH是最常用的免疫載體蛋白,適用于生物活性測定、免疫學研究;同時也可用于制備多肽抗原,以進行免疫和抗體生產,疫苗產品開發等項目。

 

與同類產品相比,biosyn公司KLH產品具有以下特點:
獨有的捕獲和處理活動物的流程,不使用冷凍動物等致命提取方法,有利于KLH分子保持穩定。
在緩沖液體系中配制,比其他凍干粉產品具有更好的溶解性。
診斷級別KLH嚴格通過生物安全性和包括甲乙丙型肝炎、弧菌、輪狀病毒、諾沃克病毒在內的無病毒檢測。
高分子量,高免疫原性(400kDa)

 

Biosyn公司于1995年創立于美國加州,是一家專業研發和生產高質量的科研和臨床級別血藍蛋白的生物醫藥公司。目前biosyn公司生產的血藍蛋白在大型國際醫藥公司中有約30%的市場占有率。biosyn公司的GMP級產品IMMUCOTHEL®率先于1997獲得荷蘭相關批文,而后也相繼被韓國、阿根廷、奧地利批準進入市場。biosyn公司另一產品VACMUNE®也已于2007年向美國FDA提交了DMF文件。

biosyn公司在海水中養殖的軟體動物大鎖孔帽貝;在GMP級別廠房生產的KLH能提供極好的免疫原性,以及出色的穩定性和溶解性。

 

KLH產品部分文獻:

Henry Milgrom, Int Arch Allergy Immunol. 2012 Feb; 157(3): 269–274. 2011 Oct 28.  PMID: 22042247.
Federico Baruffaldi,Mol Pharm. 2018 Nov 5; 15(11): 4947–4962. PMID: 30240216.
Ashwin Swaminathan, Br J Clin Pharmacol. 2014 Nov; 78(5): 1135–1142. PMID: 24833186.
Jeffrey S.Miller. Clinical Immunology. Volume 117, Issue 2, November 2005, P144-151.
Brendan D. Curti, Cancer Research.  December 2013.
Rong Shi PhD  Marek Honczarenko MD, PhD.  Clinical Pharmacology. 12 July 2016. 
Willemijn Hobo, Frans Maas, Blood 2010 116:4501-4511.
Michal Radomski, Herbert J. Zeh. Journal for ImmunoTherapy of Cancer. 19 April 2016.
 

KLH: Keyhole Limpet Hemocyanin
The hemocyanin produced from the hemolymph sera of the marine mollusk Giant Keyhole Limpet, Megathura crenulata, has been in use and marketed as a crude or partially purified product for over 40 years by some chemical companies. This molecule is well recognized and commonly abbreviated as KLH. The crude research grade KLH is used in antibody production in animals against antigens. The animal sera containing the antibody for the antigen are further processed and used as immunological reagents or in immunological assays. The partially purified product has also been used in early human trials for immune status evaluation.

KLH structure
The native KLH is a cylindrical copper containing blue protein with a molecular mass ranging from 8-32 MDa (Million Dalton). Electron microscopy structural studies reveal presence of two oligomeric forms:

Didecamers with a molecular mass of 8 MDa, are present in the form of hollow cylinders. They are approximately 35 nm in diameter and 40 nm in length.
Multidecamers with a molecular mass of 12-32 MDa, present as stacked decamers (multidecamers) of varying length, added on one or both sides of a "nucleating" didecamer. Single decamers exhibit rectangular side views.
Keyhole Limpet Hemocyanin Structure


Prof. Dr. Jürgen Markl, Direktor Zoologisches Institut der Johannes Gutenberg-Universität Mainz, Germany
The didecamer consists of 20 subunits and occurs in two different Isoforms named KLH1 and KLH2. The subunits are biochemically and immunologically distinct. Analysis of KLH by native-PAGE gives two characteristic bands, one corresponding to KLH1 with an apparent molecular mass of about 390 kDa (kilo Dalton) and the other corresponding to KLH2 with an apparent molecular mass of 350 kDa.

biosyn native KLH, low enodoxin content
The high molecular weight, native KLH, is extensively used and has been used over the last several years. Native KLH has been manufactured and supplied by some chemical suppliers. 

However, most available KLH formulations are not of the required quality and consistency suitable for use in human clinical trials. Typically, the endotoxin content in such products is very high as the hemolymph sera production is done by cutting open the animals or extraction from frozen animals. The product characteristics are also not very good and could lead to precipitation during reconstitution of lyophilized KLH. It is well documented that either freezing or lyophilization of native KLH leads to loss in activity. 

Characterization of native KLH is also an issue as the large molecular weight of 8-32 MDa does not allow use of standard biochemical methods for routine quality control, which is further complicated by the use of excessive metal ions like calcium and magnesium, presumably to stabilize the molecule. 

Right from the start of the hemocyanin development work in 1985, biosyn had identified these to be critical issues and developed appropriate strategies to overcome these limitations. To achieve the desired safety, quality and consistency of native KLH, biosyn has developed the non-lethal hemolymph sera collection procedure from live animals and instituted necessary animal handling and quarantine techniques. Quality control methodologies for routine production of this critical raw material has been developed. 

The end result of these technology developments in the animal handling, bleeding and manufacturing process was the production of biosyn low endotoxin and high concentration native KLH of consistent high quality in a phosphate buffer.

The biosyn native KLH is supplied as High Purity Grade and Research Grade (no endotoxin testing). The GMP grade material is ideally suited for use as a carrier protein in the manufacture of vaccines for human use.  It is tested for biological safety, viral clearance and heavy metal ions.

The Research Grade material is suited for use in vaccine product development activities and also for routine immunological studies, antibody production, production of activated KLH and other developmental activities. The native KLH formulations currently available from biosyn are listed in the table.

Product Grade Uses
Native KLH in Phosphate buffer low endotoxin, tested for biological safety, viral clearance and metal ions GMP
Clinical
grade
For further processing in KLH-conjugate vaccine manufacture for human and animal use
Native KLH in Phosphate buffer, or other buffers low endotoxin RG Routine immunological and antibody studies and antibody production, initial product development activity for conjugate vaccine. Preparation of Research Grade activated KLH
Native KLH in Phosphate buffer or other buffers. No endotoxin testing RG Low cost research grade material for research uses

RG = Research Grade

 

Why use biosyn KLH?
biosyn KLH has many advantages over other commercial grade material that can be procured from other vendors. The major distinction lies in biosyn's:

Environmentally friendly process used in the extraction of hemocyanin from animals.
biosyn returns the animals back to its natural habitat, as it is a humane approach as well ensures that the animals recovers readily and more impotantly assures the product quality and reproducibility leading to no potential undefined, difficult to characterize, structural alterations to the hemocyanin molecule.
biosyn uses a proprietary methodology and has built-in a test procedure for harvesting and handling of live animals, unlike the use of dead or frozen animals or lethal extraction procedure used by some commercial KLH suppliers.
biosyn hemocynin and final products are therefore of high purity and low endotoxin quality.
biosyn KLH subunits, immunocyanin, are of low molecular weight (400 kDa) and are a completely characterized product.
biosyn manufactures and packages KLH products to meet specific customer requirements including userfriendly, ready-to-use preparation and packaging.
biosyn hemocyanin and immunocyanin are formulated in either pure buffer systems like phosphate buffer or in water and packaged as small aliquots or bulk quantities. This offers customers major advantages, i.e., flexibility of use, choice of buffer in early developmental studies, and avoidance of issues associated with reconstitution of dry powder product obtained from other vendors.
biosyn Immunocyanin in water is a unique formulation and overcomes one of the major drawbacks of using KLH in clinical and commercial grade conjugate vaccine products.
biosyn clinical grade products are extensive tested for safety and lack of viruses including hepatitis A, B, C, vibrio species, rota virus and Norwalk viruses.

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