Choosing the best antibody enzyme conjugate for your application is not always easy. We can help you decide on the most suitable conjugate for your applications.
Antibody Bioconjugation Product Description
Enzyme-Antibody Conjugation Chemistry
Most antibody that can be successfully purified while maintaining activity prior conjugation also will be stable enough to withstand the rigors of chemical modification. Occasionally, some monoclonal will be partially or completely inactivated through the modification reaction. Sometimes this activity loss is caused by physically blocking the antigen binding sites during conjugation or conformational changes in the complementarity-determining regions. Sometimes site-directed chemistry may resolve this issue, but other cases, some monoclonals are just too labile to undergo modification reactions, regardless of the coupling methods. Through the use of different coupling chemistries, Bio-Synthesis will try to maintain the activity of the antibody by directing the position of the label so that it does not interfere with the antigen binding site. Bio-Synthesis can only guarantees the quality of our conjugates but not its activities. For certain conjugation, it may be necessary to modify, purify an antibody or introduce functional groups and linkers to an enzyme or antibody with an additional fee. Modification such as:
- Aldehyde Introduction via Diol Oxidation
- Aldehyde Introduction through Amine
- Thiol Introduction through Traut's Reagent
- Thiol Introduction through SATA
- Disulfide Bond Reduction
- Fab Preparation
- F(ab')2 Preparation
For conjugation of one enzyme to protein/antibody, we typically use a heterobifunctional crosslinker:
- NHS ester-Maleimide
- Reductive Amination
- Oxime
- Dydrozone Formation
Service Specification:
Our enzyme conjugates are prepared by techniques that yield an approximate 1:1 ratio of enzyme to protein of interest, unless otherwise specified by the customer. After standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule may exist.
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Procedure:
After labeling of enzyme with the crosslinking reagent, final conjugates must first be isolated from excess or unreacted reagent by gel filtration or dialysis. In many cases, simple dialysis may suffice to remove unreacted reagent from the reaction solution. Additional purification technique such as stirred cell filtration, tangential flow filtration (TFF), gel filtration chromatography may also be used to either remove excess reagent or isolate and characterized the cross-linked product. With exception of dialysis, if the protein/antibody is significantly larger (>3-fold) than the modifying or coupling reagent. For reagents (mostly protein and other biological molecules) that are similar in size or larger than the antibody, one must resort to other purification techniques such as affinity chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography.
Cross-linked target molecule may then be further characterized by biochemical or biophysical techniques. Once the product has been purified, it may be subject to many different types of studies including spectroscopic (MALDI-top, ESI, LC-MS Fluorescence), electrophoresis, immunochemical biochemical, enzymatical analysis. QC (quality control) and QA (quality assurance) procedures are also followed independently to offer you double guarantee for the highest quality possible of every delivered conjugates. Moreover, our dedicated technical account managers will guide your project through every step of the process and constantly keep you informed of the latest project progress.
Enzyme Selection for Bioconjugation
Alkaline Phosphatase
Alkaline phosphatase (AP) is a hydrolyase enzyme that is frequently conjugated to antibodies for use in immunoassays. AP has a lower catalytic rate than HRP, but has greater stability and maintains linear reaction kinetics for much longer. PNPP is a popular colorimetric substrate for AP; the product of the reaction, p-nitrophenol, can be read at 405nM. Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate; MUP) may also be employed.
Horseradish Peroxidase
Horseadish peroxidase (HRP) has long been used as a colorimetric marker for antibody-based antigen detection assays- ELISAs, Westerns, Immunohistochemistry. It may be either directly conjugated to the antibody of interest or linked to a secondary antibody of interest.
Secondary conjugates are often employed to target a species specific antibody of interest (e.g., mouse, rabbit, goat, etc.). This method may save the researcher precious time in conjugation but secondary antibodies have their drawbacks. Secondary antibodies are known to significantly increase background signal through non-specific binding to antibody/antigen surface. For producing maximum specificity, with high signal to noise, it is always best to use direct primary antibody conjugates.
β-galactosidase
Beta-galactosidase (β-gal, beta-gal) is one of the most widely used reporter enzymes in molecular biology. It is shown to be a versatile new reporter enzyme exhibits a high turnover rate and can be used with substrates yielding both soluble and insoluble products making beta-galactosidase conjugates an excellent choice for photometric and electrochemical enzyme-multiplied assay techniques (EMATs). In addition, most tissues exhibit very low levels of endogenous enzyme activity. The well-known β-gal substrate analog, o-nitrophenyl β-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl β-d-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These β-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for β-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, β-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the β-gal-estradiol conjugate up to ~75%. Reporter enzyme inhibition is relieved significantly by addition of ≤2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (β-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that β-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.
Biotin
The attachment of biotin to biomolecules is an important laboratory technique. Biotin binds to the tetrameric avidin proteins, including streptavidin and neutravidin, with exceptionally high affinity, and this interaction is exploited in various applications such as western blotting, immunohisthochemistry and ELISA. Supplied antibody will be labeled with a long spacer arm biotin derivative follow by appropriate purification method. The final average ratio of antibody to biotin will be determined by performing HABA biotin quantification assay.
Glucose Oxidase
Glucose oxidase (GOx) is an enzyme produced and purified from Aspergillus niger which catalyses the oxidation of glucose with the release of hydrogen peroxide. It's molecular weight is 80kDa, but it exists as a dimer of 160kDa. Measurement of glucose by GOx is used in the food industry, in fermentation and, most importantly, as the basis of biosensors for the diagnosis of diabetes.