Advertisement Hide. This service is more advanced with JavaScript available. Bioconjugation Protocols Strategies and Methods. Editors view affiliations Christof M. Includes supplementary material: sn. Front Matter Pages i-xi. Front Matter Pages Vladimir V. Pages Silvia Muro, Vladimir R. Gradients of either pH or salt NaCl or both may be used as eluting systems.
An excess of PEG is generally not retained in such conditions whereas the different PEGylated protein species will be sequentially eluted according to their degree of modification, with the unmodified protein eluting as the last one. Pool the protein containing fractions and concentrate by lyophylization for further analysis.
Note that high amount of salts may be present in the eluted samples. Salts must be removed before complete dryness. This can be accomplished by ultrafiltration or gel filtration.
Characterization of Products 3. Note that electrospray ionization mass spectrometry is not easily applicable for the characterization of PEG—protein adducts. In fact, the polydispersivity of PEG makes the data interpretation rather complicated. This information would be useful for the precise characterization of the product when multiple Conjugates of Peptides and Proteins 61 PEGylation sites are available.
Furthermore, SDS electrophoresis is not applicable for high-molecular-weight conjugates because of the large contribution of the highly hydrated PEG molecule to the conjugate hydrodynamic radius. Both methods require prior purification of the conjugates from unreacted PEG and nonmodified protein using gel filtration or ion-exchange chromatography.
SDS electrophoresis generally overestimates the molecular weight of PEGylated species because of the large hydration volume of the polymer chain. PEGylated standards should be used for an accurate estimation of the molecular weight. Quantitative 1H-NMR is conducted by comparing the intensity of signals from the PEG backbone or from the terminal methoxy residue to the one of an internal standard e. PEG samples of know concentration 0.
Calculate protein concentration by UV absorbance. Again, calculate the ratio between 3. Compute the PEG concentration form the calibration curve obtained as above. Quantitative information can be obtained provided that a calibration curve is properly built in parallel with sample measurements.
It is important to note that the absorbance values in the assay change significantly with time, even seconds. When quantitative data are searched, the assay should be conducted with the aid of a timer in order to allow the exact time of interaction between each PEG sample and the reagent mix before the spectrophotometric evaluation.
In parallel, build a calibration curve by using between 2. Wait for 15 min at room temperature. Read the Abs at nm against a blank solution prepared with the same buffer or solution containing the PEG samples. Extraction of the chromophore into the organic phase occurs only in the presence of PEG, and the efficiency of extraction depends on polymer concentration. Therefore, a linear correlation between the organic phase color intensity Abs at nm and the PEG concentration in the assay is obtained.
Each unknown samples should be analyzed in parallel to solutions at known PEG having the same molecular weight concentration. This assay was never tested in our laboratory. Therefore, we only provide the protocol as described in the original publication Prepare a series of microcentrifuge polypropylene tubes and, to each of them, add equal volumes 0.
Mix vigorously for 30 min, and then centrifuge the tubes for 2 min at g. Separate the organic and aqueous phases and read spectrophotometrically the Abs nm of the organic one. Calculate the PEG concentration in the unknown sample on the basis of a calibration curve that is built in parallel. An aliquot of the Conjugates of Peptides and Proteins 63 purified sample is hydrolyzed by acidic treatment in a closed vial e. The first is an indirect one Subheading 3.
The second method Subheading 3. The main advantage of the first method is its rapidity of execution. However, risks of false positives exist because of the incomplete protein digestion inferred by the polymer surrounding the protein surface.
The second method is more precise because it is based on a standard procedure of sequence analysis, historically developed to reveal posttranslational modifications of proteins. Perform a proteolytic digestion on both nonmodified and PEG-conjugated protein. Use a few milligrams of each sample. Other proteolytic enzymes with different digestion specificity can be used according to the sequence requirements of the protein under investigation. Analyze and fractionate both native and PEG-conjugated digestion mixtures by reverse-phase or ion exchange-HPLC, as commonly conducted in protein sequence analysis.
Collect the peaks identified by UV. Compare the elution patterns of the modified and native digests. The identity of those peptides that are missing in the PEGylated protein digest can be identified by analyzing the corresponding peak in the non-PEGylated digest.
For this purpose, amino acid analysis, sequence, or mass spectrometry can be used. In the case of simple peptides of up to 30—40 amino acids, identification can be performed by 64 Morpurgo and Veronese direct sequence analysis Edman degradation of the PEG—peptide. Perform a CNBr treatment on a few milligrams of nonmodified and PEGylated samples that were previously freeze dried Dilute each mixture with water and dry it by evaporation.
Repeat this procedure two- to threefold. Perform chromatographic analysis and fractionation as described in Subheading 3. Analyze by MALDI or ion-spray mass spectrometry the new peaks appearing in the modified protein digest as compared with the nonmodified sample. Alternatively, perform amino acid analysis to reveal the presence of the reporter amino acid.
PEG reagents used in protein modification must be monofunctional, namely only one extreme of the polymer is reactive whereas the other one is capped with a stable methoxy residue mPEG.
Nevertheless, for simplicity, we are using the term PEG here to define any poly ethyleneglycol molecule, independently of its terminal functionality or shape. The polymer is formed by the anion polymerization started by CH3O— of ethylene oxide in dry conditions. When traces of water are present in the polymerization reaction, some of the polymer chains grow at both ends leading to a bifunctional product HO-PEG-OH as a contaminant having twice the molecular weight of the desired mPEG.
The presence of the bifunctional form can be detected by size-exclusion HPLC using a refraction index detector. Huntsville, AL. The last company is the most well-known and traditional supplier from which custom-made products are also available. The great majority of the hydroxyl and activated PEGs used so far in research or for the production of already-approved PEGylated drugs come from this source. Abundant literature Conjugates of Peptides and Proteins 65 and several recent reviews are available on this subject that can help a reader interested in preparing his or her own PEG reagents 5— The polymeric backbone of PEG is relatively stable at room temperature.
However, upon long storage oxidation can occur with formation of peroxide groups ending by breaking of the polymer chain. Moreover, because most of the reactive PEGs are also sensitive to moisture, we generally recommended storing any PEG in a dry, oxygen-free environment at low temperature.
Furthermore, those reagents that are sensitive to moisture or oxygen can rapidly loose their reactivity upon storage, especially if the compounds have been kept in the wrong environment. The amount of reagent to be added to the protein in the conjugation reaction mixture depends on the reactivity of the PEG derivative and the functional group on the protein.
As a general guideline, independently of the type of reactive function in the PEG, the larger and more sterically hindered is the polymer chain, the less reactive is its functional end-group. Polymer Sci: Polymer Chem. Farmaco 54, — Biomaterials 22, — Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase.
Jackson, C. Bioconjug Chem. Dolence, E. Conjugates of Peptides and Proteins 67 Anal Biochem. Lehninger, A. Worth Publishers Inc. Harris, J. Proceeding s of the 7th International Symposium on Recent advances in drug delivery systems. Sacca, B. Orsatti, L. Bioactive Compatible Polymers 14, — El-Tayar, N. US Patent Application no. Biochemistry 15, — Wang, Y. Biochemistry 39, 10,—10, Sivakolundu, S.
Sherman, M. Biotechnology 8, — Benhar, I. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.
Zalipsky, S. Song, S. Caliceti, P. Bioactive Compatible Polymers 8, 41— Bioactive Compatible Polymers 9, — Conjugates of Peptides and Proteins 69 Clark, R. B Biomed. Vestling, M. A mass spectrometric analysis of the attachment sites in polyethylene glycol-derivatized superoxide dismutase. Drug Metab. Biotechniques 16, — Chowdhury, S.
Nag, A. Sartore, L. Enzyme modification by MPEG with an amino acid or peptide as spacer arms. Biotechnol 31, — Udenfriend, S.
Science , — Stocks, S. Mabrouk, P. Lee, K. Fang, J. Cancer Res. Morpurgo, M. Both methods require the generation of purified F ab' 2 fragments of each antibody and use reagents that react with the free thiols generated upon reduction of interheavy chain disulfide bonds of the F ab' 2 fragments. After coupling, the bispecific antibody is purified from the uncoupled Fabs by size-exclusion chromatography. The advantages and disadvantages of each conjugation method are discussed.
Introduction Over the past two decades, bispecific antibodies BsAb —molecules combining two or more antibodies with different antigenic specificities—have been developed as tools for basic research as well as for clinical studies for reviews, see refs. A number of methods for producing BsAb have been developed. BsAb can be produced biologically by fusing two hybridoma lines, yielding quadromas that are capable of secreting BsAb. However, because of the various potential combinations of heavy and light chain pairing, only a small percentage of the molecules being secreted will have the appropriate bispecificity 9.
BsAb can also be generated genetically, and a variety of genetic techniques have been used to create bispecific molecules A third way to create BsAb is by chemical means. Nisonoff and Rivers pioneered the production of chemically linked BsAb over 40 yr ago Since then, many methods using a variety of homobifunctional and heterobifunctional chemical From: Methods in Molecular Biology, vol.
Both methods use reagents that react with the free thiols generated upon reduction of inter heavy chain disulfide bonds. The advantages and disadvantages of each of these methods will be discussed. Phosphate-buffered saline PBS , pH 7. Dimethyl formamide. PE: 0. HPLC equipment. Chromatography equipment. Sephadex G gel Pharmacia. Superdex gel Pharmacia. Purified F ab' 2 fragments of antibodies to be coupled. Both start from purified F ab' 2 fragments of each antibody to be coupled. Generation of F ab' 2 fragments is normally performed by pepsin digestion of the whole antibody; this method has been well-described and is beyond the scope of this chapter.
Chemical Production of Bispecific Antibodies 73 73 Fig. Before beginning the reduction, remove a sample of the F ab' 2 fragment and analyze it by size-exclusion HPLC to obtain baseline retention time Fig. Chemical Production of Bispecific Antibodies 75 Fig. B Creating a bispecific antibody using o-PDM that is bivalent for one specificity and monovalent for the second.
To monitor the progress of the reduction, remove an aliquot of the mixture, mix it with an equal volume of mM IAA solution, and inject onto the TSK analytical column. The IAA will serve to alkylate the free sulfhydral groups and prevent reoxidation of the Fab'.
The mixture is loaded onto the column and the protein peak is collected. Incubate for 30— 60 min at room temperature. Chemical Production of Bispecific Antibodies 77 Fig. Conjugation Reaction 1. Monitor the conjugation reaction by analytic HPLC. Purify the bispecific fraction from the uncoupled Fabs by running it over a Superdex column that has been equilibrated in PBS see Note 3. Gels were run under nonreducing A or reducing conditions B.
Under reducing conditions the BsAb migrates as two bands, one representing the Fd fragment at apparent MW of 31—33 kDa and the second representing the light chain at an apparent MW of 25 kDa Fig. One significant disadvantage of using o-PDM is its requirement of having an odd number of interheavy chain bonds in the Ab to be maleimidated see Fig.
The condition for reduction of the F ab' 2 fragments of the antibodies and buffer exchange to be coupled are essentially the same as for the DTNB reaction with the exception that the G columns are equilibrated with SACE buffer instead of PE buffer.
To monitor the progress of the reduction remove an aliquot of the mixture, mix it with an equal volume of mM IAA solution and inject onto the TSK analytical column. Store the Fab' on ice.
Measure the volume of the buffer exchanged Fab' Ab B and store on ice. Make a 12 mM 3. The final o-PDM concentration will be 4 mM. Incubate for 30 min on ice. The column should be chilled by having ice water pumped through the jacket.
Determine the protein concentration of the derivatized Fab' by taking the OD Incubate for at least 12 h on ice. Monitor the progress of the conjugation reaction by size-exclusion HPLC. Purify the bispecific fraction from the uncoupled Fabs by running it over a Superdex column that has been equilibrated in PBS see Note 6. Two major peaks are shown in the figure at retention times of The identity of these peaks is discussed in Note 7. The primary species migrates at an apparent MW of — kDa under nonreducing conditions and represents the F ab' 2 BsAb.
However, several bands appear on the nonreduced gel. The nature of these species and the bands shown on under reducing conditions are discussed in Note 5. Small-scale trial reduction should be performed to determine the optimal 82 Graziano and Guptill Fig. Conditions should be chosen such that efficient reduction of the inter heavy chain disulfides in achieved without extensive reduction of heavy-light chain disulfide bonds. These results confirm efficient separation of the Fab' from free MEA.
G and Superdex columns can be sanitized by running at least three column volumes of 0. The columns should be adequately equilibrated in the appropriate buffer before loading the sample.
Sanitizing should remove undesirable contaminating endotoxin. Of course, care should be taken to make buffers using endotoxin-free reagents. Both methods create BsAb that are coupled at a defined site, the hinge region sulfhydral, which should not affect the affinity of the respective Fabs. BsAb created using the o-PDM method may be more stable because of the formation of a thioether bond 12 , and yields are Chemical Production of Bispecific Antibodies 83 Fig.
Another distinct disadvantage of the o-PDM method is the necessity to have an odd number of inter heavy chain disulfide bonds in the antibody molecule to be maleimidated. This prevents its application in the construction of human—human BsAb unless the hinge region of the human antibody is altered. Other species may represent F ab' 3 or F ab' 2 species that may have lost a noncovalently linked light chain Under reducing conditions, four bands are observed with this BsAb.
The band that migrates at an apparent MW of approx 90 kDa most likely represents three heavy chains from the F ab' 3 BsAb species, which are linked covalently by thioether bonds. The second band that migrates at an apparent MW of approx 65 kDa most likely represents two heavy chains from the F ab' 2 BsAb species, which are linked covalently by thioether bonds. The bands running at 25 kDa and at 28 kDa represent the light chains from each of the Abs that were coupled, which, in this instance, run at slightly different apparent MW.
Purification of bispecific antibody by size-exclusion chromatography using Superdex gel normally gives adequate separation of the bispecific Ab from uncoupled free Fabs and small molecules o-PDM, IAA. However, other methods that have been developed to purify bispecific of interest such as affinity or ion exchange chromatography may be employed. Acknowledgments We are grateful to Dr. Aditya Mandel for providing data shown in Figs.
Fanger, M. Landes Co. Cancer Immunol. Chemical Production of Bispecific Antibodies 85 4. Immunobiology , — Today 21, — Cancer J. Chamow, S. Graziano, R. Landes Company, Austin, TX, pp. Science , 81— Tutt, A. Niemeyer Antibody Oligosaccharide Derivatization 87 6 Preparation of Immunoconjugates Using Antibody Oligosaccharide Moieties Carl-Wilhelm Vogel Summary Heterobifunctional crosslinking reagents are small molecular weight chemicals containing two different reactive groups that have become important tools in generating conjugates of two different biomolecules, such as two proteins.
The resulting bioconjugates are hybrid molecules or proteins, a new category of biomolecules that exhibit the combined functions of the two parent biomolecules. An important category of hybrid proteins are conjugates of antibodies with other effector molecules, such as drugs or toxins. These antibody conjugates or immunoconjugates have a variety of the applications in medicine, with particular emphasis on the treatment of cancer. The most commonly used heterobifunctional crosslinking reagents for the synthesis of antibody conjugates contain an N-hydroxysuccinimide ester moiety, which allows derivatization of amino groups in proteins.
The chemical modification of a functionally important amino group in the antigen-binding region of an antibody causes impairment or loss of the antigen binding function, resulting in a defective antibody conjugate that lacks one of its component functions. Furthermore, even if the chemical derivatization does not affect the antigen binding function, the subsequent coupling of an effector protein at or near the antigenbinding region can also cause the loss of the antigen binding function for steric reasons.
In this chapter, heterobifunctional crosslinking reagents are described that allow the generation of antibody conjugates where the effector proteins are coupled to the antibody carbohydrate moieties. Because antibody carbohydrate moieties are distal from the antigen-binding region, the use of carbohydrate-directed heterobifunctional crosslinking reagents, such as S- 2thiopyridyl -L-cysteine hydrazide TPCH , prevents inactivation of the antigen-binding function.
The synthesis of two carbohydrate-directed heterobifunctional crosslinking reagents is described. Coupling protocols for the preparation of antibody conjugates with effector proteins of different sizes using carbohydrate-directed heterobifunctional crosslinking reagents are also provided.
Key Words: Antibody conjugates; carbohydrate-directed derivatization; crosslinking; crosslinking reagents; heterobifunctional crosslinking reagents; hybrid proteins; immunoconjugates; immunotoxins; oligosaccharide moieties; protein derivatization; protein—protein conjugation; site-directed conjugation; regio-specific conjugation.
Introduction Derivatization, coupling, and immobilization of biomolecules—and biological macromolecules in particular—have been the subject of intense research for at least two decades with the intent of developing new applications for biological molecules in biotechnology and medicine.
Conjugates consisting of two or more biological macromolecules can be created by recombinant means if the biomolecules involved are proteins, or they can be generated by chemical means. Bioconjugates represent a novel and interesting category of chemicals because they represent hybrids of biological molecules that do not exist in nature but are synthesized by combining two or more naturally occurring biological macromolecules into a new chemical compound.
An important category of semisynthetic hybrid proteins are conjugates of antibodies with a host of other molecules, such as drugs, toxins, chelating reagents, enzymes, and biological response modifiers 1—3.
These antibody conjugates, often referred to as immunoconjugates, have a variety of applications in medicine, with particular emphasis on the diagnosis or treatment of cancer. Several antibody conjugates are used successfully in cancer therapy 4,5.
The field of immunoconjugate research has received a significant boost with the availability of heterobifunctional crosslinking reagents. These are smallmolecular-weight chemicals that contain two different reactive groups, each of which is able to react with a chemically different functional group in a biological macromolecule 6,7.
Approximately heterobifunctional crosslinking reagents have been prepared and, for the most part, are commercially available. The vast majority of heterobifunctional reagents contain a chemical moiety that reacts with amino groups in proteins and a second chemical moiety that reacts with free sulfhydryl groups.
The amino-reactive group is almost exclusively an N-hydroxysuccinimide ester, whereas three sulfhydryl-reactive groups are commonly used: the pyridyldithio group, the maleimide group, and an aliphatic halide iodide. This is not the place to provide a detailed review of the various properties of the different heterobifunctional crosslinking reagents, the chemical nature of the resulting intermolecular crosslinks, and their chemical and biochemical properties.
Suffice it to say that heterobifunctional crosslinking reagents containing the pyridyldithio group generate an intermolecular crosslink with a disulfide bond, whereas reagents containing maleimide or halide groups result in the formation of a thioether bond.
Other differences relating to the chemical nature Antibody Oligosaccharide Derivatization 89 of the intermolecular crosslink include length, charge, solubility, aromaticity, and stability to reduction, enzymatic cleavage, and pH 6,7.
One advantage inherent to all heterobifunctional crosslinking reagents is the fact that they result in the formation of heteroconjugates, which means that the resulting conjugates contain at least one molecule each of the two biomolecules to be coupled. The design of the heterobifunctional reagents prevents the formation of homoconjugates, that is, the formation of conjugates consisting of only one of the two protein species intended to be coupled.
However, heterobifunctional crosslinking reagents do not generate hybrid proteins consisting of only one protein molecule each of the two molecular species to be coupled. For example, an immunoglobulin G antibody molecule has at least 70 amino groups. Accordingly, derivatization of a protein with an amino groupdirected heterobifunctional crosslinking reagent results in the modification of several or even many amino groups which, in turn, allows for the subsequent coupling of multiple protein molecules of the second coupling partner with free sulfhydryl groups.
If the free sulfhydryl group-containing protein contains only one usually naturally occurring free sulfhydryl group, its coupling to the amino group-derivatized protein results in a mixture of hybrid proteins of the molecular composition , , , , , and so on. If a protein does not contain one or more natural free sulfhydryl groups, these can be introduced by a crosslinking reagent e.
SPDP derivatizes amino groups, resulting in the introduction of pyridyldithio groups. Subsequent reduction of the pyridyldithio groups results in free sulfhydryl groups 8.
When a protein with introduced free sulfhydryl groups is coupled to another protein derivatized with sulfhydryl-reactive groups, the resulting conjugates represent mixtures of hybrid proteins of the molecular composition , , , , , , , , , and so on. As much as heterobifunctional crosslinking reagents with one of the reactive groups being an amino-reactive group allow for easy and multiple derivatization of proteins, they exhibit one inherent drawback. Chemical structures of corresponding carbohydrate-directed left and amino group-directed right heterobifunctional crosslinking reagents.
Upper panel, crosslinking reagents introducing a pyridyldithio group. Lower panel, crosslinking reagents introducing a maleimide group. Whereas a protein derivatized at a functionally important amino group can still be incorporated into a hybrid protein, the derivatized protein has lost its function, resulting in the creation of a hybrid protein that lacks one of its component functions. Antibody Oligosaccharide Derivatization 91 In addition to functional inactivation of a protein by direct chemical modification of functionally important amino groups, additional functional inactivation of a coupling partner can be caused by steric hindrance after incorporation of a protein into a hybrid protein.
For example, in the case of antibodies, both chemical derivatization with the crosslinker at the antigenbinding site and conjugation of the coupling partner at or near the antigenbinding site will impair the antigen-binding function of the particular Fab component of the resulting antibody conjugate. One successful approach to avoid both chemical and steric inactivation of the antigen-binding function of an antibody is to couple the other protein to the oligosaccharide moieties of antibodies, which are located distal to the antigen binding sites.
In this chapter, the generation of antibody conjugates with other proteins is described using heterobifunctional crosslinking reagents where one reactive group is a hydrazide that binds to aldehyde groups generated in the oligosaccharide moieties of antibodies by periodate oxidation of cis-diol groups e.
This crosslinking approach prevents the functional inactivation of antibody-binding sites as will be shown further below 9, Chemicals 1. All chemicals were obtained from Aldrich Milwaukee, WI. With the exception of TPCH, all heterobifunctional crosslinking reagents mentioned in the manuscript are commercially available from Pierce Rockford, IL.
N,N'-bis- tert-butyloxycarbonyl -L-cystine dimethyl ester was obtained by protecting the amino groups of L-cystine methyl ester with di-tert-butyl pyrocarbonate Proteins 1. Human monoclonal IgM antibody 16—88, derived from a patient immunized with autologous human colon carcinoma cells, was used in this study 12, The antibody is obtained from hollow fiber culture and purified by gel filtration and ion-exchange chromatography A solution of 2. The solution is maintained at room temperature for 2 h, over which time a fine white material precipitates.
The yield is 2. A suspension of The suspension dissolves upon warming. The clear, colorless solution is refluxed for 30 min and then allowed to cool to room temperature. After 20 min, the product begins to crystallize from solution. The white crystalline product is collected by filtration and washed with ice-cold ethanol. The yield is At least three rotamers can be identified in the spectrum.
Zinc dust 3 g is added in portions over 2 h to a suspension of Gradually, the suspension dissolves. Antibody Oligosaccharide Derivatization 93 2. After 2 h the solution is concentrated under reduced pressure, and the residue is partitioned between methylene chloride and saturated aqueous sodium bicarbonate.
The methylene chloride is dried over sodium sulfate and concentrated to a viscous glass. The yield is 10 g This solution is maintained at room temperature for 24 h and then concentrated in vacuo to a yellow syrup.
The crude product is taken up in mL of methanol, and 20 g of silica gel 32—60 mesh is added. Fractions mL each containing product are pooled and concentrated to provide a colorless glass. The yield is 3. Preparation of TPCH 1. A solution of 1. After 30 min, a white crystalline material begins to separate. The mixture is stirred at room temperature for 4 h. The mixture is filtered under argon, washed with ethyl acetate, dried under argon, and then under vacuum to provide hygroscopic white crystals.
S- 2-thiopyridyl mercaptopropionic acid 4 mmol; 0. After the addition of 4 mmol 0. N-tert-butyloxycarbonyl-S- 2-thiopyridyl mercaptopropionic acid hydrazide 0. The yield is 0. Antibody Derivatization With Heterobifunctional Crosslinkers 3. The eluted antibody 1. The extent of antibody derivatization with TPCH has virtually no measurable effect on the antigen binding activity of the antibody see Note 1. It is important to perform the periodate oxidation in the presence of the TPCH crosslinker see Note 3.
Other carbohydrate-directed heterobifunctional crosslinking reagents with a maleimide function as sulfhydryl-reactive group have been described see Note 5. The IgM antibody 2 mg, 2. The extent of antibody derivatization with SPDP affects the antigen-binding activity of the antibody see Note 1. The SPDP concentration needs to be adjusted depending on the molecular weight of the protein to be derivatized see Note 4. Preparation of Antibody Conjugates 3. The reaction mixture containing crosslinker-derivatized antibody 1.
After purification of the antibody conjugates by size-exclusion chromatography using a Fractogel HW65F column 1. Once free sulfhydryl groups are introduced, the proteins should be immediately subjected to coupling. The coupling reaction vial should be flushed with nitrogen. Once coupled, the antibody conjugates exhibit good stability see Note 6.
Ricin A-chain 1. The coupling efficiency of ricin A-chain is relatively low compared to barley toxin, a protein of similar size see Note 7. Subsequently, the pyridyldithio groups of the SPDP-derivatized barley toxin 4. Sulfhydryl-derivatized barley toxin 1. The incubation and subsequent purification of the antibody conjugates is performed as described above for ricin A-chain 9.
Antibody Oligosaccharide Derivatization 97 3. The extinction coefficient of pyridinethione at nm is 8. Determination of the Stoichiometry of Effector Proteins to Antibody Sodium dodecyl sulfate SDS gradient polyacrylamide gel electrophoresis is a good method to get a rough estimate of the molecular composition of the hybrid protein mixture after conjugation 18— To determine the average ratio of effector molecule per antibody molecule, I-labeled effector protein at approx 1. Stoichiometries can then be determined from the difference in specific radioactivity before and after conjugation.
The toxin to antibody stoichiometry for smaller effector molecules such as ricin A-chain or barley toxin can also be obtained by scanning gel densitometry after SDS polyacrylamide gel electrophoresis under reducing conditions 9, Other Methods 1.
The antigen binding activity of IgM antibody 16—88 is determined in a competitive binding assay with I-labeled antibody using microtiter plates with immobilized tumor antigen The complement activating activity of IgM antibody 16—88 is determined in a modified complement fixation assay using immobilized tumor antigen extract and human serum as a complement source. Sensitized sheep erythrocytes are used to determine the remaining serum complement activity The CVF hemolytic activity is determined in a bystander lysis assay using guinea pig erythrocytes Barley toxin activity is determined in a cell-free reticulocyte assay based on the inhibitory activity of barley toxin on protein translation 9.
Protein concentrations are determined by the Lowry method see Note 9; ref. Shown is the ability of unmodified antibody open circles and derivatized antibody filled symbols to bind antigen in a competition binding assay with I-labeled antibody.
SPDP-derivatized antibody has 2 filled circles , 8. TPCH-derivatized antibody has 1. Modified from ref. Figure 2 shows the effect of derivatization of human monoclonal IgM antibody 16—88 with the carbohydrate-directed crosslinker TPCH compared to the amino group-directed crosslinker SPDP. Samuel H. Weisbrod, Anna Baccaro, Andreas Marx.
Musser, Andreas Herrmann. Chemically Selective Liposome Surface Glyco-functionalization. Page 1 Navigate to page number of 3. Biofunctionalization Biomaterials engineering Biomedical diagnostics Labeling techniques Nanomaterials Organic synthesis Surface biotechnology Therapeutics. Editors and affiliations. Sonny S.
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