Hapten-carrier protein conjugated

Since, the broad protein and carbohydrate band overlapped each other in the 42 - 200 kDa range of SDS-PAGE and in the pH 3.0 - 4.3 range of IEF-PAGE (data not shown), MALDI MS of ARS2 was performed as previously reported for hapten-carrier protein conjugates For more detail MW determination [29-31],... 

Polyclonal antibodies Antibodies derived from a mixture of cells, hence containing various populations of antibodies with different amino acid sequences. They are of limited use in that they will not all bind to the same epitopes following immunization with a hapten carrier protein conjugate. They are also difficult to purify and characterize, but have been used with success in the catELISA system. 

Standard immunizations of mouse involve two injections, or boosts , of a hapten-carrier protein conjugate together with an adjuvant at a 15 days interval. At least eight weeks after the second boost, immune cells are stimulated by a final intravenous injection of hapten-conjugate, and the fusion carried out 4 days later to generate hybridoma from spleen cells (Fig. 7). Experiments with catalytic polyclonal antibodies [103] have shown that over-immunization re-... 

The epitope, considered as an allergenic determinant, may also consist of the shape of the hapten-carrier protein conjugate. The identical or very close, three-dimensional shape of the different epitopes explains the possibility of cross-immune response. On the contrary, a geometric conformation change, which can sometimes be secondary, may prevent a specific recognition by the immune system. Some examples will illustrate this concept. First, let s study some examples of similarities. 

Haptens with an amino group. Amine groups in haptens, carrier proteins or both can be modified for conjugation through homo- or heterobifunctional crosslinkers such as acid anhydrides (e.g., succinic anhydride), diacid chlorides (e.g.. 

We used 1,3-diketone hapten 1-carrier protein conjugate for immunization in our experiments to generate aldolase antibodies (Scheme 6.1) [4]. Class I aldolases use the e-amino group of a lysine in their active site to form a Schiff base vith one of their substrates and this substrate becomes the aldol donor substrate. Schiff-base formation reduces the activation energy for proton abstraction from the Ca atom and for subsequent enamine formation. The enamine, a carbon nucleophile, then reacts vith an aldehyde substrate, the aldol acceptor, to form a ne v C-C bond. The Schiff base is then hydrolyzed and the product is released [2]. The 1,3-diketone hapten acts as a mechanism-based trap of the requisite lysine residue in the active site and this lysine is necessary for formation of the essential enamine intermediate. The molecular steps involved in trapping the requisite lysine residue are essentially the same chemical steps as are involved in activating... 

Antibodies prepared from hybridomas derived after immunization vith 1,3-diketone hapten 1-carrier protein conjugate vere screened for their capacity to form the stable enaminone (absorption maximum = 316 nm)... 

Hapten density is important for both immunization and assay performance, and hence the extent of conjugation or hapten density should be confirmed by established methods. A characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or use of a radiolabeled hapten can be used to determine the degree of conjugation. If the hapten has a similar A. iax to the protein, the extent of incorporation can still be estimated when the concentration of the protein and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to... 

Raising the pi of macromolecules also can significantly alter the immune response toward them upon in vivo administration. Cationized proteins (those modified with diamines to increase their net charge or pi) are known to generate an increased immune response compared to their native forms (Muckerheide et al., 1987a, b Apple et al., 1988 Domen et al., 1987 Domen and Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can result in a dramatically higher antibody response toward a coupled hapten (Chapter 19). 

The Mannich reaction can be used for the immobilization of certain drugs, steroidal compounds, dyes, or other organic molecules that do not possess the typical nucleophilic groups able to participate in traditional coupling reactions (Hermanson et al., 1992). It also can be used to conjugate hapten molecules to carrier proteins when the hapten contains no convenient nucleophile for conjugation (Chapter 19, Section 6.2). In this case, the carrier protein contains the primary amines and the hapten contains at least one sufficiently active hydrogen to participate in the condensation reaction. 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

Figure 19.6 Peptide haptens are easily conjugated to carrier proteins using the water-soluble carbodiimide EDC.

Figure 19.13 Conjugation to KLH often can cause precipitation due to the high-molecular weight of the carrier protein. The conjugation of [Met5]-enkephalin to KLH yields a soluble immunogen if the level of EDC addition is about 0.1 times that typically used with BSA as a carrier. This figure shows the gel filtration pattern on Sephadex G-25 after completion of the crosslinking reaction. The first peak is KLH and the second peak is excess peptide. Depletion of the peptide peak correlates to hapten-carrier conjugation.

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