Two-step conjugation procedure

Figure 5.5 The two-step conjugation procedure for the MBS crosslinking of an amine-containing molecule with a sulfhydryl-containing molecule.

Fig. 11.7. One- and two-step conjugation procedures by glutaraldehyde. The one-step procedure, in which reagents and proteins are mixed, is poorly controlled and generally leads to excessive polymerization of antibody or of enzyme and antibody. In the two-step procedure, used primarily for POase which is relatively unreactive with GA, the enzyme is first treated with an excess of eross-linker, which is removed prior to admixture with the antibody so that no polymers of the latter are formed. However, if the enzyme has several activated groups, it can act as a bridge between antibodies and cause large polymers.

Two-step conjugation procedure with p-benzoquinone (Avrameas et al., 1978) A. If enzyme is activated in the first step ... 

Figure 9.62 Sulfo-SMCC can be used to conjugate amine-containing QDs with thiol-containing proteins or other molecules using a two-step coupling procedure.

HRP is also conjugated to (strept)avidin using the two step glutaraldehyde procedure and is used in this form in the Labeled... 

Alkaline phosphatase can also be coupled to antigens and antibodies using a two-step glutaraldehyde procedure. In the case of coupling to IgG, a two-step procedure does not seem to be of any advantage. However, in the case of labeling of staphylococcal protein A with alkaline phosphatase, a two-step procedure was found to be the only way to obtain workable conjugates, and with fibronectin it was the most efficient way. ... 

The GA method is most suitable for APase (one-step procedure), and for the antibody-chimera method (Section 11.5), whereas for EIH the POase-Fab or POase-IgG conjugates are frequently prepared by the two-step GA procedure. 

A result equivalent to an allylic substitution reaction with a chiral leaving group can also be achieved by a two-step procedure involving a conjugate addition reaction and a subsequent elimination reaction, as demonstrated by Tamura et al., wbo studied the reaction shown in Scheme 8.15 [27]. 

The two-step procedure includes formation of a N-substituted phthalimide 3, and its subsequent cleavage to the primary amine 5. Phthalimide (which can be obtained from reaction of phthalic acid with ammonia) shows NH-acidity, since the negative charge of the phthalimide anion (the conjugated base) is stabilized... 

Figure 5.7 SMPB may be used in a two-step procedure to conjugate an amine-containing molecule to a sulfhy-dryl compound, forming amide and thioether bonds, respectively.

The following procedure utilizes the one-step glutaraldehyde method. A two-step method may be used to somewhat limit polymerization of the conjugate (Chapter 20, Section 1.2). Varying the pH and the amount of glutaraldehyde added to the reaction can control the yield and molecular weight of the conjugates formed. 

The one- and two-step procedures for enzyme activation and conjugation using glutaraldehyde can be found in Chapter 20, Section 1.2. 

The two-step nature of SPDP crosslinking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensively cited in the literature, perhaps making it one of the more popular crosslinkers available. It is commonly used to form immunoto-xins, antibody-enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

Flash vacuum pyrolysis of oxiranes 156, accessible by a two-step procedure, results in carbonyl ylides 157, conjugated with a diene system formed by benzene and heterocyclic substituents R. They further undergo 1,7-electrocyclization and subsequent 1,5-sigma tropic hydrogen shift to give oxepines 158 and 159 (Scheme 31 (1997JCS(P1)3025, 1996JCS(P1)515)). 

A new type of spiro-conjugated aminopyrans, pyrans, containing a cyclophane moiety 254, were obtained from a carbonyl[2.2]paracyclo-phane derivative 255 in a two-step procedure with isolation of UN 256 (03T1739) (Scheme 98). 

---