Chemokine Receptors by Richard Horuk (Eds.)

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By Richard Horuk (Eds.)

Chemokines are significant mediators of immune cells and are occupied with a variety of proinflammatory human ailments, together with rheumatoid arthritis, a number of sclerosis, and organ transplant rejection. It has lately been chanced on that their receptors are excited about HIV an infection. The characterization of those molecules and their receptors is therefore of basic significance in knowing a couple of human illnesses and infections. This quantity and its spouse quantity 287 Chemokines supply entire experimental protocols utilized in this box of analysis.

Key Features
* Chemokine receptors
* Chemokines in disease
* sign transduction of chemokines

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13 Polyclonal antisera to CCR1 and CCR5 are raised in New Zealand White rabbits by subcutaneous and intramuscular injection with the corresponding amino-terminal domains for CCR1 conjugated to keyhole limpet hemocyanin (KLH) and for CCR5 conjugated to glutathione S-transferase (GST). Following primary immunization and six challenges with peptide, CCR1 antiserum from several pooled bleeds is collected and purified over a 5 ml protein A-Sepharose HiTrap column (Pharmacia, Uppsala, Sweden). Following initial immunization with CCR5-conjugated peptide and three challenges with the antigen, aliquots of anti-CCR5 serum are absorbed with GST-glutathione-Sepharose to remove antibodies to the GST moiety of the fusion protein.

5% sucrose in PBS, 13M. E. Nichols, P. Rubinstein, J. Barnwell, S. R. de Cordoba, and R. E. Rubinstein, J. Exp. Med. 166, 776 (1987). 14A. R. Glabinski, M. Tani, V. K. Tuohy, and R. M. Ransohoff, Methods Enzymol. 288, 182 (1997). 3. The fixative is removed after 1 hr, and the cells are carefully washed twice in PBS and then stored in PBS at 4 ° in an airtight container until the time of immunostaining (never store for longer than 2 to 3 weeks unless PBS is replaced because evaporation will alter osmolarity).

However the homology of these two receptors is only 29% in the N-terminal regions that were shown to be involved in ligand binding. 19'2° Our results with polyclonal antibodies and MAbs suggest that the N-terminal portion of IL-8R is the most immunogenic region of the receptors. Therefore, it is reasonable to try to generate MAbs by immunizing with N-terminal synthetic peptides of chemokine receptors. This has been demonstrated by our laboratory 4 and by Quan et aL, 7 although we were able to generate the most potent blocking monoclonal antibodies by immunizing with cells expressing IL-8RA and IL-8RB rather than with peptides.

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