Detection of Legionnaires' Disease Bacteria in Clinical Speciments by Direct Immunotluorescence 



ADDENDUM 

 I. PREPARATION OF FLUORESCENT ANTIBODY REAGENTS 



A. Production of Antisera 



1 . Preparation of vaccine for antisenmi production 



The bacterial cell vaccine is prepared as a suspension of cells harvested from solid 

 media, killed by suspending overnight in 1% Fomialin-0.85% saline, centrifuged, and 

 resuspended and adjusted with 0.5% Formalin-0.85% saline to give a turbidity reading 

 of 40 International Units (Maaloe, 0. 1955. The International Reference Preparation 

 for Opacity - Notes and Description. Bull. World Health Organ. 12: 769-775). This is 

 roughly comparable to 4 x lO"* bacterial cells/ml. For injection, a portion of the 

 suspension is mixed with an equal volume of Freund's complete adjuvant. 



2. Immunization schedule 



Day 1 - Pre-bleed the rabbits for control sera. Distribute 2 ml of the cell suspension- 

 Freund's adjuvant mixture by intracutaneous injection into 20-40 sites along the 

 shaved back of each rabbit. 



Day 31 - Inject 1 ml of the cell suspension-Freund's adjuvant mixture deep into the 

 thigh muscle of each hind leg of the rabbit ( 2 ml total). 



Day 38 - Take 50 or 60 ml of blood from the ear for antiserum. Inject 2 ml of the 

 Formalinized cell suspension intravenously into each rabbit (no adjuvant). 



Day 45 - Take 50 or 60 ml of blood from the ear. Inject 2 ml of FomTalinized cell 

 suspension intravenously into each rabbit. 



Day 52 - Exsanguinate the rabbits. 



B. Preparation of Conjugates 



1. Specific LDB conjugates 



Specific antisera for the four known serogroups of LDB were raised in rabbits by 

 immunizing with cells of the representative strains according to the above schedule. 

 Sera were fractionated and labeled with fluorescein isothiocyanate according to the 

 methods of Hebert et al. (7). Serogroup 1 comprises strains of LDB, such as Knoxville 

 1, that are stained brightly (3+ to 4+ intensity) with relatively high dilutions of the 

 Knoxville 1 conjugate, and others, such as the Bellingliam 1 strain, that are only 

 stained to a 3-l- to 4+ intensity when relatively low dilutions of the Knoxville 1 

 conjugate are used. Thus, serogroup 1 conjugates must be titered against both types of 

 the group 1 strains to insure that a sufficiently low working dilution is used to detect 

 all serogroup 1 LDB organisms. A polyvalent conjugate was prepared by combining 

 appropriate volumes of the four serogroup-specific conjugates so as to give 3-1- to 4-1- 

 staining of all of the available isolates of the four known serogroups of LDB. The 

 number of serogroup-specific conjugates that can be practically included in a poly- 

 valent preparation is limited because the total fluorescein concentration is higher in a 

 polyvalent reagent. Eventually, as the number of group specific conjugates is increased, 

 nonspecific or background fluorescence of the stained tissue specimen becomes a prob- 

 lem. Thus, if additional serogroups of LDB are found it will probably be necessary to 

 use two polyvalent conjugates for diagnostic staining. 



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