Primary Immune Response to Sheep Red Blood Cells (SRBC) as the Conventional T-Cell Dependent Antibody Response (TDAR) Test

Figures & data

TABLE 1 Antibody forming cell assay

• Pros

• Well-characterized across multiple labs

• Most validated endpoint in immunotoxicology – Large database (NTP)

• All reagents are commercially available

• Adapted to work with other antigens (DNP-Ficoll)

• Can be conducted entirely in vitro (separation- reconstitution studies; direct vs. indirect effects)

• Cons

• Animal sacrificed; tissue (spleen) harvested and cells must be assayed within 18–24 hrs after collection. – Limits experimental design relative to exposure

• Not easy to extrapolate to human tests

• Labor intensive and does not lend itself to automation

TABLE 2 SRBC-specific ELISA

• Pros

• Does not require animal to be sacrificed; collected sera can be stored frozen and tested at a later time.

• Measured in serum (reflection of systemic immunity)

• All tissues available for histopathology (‘piggy-back’ on existing protocols)

• Can extrapolate to human tests

• Can be automated

• Cons

• Critical reagent is not commercially available – SRBC membrane

• Limited mechanistic utility

• Not well characterized (few labs/chemicals)

• Not well validated (few studies-ELISA vs AFC assay)

TABLE 3 Lowest dose of cyclophosphamide at which statistical significance was observed

	Lab 1	Lab 2	Lab 3	Lab 4
	mg/kg	mg/kg	mg/kg	mg/kg
	(Inhibition)	(Inhibition)	(Inhibition)	(Inhibition)
Rat AFC	1 (44%)	10 (99%)	10 (98%)	10 (71%)
Mouse AFC	5(36%)	5(64%)	10 (55%)	20 (86%)
Rat ELISA	30 (47%)	10 (40%)	10 (69%)	10 (30%)
Mouse ELISA	10 (37%)	10 (6%)	5(11%)	20 (22%)

*10⁶ splenocytes.

TABLE 4 Lowest dose of dexamethasone at which statistical significance was observed N.S.- not significant

	Lab 1	Lab 2	Lab 3	Lab 4
	mg/kg	mg/kg	mg/kg	mg/kg
	(Inhibition)	(Inhibition)	(Inhibition)	(Inhibition)
Rat AFC/10^6	1(58%)	1(59%)	0.1 (47%)	1(76%)
Rat AFC	0.1 (61%)	1(80%)	0.1 (60%)	0.1 (66%)
Mouse AFC/10^6	3(55%)	1(52%)	N.S.	N.S.
Mouse AFC	0.1 (43%)	1(65%)	3(65%)	1(55%)
Rat ELISA	N.S.	5(20%)	3(43%)	5(35%)
Mouse ELISA	N.S.	3(7%)	N.S.	N.S.

*AFC/spleen.