Biological system to test in vitro if a substance is immune reactive

Abstract

Preparations containing deep-frozen blood are used for test procedures for determining blood response.

Claims

1. A method of testing whether a substance planned for use in human therapy would cause an immune reaction in humans comprising the steps of: selecting a cryopreserved unit dose comprising whole blood including viable leukocytes and a cryopreservative from among a plurality of identical cryopreserved unit doses obtained from a single or pooled sample of blood taken from a human or animal, which single or pooled sample was pre-tested to confirm immune reactivity; thawing the cryopreserved unit dose; contacting the thawed, cryopreserved unit dose with the substance; and determining whether the whole blood in the unit dose has an immune reaction to the substance.

2. The method of claim 1 wherein the whole blood further comprises clotting inhibitors, diluents, or a combination thereof.

3. The method of claim 1, wherein the identical cryopreserved unit doses are standardized.

Description

EXAMPLE

(1) Citrated blood from healthy donors was mixed with 10% dimethylsulfoxide (Merck, Darmstadt) immediately after it was drawn. Aliquots of 100 microliters were distributed in 2 milliliter reaction vessels (Eppendorf, Hamburg) and transferred to a −70° C. cold box in a commercial freezing system (Mr. Freezy, Nalgene). Thawing was done in a thermoshaker (Eppendorf, Hamburg) at 37° C. Immediately after thawing, 900 microliters of RPMI 1640 (Gibco, Eggenstein), warmed to 37° C., was added. Then the test substances, such as dilutions of a pyrogen, in this case, lipopolysaccharide of Salmonella abortus equi (Sigma, Deisenhofen) were added. After a four-hour incubation at 37° C., for example, in an incubator (Heraeus, Fellbach) with 5% carbon dioxide the incubated samples are shaken and centrifuged. Endogenous pyrogens, IL-1β in this case, are determined in the cell-free supernatant, after freezing, if desired. FIG. 1 shows formation of IL-1β in the cryopreserved blood from four healthy donors, depending on the amount of pyrogen added. IL-1β was determined by ELISA (enzyme-linked immunosorbent assay) from antibodies from the company Pharmingen (Hamburg). The test shows release of endogenous pyrogens in the presence of minimal amounts of pyrogens.

(2) FIGS. 2 and 3 show dependence of release of IL-1 in frozen whole blood stimulated with LPS on the amounts of azathioprin or dexamethasone administered.