Quantification of soluble HLA class I gene products by an enzyme linked immunosorbent assay

Soluble HLA class I antigens in human plasma preparations possibly play a role in HLA sensitization and modulation of the immune response. We therefore have determined their concentration in albumin and immunoglobulin preparations from several commercial sources and compared these values to the concentration in normal human sera. For this purpose we used a newly developed solid-phase enzyme immunoassay employing rabbit anti-mouse antibody, monomorphic HLA class I monoclonal antibody and a polyclonal enzyme-linked β(2)-microglobulin-specific antiserum. Soluble HLA antigen concentration in 14 albumin batches from 6 manufacturers and in 16 immunoglobulin batches from 11 manufacturers ranged from 0 to 9.6 and from 0 to 20.9ng/ml. The concentration in normal human sera 1,328 ± 954 ng/ml (n = 54). We conclude that the concentration of soluble HLA concentration in albumin and immunoglobulin preparations is more than 50 times lower than in normal human serum, but considerable differences exist between products of various manufacturers.

Section: Purification Of Hla Antigenssupporting

confidence: 68%

Section: Purification Of Hla Antigensmentioning

confidence: 99%

Quantitation of Soluble HLA Class I Antigen in Human Albumin and Immunoglobulin Preparations for Intravenous Use by Solid-Phase Immunoassay

Santoso¹,

Kiefel²,

Volz³

et al. 1992

“…This prompted us to investigate the con tamination of therapeutically used hemostatic preparations with soluble HLA class I (sHLA-CI) and class II (sHLA-CII) antigens. We studied 11 factor VIII and prothrombin complex concentrate (PCC) preparations for the presence of these molecules by enzyme-linked immunosorbent assays (ELISA) [8,9], Quantitation of sHLA-CI Soluble HLA-CI molecules in the above-mentioned preparations were detected using a modified ELISA-system [8], Nunc Maxisorp immunoplates were coated overnight at 4°C with 100 pi of monoclonal antibody (MAB) W6/32 (Seralab) diluted at 1:500 with phosphatebuffered saline (PBS) instead of MAB COB6-3. MAB W6/32 is known to recognize a monomorphic epitope on native HLA class I 4°C with 100 pi of HLA class II specific capture MABs (TÜ22 and TÜ35, Biotest) diluted at 1:1,000.…”

Section: Factor VIII and Pcc Preparationsmentioning

confidence: 99%

Soluble HLA Class I and Class II Concentrations in Factor VIII and PCC Preparations

Westhoff¹,

Grosse‐Wilde²

1995

Self Cite

Soluble HLA class 1 (sHLA-CI) and class II (sHLA-CII) molecules were quantitated in 11 commercially available factor VIII and prothrombin complex concentrate (PCC) preparations by enzyme-linked immunosorbent assays (ELISA). In 4 preparations, we detected traces of sHLA-CI (0.01-0.07 mg/l), and in 7 hemostatic preparations small amounts of sHLA-CII molecules (0.02-0.28 mg/l). The concentrations of these contaminant molecules are unequivocally below the mean values of sHLA in human plasma (sHLA-CI: 1.01±0.72 mg/l; sHLA-CII: 1.53±2.44 mg/l). Based on the total amount of chronically administered factor VIII or PCC, contaminating sHLA molecules may be in principle able to exert immunomodulatory effects in patients treated with such preparations.

“…The range of values at day 1 was slightly less than the range measured in serum from 61 normal blood donors (0.16-3.13 (.tg/ml) [14], probably as a result of dilution by the anticoagulant-preservative solution. As expected from the findings of previous studies, the concentration of solu ble HLA was not normally distributed, and higher levels (> 1.75 pg/ml) were recorded in individuals with the HLA-A9 phenotype [15][16][17][18]. The ten paired half units of leukoreduced or standard RCCs were stored at 1-6 °C for 35 days.…”

Section: Resultsmentioning

confidence: 68%

“…Several investigators have previously used either sand wich [15,16,28,30,31] or competitive [17,18] EIA assays to measure the concentration of soluble class I HLA in plasma or serum. Although different investigators have found different normal ranges depending on the assay used, a consistent finding has been that individuals of cer tain HLA phenotypes -notably HLA-A23(9) and HLA-investigateci the immunogenicity of leukocyte fragments in a rabbit model.…”

Section: Mlrs Using Primed Lymphocytes With and Without Added Il-2mentioning

confidence: 99%

HLA Antigens on Leukocyte Fragments and Plasma Proteins: Prestorage Leukoreduction by Filtration

Dzik¹,

Szuflad²,

Eaves³

1994

HLA alloimmunization following blood transfusion results from recipient exposure to donor alloantigens. Numerous studies have documented that intact donor leukocytes are capable of provoking primary alloimmunization and that leukoreduction can decrease the incidence of primary HLA alloimmunization. HLA antigens also exist in soluble form and are present on leukocyte cell fragments. We measured the concentration of soluble HLA class I antigen in both standard and leukoreduced blood components during storage. Although the concentration of soluble class I HLA protein varied widely among different individuals, the concentration was stable during refrigerated storage of red cell concentrates and was not affected by leukocyte reduction by filtration. We also investigated whether or not HLA antigens present on leukocyte fragments were capable of stimulating either resting or in-vitro-primed lymphocytes in the mixed lymphocyte reaction (MLR). Leukocyte fragments prepared by repeated freeze-thaw were found to express both class I and class II HLA antigens, but fragments were not able to stimulate in primary or secondary MLR even in cultures supplemented with recombinant interleukin-2. These studies provide in vitro evidence to support the hypothesis that HLA antigens are not shed from intact cells to soluble forms during storage and that HLA alloantigens on cell fragments do not elicit a cellular immune response.