A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9.

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immunobiology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 transfectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1-145 was established. 1-145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1-145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1-145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that against HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1-145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxicity assay in which 1-145 culture supernatant lysed all PBLs expressing HLA-A2,-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class 1 molecules indicate that Lys in position 127 may be critical for 1-145 binding.

Immunization of mice by injection with a recombinant retrovirus vector containing human factor IX for production of monoclonal antibody against factor IX.

A novel immunization procedure for eliciting murine monoclonal antibodies (mAb) is described. A murine leukemia virus (MLV)-based retroviral vector, designed as a marker protein for the expression of human factor IX (FIX), was constructed. Direct injections of mice with 0.8-1.3 x 10(4) retroviruses were carried out three times at 4-week intervals. Three out of 4 mice that had the viruses injected subcutaneously produced the antibodies against FIX, and 2 out of 3 mice injected intraperitoneally produced the antibodies. From 1 of the mice with the antibodies, an anti-human FIX murine monoclonal antibody, designated ACTIX (IgMk type), was produced. The immunization of mice by direct injection of viruses facilitated mAb production in many instances in which cDNAs are available.

Adsorption of anaphylatoxins and platelet-specific proteins by filtration of platelet concentrates with a polyester leukocyte reduction filter.

Anaphylatoxins generated during storage of platelet concentrates (PCs) may potentially have side effects on platelet transfusion. We evaluated the anaphylatoxin-scavenging abilities of white blood cell reduction filters. Among the commercially available filters for PCs, one made with polyester fiber (PL50) dramatically adsorbed C3a and C4a anaphylatoxins to the respective mean level of 1,721-208 ng/ml and 1,240-141 ng/ml in 3-day-old PCs. C3a and C4a were measured as the native and des Arg form of each complement by radioimmunoassay. C3a and C4a anaphylatoxins in the supernatant plasma fraction from 3-day-old PC again decreased from 1,136 to 114 ng/ml and from 1,086 to 65 ng/ml, respectively. The filter also adsorbed 85% of platelet factor 4 (PF4) and 31% of beta-thromboglobulin (beta-TG), which had been released from platelets into the plasma during storage. The plasma levels of adhesive proteins such as fibronectin, fibrinogen, and von Willebrand factor, and plasma lactate dehydrogenase activity did not decrease after filtration. Another polyester filter (PL5A), on the other hand, significantly increased C3a and C4a levels with filtration. In addition, there was no PF4 adsorption ability during the filtration. The filters for red cells (RC50, BPF4, and R500A) had no anaphylatoxin adsorption capabilities. The observed specific adsorption of anaphylatoxins might be attributed to the electrostatic force between the positively charged anaphylatoxins with high pI and the possibly negatively charged filter membranes. Since PF4 and beta-TG have positively charged moieties in the C-terminal position, the same adsorption mechanism might operate.(ABSTRACT TRUNCATED AT 250 WORDS)