Extraction of cultured human lymphoid cells with hypertonic salt solutions (3 molar potassium chloride) resulted in high recoveries of membrane-associated histocompatibility (HL-A) antigens in soluble form with potent activity and marked immunologic specificity. The active principle was purified by preparative acrylamide-gel electrophoresis. Application of the hypertonic salt extraction method is now yielding sufficient HL-A antigen to begin the elucidation of the molecular basis of transplantation individuality.

Salt extraction of soluble HL-A antigens.

/Js-microglobulin {[^T/I) is a low molecular weight protein (MW 11.800) originally isolated by Berggard & Beam (1968) from human urine. /?2-/i was later found in human serum, cerebrospinal fluid, saliva and colostrum (Berggard & Bearn 1968, Evrin et al. 1971). The serum levels have been shown to vary within a narrow range for normal individuals (Evrin & Wibell 1972). High levels are recorded regularly in sera from patients with renal diseases (Bemier et al. 1968, Peterson et al. 1969, Wibell et al. 1973) and irregularly in patients with malignant diseases (Evrin & Wibell 1973, Kithier et al. 1974). Studies of the primary structure have shown a high degree of homology of p2-u with the constant domains of human IgG (Peterson et al. 1972, Smithies & Poulik 1972). This finding and the fact that ^2-/^ has an intrachain disulphide loop suggested that /?2-/( was a free immunoglobulin domain with effector function similar to that of the C H 3 domain of IgG (Peterson et al. 1972). Consequently early work on the production of ^2-^ by human cells was concentrated on lymphocytes. Peterson et al. (1972) and Evrin & Pertoft (1973) showed that ^2-fiwas present at the cell surface of

Production of beta 2-microglobulin by normal and malignant human cell lines and peripheral lymphocytes.

The human major histocompatibility system.

Platelets and human serum have been evaluated as sources for extraction of soluble HL-A antigens. 3 M KCl was found to efficiently solubilize HL-A antigens from platelets with a recovery ranging between 50 and 100 %. However, because of the low density of HL-A determinants on platelets, the yield of soluble antigens is low, as only 1 mg of protein can be recovered from 1 × 109 platelets. Thus, while it is difficult to solubilize sufficient antigens from platelets for chemical characterization, it is possible to use these materials for biological applications such as pretreatment of kidney recipients. Soluble HL-A antigen is present in human serum, since following extensive ultracentrifugation, it can still effectively inhibit the cytotoxic activity of HL-A alloantisera. The same HL-A specificities were found to be present in varying amounts among different donors. Partial purification of such antigens can be achieved by ion-exchange chromatography of serum on QAE-Sephadex.

Evaluation of two sources of soluble HL-A antigens: platelets and serum.

This quotation seems particularly appropriate to the way that knowledge of the complexity of human histocompatibility antigens (HLA) has progressed. At one time leukocyte antigens were a tangle of cell surface markers many of which could be identified in only one laboratory; they then took on the appearance of a well-regulated and orderly series of membrane antigens of little interest outside transplantation. With little warning HLA became one of the most precise of all tools for the anthropologist, and now a new horizon of biologic functions and involvements in disease is opening up. Like Topsy, it has “just growed,” and like Pandora’s box it continues to offer unlooked for and sometimes unwanted surprises.

HLA--a central immunological agency of man.

Water soluble HL-A antigens isolated from a cultured human lymphocyte cell line derived from the peripheral blood of a normal donor inhibited cytotoxic reactions of monospecific anti-HL-A antisera in a pattern identical to that found upon direct and absorption typing of the donor's peripheral lymphocytes. The possibility of obtaining large quantities of cells from mass cultures now makes the chemical and biologic characterization of HL-A antigens a reality.

HL-A Antigens from a Continuous Lymphoid Cell Line Derived from a Normal Donor: I. Solubilization and Serologic Characterization

Quantitative serologic parameters of purified HL-A antigens.

http://link.springer.com/content/pdf/10.1007/978-1-4612-9843-4_16.pdf

B. W. Papermaster

Papers

HL-A Antigens from a Continuous Lymphoid Cell Line Derived from a Normal Donor: I. Solubilization and Serologic Characterization

Quantitative serologic parameters of purified HL-A antigens.

Characterization and Isolation of HL-A Antigens from Continuous Cultured Human Lymphocyte Cell Lines: A Report of Current Progress

Probit analysis of cytotoxicity data.