Immunofixation as an adjunct to immunoelectrophoresis in characterization of serum monoclonal immunoglobulins

We evaluated quantitatively the usefulness of immunofixation (IF) as an adjunct to immunoelectrophoresis (IEP) in characterizing serum monoclonal proteins. In 33 out of 97 consecutive patients with one or more homogeneous bands on cellulose acetate serum protein electrophoresis, a monoclonal immunoglobulin could not be characterized with certainty by IEP. Of these 33 cases, in 76% a monoclonal immunoglobulin was subsequently characterized by IF on agarose gel. In 18% IF provided additional suggestive information, and in 6% it provided no additional or less information than IEP. The mean concentration of monoclonal proteins (other than free light chains) that could be characterized by IEP was 25 g/l. The monoclonal proteins that required IF for characterization were all less than 10 g/l. Of all monoclonal proteins below 10 g/l, 53% required IF for complete characterization. IF is a useful adjunct to IEP, primarily in cases where the concentration of monoclonal immunoglobulin is less than 10 g/l.     

Immunofixation. I. General principles and application to agarose gel electrophoresis.

Immunofixation offers the worker an economical means of physically locating a protein in an electrophoretic strip and is ideally suited to forensic medicine, genetic studies, or research. The method is as simple and economical as the commonly used one- or two-dimensional immunoelectrophoresis, yet yields considerably more information.     

A novel method for generating region-specific monoclonal antibodies to modified proteins. Application to the identification of human glucosylated low density lipoproteins.

Modifications of plasma lipoprotein structure and function resulting from in vivo post-translational nonenzymatic glycosylation may play a role in the premature atherosclerosis of patients with diabetes mellitus. This report describes the generation and characterization of six unique murine monoclonal antibodies that bind glucosylated human plasma lipoproteins, but do not react with normal plasma lipoproteins. This was accomplished by immunizing mice with homologous glucosylated low density lipoprotein. In competitive inhibition radioimmunoassays, the dominant epitope recognized by these antibodies on glucosylated low density lipoprotein was identified as glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine. Each of these antibodies was capable of identifying glucitollysine epitopes on all reduced glucosylated proteins studied, including high density lipoprotein, albumin, hemoglobin, and transferrin. These antibodies were also capable of identifying and quantitating glucitollysine residues on the total plasma proteins and isolated lipoproteins of normal and diabetic individuals after reduction of the proteins with NaBH4. Preliminary data suggest that diabetic total plasma proteins and isolated lipoproteins contain at least threefold more immunochemically detectable glucitollysine residues than nondiabetic plasma proteins and lipoproteins. The technique described in this report should allow production of region-specific antibodies to any immunogenic modification of a protein.     

Immunofixation to quantify beta 2-transferrin in cerebrospinal fluid to detect leakage of cerebrospinal fluid from skull injury.

beta 2-Transferrin, the desialated form of transferrin normally found only in cerebrospinal fluid (CSF) and aqueous and vitreous humor, is detected by high-resolution immunofixation (IFE). It is not normally found in nasal or aural fluids, saliva, tears, or serum. Detection in nasal fluid has been suggested to document CSF leakage into the nose after skull injury. We measured beta 2-transferrin in 48 samples of CSF. IFE of the CSF was performed on high-resolution agarose gels and stained with Coomassie Blue. beta 2-Transferrin was estimated by quantifying the total transferrin by rate nephelometry and then determining the percentage of transferrin in the beta 2 vs beta 1 region by densitometric scanning of the IFE pattern. We accurately quantified as little beta 2-transferrin as 2.5 mg/L in the CSF samples. The beta 2-transferrin fraction was clearly visible by IFE at concentrations less than 2.5 mg/L, but accurate quantification was difficult. In the samples assayed, the range of beta 2-transferrin was 4.6 +/- 1.9 mg/L. Use of this technique to examine rhinorrhea in a motor-vehicle-accident patient confirmed leakage of CSF into the nasal cavity through a vent in the left olfactory groove.     

Computer-controlled instrument system for sequential chemical testing III. Application to liver assessment.

We used the previously described [Clin. Chem. 19, 1114 (1973)] and evaluated [Clin. Chem. 19, 1122 (1973)] computer-controlled instrument system for sequential chemical testing to select and perform tests of hepatic status, to aid the clinician in the diagnosis of liver disease. Results for total bilirubin, aspartate aminotransferase, and alkaline phosphatase obtained from the continuous-flow analysis (SMA 12/60) admission screen were used by the instrument system to determine selectively the values for gamma-glutamyltransferase, alanine aminotransferase, creatine kinase, and total and direct bilirubin. Kit methods for the latter four tests were evaluated on the system; results were similar to manual procedures. A software, enzymatic ratemeter was found to be better than the previously described hardware ratemeter. The follow-up tests of serum prescribed by the system are compared to clinician-prescribed follow-up tests and discharge diagnoses. In 10 of 19 cases, the system and clinician ordered similar follow-up tests; in three cases follow-up differed, and in six cases, the system ordered follow-up tests and the clinician ordered none.     

Human monoclonal antibodies to the S glycoprotein and related proteins as potential therapeutics for SARS

Polyclonal antibodies have a century-old history of being effective against some viruses and, recently, monoclonal antibodies (mAbs) have also shown some clinical success. Human mAbs to the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein have been developed by several research groups at an amazing pace. These antibodies potently neutralize infectious virus in tissue cultures and animal models, and, alone or in combination with vaccines and other drugs, may have potential for the prevention and treatment of SARS.     

A comparative study of three approaches to the routine quantitation of human serum proteins.

The Laser Nephelometer PDQTM (Hyland Division, Travenol Laboratories Inc.) and the Abbott Bichromatic Analyser 100 (Abbott Laboratories) were compared with a radial immunodiffusion method. Seventy-eight serum specimens collected during routine blood testing were aliquoted and quantitated by the three procedures described. The nephelometric system was used as described by Hyland in their instruction accompanying the LAS-R Nephelometric test kits. The ABA-100 was used with a filter unit transmitting at 340 and 650 nm and at a water bath temperature of 30 degrees C. The Laser Nephelometer correlated well with the RID system giving a correlation coefficient varying from 0.94 for IgA to 0.79 for C3. It was not possible to quantitate IgA by the turbidometric method using the ABA-100. Results obtained for the other proteins were satisfactory and the correlation coefficient with the RID method varied from 0.88 for IgG to 0.90 for C3. The RID procedure in routine use takes 5 hours technologist time and a 16-hour incubation period to produce results for IgG, IgA and IgM on nine patient specimens. Using the Laser Nephelometer to obtain the same results on 20 patient specimens took two to three technologist hours. Nephelometry, therefore, appears to be a satisfactory alternative to RID with a comparable precision and a great saving in technologist time.     

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.     

Analysis of peroxidase negative acute leukemias by monoclonal antibodies: III. Acute lymphoblastic leukemia

Peripheral-blood leukemic cells from 45 patients with peroxidase negative acute lymphoblastic leukemia (ALL), which did not express either myeloid or megakaryocyte-platelet-related cell surface antigens, were analyzed by using monoclonal antibodies capable of recognizing T- or B-cell-associated and/or T- or B-cell-restricted antigens. Numerous subclasses of ALL, including B-cell lineage leukemias and T-cell lineage leukemias, were identified phenotypically and immunophenotypically in an effort to more accurately characterize the heterogeneous ALLs, their states of differentiation, and their relationships to normal B- and T-lymphoid cells. Among the cases studied, only seven (15.6%) were found to have stem cell (undifferentiated) leukemia (Ia+, CD24+, CD9+, CD34+). It is concluded that the use of monoclonal antibodies for the characterization of heterogeneous ALLs improves the specificity of leukemia classification, which may contribute to the selection of more effective forms of therapy for the types of leukemia identified.     

Detection of oncogene-related proteins with site-directed monoclonal antibody probes

Sequences of oncogenes define related families and provide a large data bank for the production of synthetic peptides. These peptides can be used to produce site-directed monoclonal antibody probes, which identify oncogene-encoded and oncogene-related proteins. The creation and utilization of these probes for clinical laboratory analysis are discussed.     

Heterogeneity of monoclonal immunoglobulin-G proteins studied by isoelectric focusing

Heterogeneity of myelomatous and nonmyelomatous monoclonal IgG₁ proteins was investigated by isoelectric focusing experiments in thin-layer polyacrylamide gels.It appears from this study that nonmyelomatous monoclonal IgG₁ proteins possess the same individuality and limited heterogeneity as myelomatous monoclonal IgG₁ proteins.     

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. Delivery of ribosome-inactivating proteins to spleen and lymph node T cells.

The selective delivery in vivo of a T lymphocyte-specific monoclonal antibody and immunotoxin conjugates to T cells in lymph node and spleen was assessed in rhesus monkeys. A transient coating of all T lymphocytes in the lymph nodes and spleens of healthy rhesus monkeys could be achieved after infusion of unconjugated anti-T11. Because derivatized antibody is cleared more rapidly than unconjugated antibody, it was necessary to infuse a higher dose of immunotoxin than antibody alone to achieve saturation of the lymphocyte binding sites with anti-T11. When sufficient antibody-toxin conjugate was infused, toxin was readily demonstrable on lymph node and spleen T cells by 16 h after infusion. This demonstration that toxins can be successfully delivered with specificity to target T cell populations in the monkey suggests that killing of restricted cell populations in vivo should be feasible.