Richard S. Farr

Deoxyribonucleic Acid Antibody: A Method to Detect Its Primary Interaction with Deoxyribonucleic Acid

Antibody to DNA in human serums can be detected by the ammonium sulfate method. This sensitive and specific technique, which measures the primary interaction between DNA and antibody to DNA, is based on the observation that free DNA is soluble in 50-percent saturated ammonium sulfate whereas antibody-bound DNA is insoluble.

Quantitative comparison of immunoglobulins in atopic (reaginic) and nonatopic (nonreaginic) individuals: Higher γD levels in atopic sera

Abstract A quantitative comparison of γG, γA, γM, and γD was made between 67 sera from persons with high titers of reaginic (skin-sensitizing) antibody and 51 sera from individuals lacking detectable reagin. A statistically significant elevation of γD and slight increases of γG and γA were present in the reaginic sera when considered as a group. In addition, a correlation between age and γA levels was present with increasing concentrations throughout adult life. The increase in γD is probably not a consequence of the presence of reaginic antibody per se but rather represents an additional quantitative difference in the immunologic makeup of atopic persons. The basis for this quantitative alteration is uncertain at the present time since the functional role of γD has not been determined. The distribution of immunoglobulins in the 118 subjects studied followed a log normal rather than arithmetic normal distribution, similar to the antibody titers of experimental animals immunized with standard amounts of antigens.

THE MEASUREMENT OF ANTIBODIES

The purpose of this report is to review some of the currently available methods used for detecting and measuring antibodies in human serum. In order to do so, it is necessary to draw heavily upon observations made with antigen-antibody systems which have nothing to do with mycobacteria, because most of the procedures emphasized in this paper have not as yet been employed for this purpose. What we would like to do first is to discuss some important general considerations regarding the problem of detecting and measuring antibodies in general; and second, to draw attention to a group of antibody detection systems as potentially powerful tools for studying the humoral antibody response of humans and animals during the course of immunization or infection with mycobacterial organisms. There has been a tremendous burst of activity in immunologic research over the past two decades, and the body of knowledge concerning humoral antibodies has become complex. However, as frequently happens when knowledge accumulates in a given area in science, certain simplicities and generalizations sometimes emerge. Such is the case concerning antibodies, and it is currently possible to make four and only four generalizations about antibodies as a class. First, under normal circumstances, antibodies are produced in response to antigenic stimulation. This property functionally separates antibodies from other binding proteins in sera such as haptoglobin and transferrin. Also to be kept in mind is the fact that antigenic stimulation can come from very subtle exposures, such as bacteria in the intestinal tract stimulating the production of isoagglutinins.1 Second, much has been learned about the structure of the several classes of immunoglobulins, and despite major differences among the various heavy peptide chains, all known antibody molecules have either kappa or lambda light chains.2 The third characteristic of antibody populations raised following exposure to even the purest antigens is their heterogeneity. They are heterogeneous not only regarding their structure, as alluded to above, but are also heterogeneous with respect to the strength of the bond they can form with their corresponding antigen sites and with respect to their function in vivo and in vitro. The fourth and last generalization that can be made regarding antibodies as a broad class of molecules is that all antibodies have the capacity to bind with their respective antigens. Noticeably absent from these generalizations regarding antibodies are statements that all antibodies precipitate with antigen, fix complement in the presence of antigen, or that all antibodies have the capacity to agglutinate antigen-coated erythrocytes. The only one of these characteristics which can be approached from a physicochemical standpoint is that antibodies bind to antigens. This binding of antigen to antibody is represented by the following equation:

Antibodies in rabbits fed milk and their similarities to antibodies in some human sera

Four of six adult rabbits drinking skimmed cows milk produced antibody against BSA and ALA. Anti-BSA was first detected in some sera 14 days after the initiation of milk feedings and anti-ALA was present when first studied on the thirty-fifth day. Three of the four antisera bound more I*BSA than I*ALA during the entire study. The specificities of the antibodies which bind ALA and BSA in human serum were very similar to the specificities of the respective antibodies in the sera from rabbits drinking milk and in the sera from rabbits immunized with purified BSA or ALA. No shared antigenic groups could be demonstrated between ALA and BSA, HSA or ovalbumin. As measured by immunological methods, commercial skimmed milk contained an average of 0.29 mg. BSA per milliliter and an average of 1.3 mg. ALA per milliliter. Raw beef contained 1.36 mg, extractable BSA per gram and cooked beef contained only 0.08 mg, extractable BSA per gram. Cows milk is probably the major source of antigenie stimulation for both the anti-BSA and anti-ALA in human serum. Even when compared on a weight-for-weight basis rather than on a mole-for-mole basis, BSA appears to be a generally more effective oral antigen than ALA in both humans and rabbits. As judged by the few parameters studied, the immune response of rabbits drinking milk was similar to the human immune response to milk.

An analysis of the immunological specificity of antiserum against human IgG F(ab)2

Inhibition of the interaction between 131I human Fab and a standard dilution of rabbit anti-human F(ab)2, as measured in the ammonium sulfate technique, has been utilized to study and quantitate differences in antigenic determinants associated with pooled IgG, isolated IgG from normal individuals, IgG myeloma proteins of the four known subclasses and IgA and IgM paraproteins. Isolated IgG from four normal individuals was as effective as pooled IgG as an inhibitor of the standard anti-F(ab)2-131I Fab system, indicating that the inhibition system may be used to quantitate small amounts of normal IgG, assuming a normal distribution of K and λ light chains. When K and λ type IgG paraproteins of the four known subclasses were assayed as inhibitors, the K type paraproteins were more effective than the λ type paraproteins. However, the two were additive when mixed in the proportion of 23 K and 13 λ. When K and λ type IgA and IgM paraproteins were used in the inhibition system, neither were as effective as IgG; however, the K type paraproteins were more effective as inhibitors than the λ type. The mixture of 23 K and 13 λ IgA and IgM paraproteins were not additive but instead were dilutional. The test offers a sensitive method for studying antigenic determinants of protein molecules, previously evaluated only by the precipitation reaction or hemagglutination.

Biological and chemical differences among proteins having reaginic activity

Abstract The reaginic properties of eleven human sera and/or their chromatographic fractions were examined with the use of physicochemical and biological methods. Proteins with reaginic activity were found to be markedly heterogeneous with regard to their elution characteristics from DEAE columns and with respect to their skin-fixing properties. Certain differences were also noted with respect to the degree of sensitivity of these proteins to sulfhydryl splitting agents. It remains to be learned if the heterogeneity demonstrated in these experiments is compatible with the concept that reaginic activity is associated with a single class of immunoglobulins.

A clinical study of an unusual case of asthma associated with urticaria pigmentosa

Abstract Studies were made on a 3-year-old boy with urticaria pigmentosa who has the unique feature of asthma associated with his flushing episodes. The flushing episodes were frequently followed by bulla formation and appeared to occur in three week cycles. Fluid from the bullous lesions was similar to the patients serum in protein content but contained approximately five times more histamine and ten times more serotonin than did the patients serum. The urine 5-hydroxy indol acetic acid collected following flushing episodes and during control periods was within normal limits. The combination of an antihistamine (chlorpheniramine) and a serotonin inhibitor (methysergide maleate) suppressed his skin reactivity after intradermal injections of histamine, serotonin, and 4880 better than either drug alone. The serotonin inhibitor did not have a demonstrable effect upon anaphylaxis induced in guinea pigs following the intravenous injection of histamine. Clinically, this child has done well on long-term antihistamine and serotonin inhibitor therapy. The number of urticarial lesions has dramatically decreased, and he has had no episodes of flushing, asthma, or bulla formation. The antihistamine alone was able to block these symptoms only partially. Codeine was shown to be capable of inducing flushing episodes and bulla formation, even when the child was taking the above medication.