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Paper disc-electroimmunodiffusion in agarose gel provides a rapid and sensitive quantitation of the immunoprecipitin reaction.

A semi-automatic device is described for harvesting cells or precipitates from microplates on to disposable microfilters (‘C.R.C. thimbles’). The use of this harvesting system for measuring incorporation of [ 3 H] thymidine into phytohaemagglutinin-stimulated lymphocytes is investigated. The thimbles themselves do not complicate beta counting compared with counting on glass fibre filter paper itself. An estimated counting efficiency of 25% was obtained with trichloroacetic acid washing. This could be increased to 43% by dissolving the residue off the filter with NCS Solubiliser. If a trichloroacetic acid wash is not used efficiency is lower because of increased internal absorption. At least 10 6 lymphocytes/microfilter could be harvested. The system could have wide application in immunology and cell biology.

This paper describes a new technique, reversed one-dimensional single electroimmunodiffusion (REID), for rapid and simple quantitation of antiserum precipitins on a continouous scale, reports results of experiments on factors affecting use and interpretation of this new method, and describes a new device for accurately and simply metering microliter quantities of antisera to be analyzed and a simple method for calibrating the device. Results can be obtained within a few hours, and the technique readily detects ≦ 50 ng of precipitins and can resolve differences of 50 μg or less per ml in antisera containing 150 μg or more precipitants per ml. Aside from being useful and convenient for quantitating antibodies, this technique may prove especially valuable for studying the currently little-undestood mechanisms of antigen-antibody precipitation in electroimmunodiffusion tests.

This paper provides evidence that it is possible to prepare facilitating anti-mouse immunoglobulin (that is, anti-mouse immunoglobulin which facilitates complementary lysis of red cells sensitized with mouse-produced haemolysin) which, when injected into mice 24 hours before an injection of sheep red cells, very markedly reduced the number of haemolysin-producing cells detectable in spleen four days later. The diffusion-lysis method was used to recognize this and other anti-Ig's in heterologous antiserum and fractions thereof. The effective antibody was in the gamma2 fraction of antiserum produced in guinea pigs by injecting them with guinea pig red cells sensitized with mouse-produced haemolysin. This method of immunizing was used in order to stimulate the production of antibody against immunoglobulin which had undergone the configurational change characteristically occurring when antibody unites with antigen. The 19S fraction of the antiserum contained inhibiting anti-mouse immunoglobulin (anti-mouse immunoglobulin which inhibits complementary lysis of red cells sensitized with mouse-produced haemolysin) and interfered with immune depression by the gamma2 fraction. It is postulated that the gamma2 fraction induces complementary lysis only of lymphocytes whose surface immunoglobulin receptors have bound antigen and undergone configurational change. It is suggested that facilitating anti-immunoglobulin of the type described is responsible for immune suppression by anti-lymphocyte serum (ALS). Facilitating anti-mouse immunoglobulin was demonstrated in two samples of ALS (anti-mouse) which were active in suppressing graft rejection, but inhibiting anti-mouse immunoglobulin only was found in a sample which was ineffective in suppressing graft rejection.

The present paper shows that different tests give different pictures of the antibody response to bacterial endotoxin. In a study of the antibody response in rabbits to O antigen from Vibrio cholerae it was found that measured with indirect hemagglutination IgM antibodies predominated in the primary as well as in the secondary response. In contrast, when measured with a bactericidal technique, O antibodies of the IgG class were generally found in titres equal to the IgM titres during the primary response and during the secondary response the IgG antibodies dominated. The differences could be ascribed to a superior detection of IgG antibodies in the bactericidal as compared to the indirect hemagglutination method, while the techniques were about equally apt for detection of IgM antibodies. In addition, absorption experiments indicated that minor determinants on the endotoxin participating in the bactericidal reaction might be blocked by the attachment of the antigen to the erythrocytes in the indirect hemagglutination test.

The serial dilutions commonly employed in hemagglutination titrations give end-points of low precision. This weakness can be overcome by performing sufficient observations to yield mean titers of the desired precision. Statistical analysis thus becomes an important part of experiments depending on this assay. Such analysis is needed not only to interpret objectively available data but also to design future experiments. These two interrelated matters are discussed in detail here but from an introductory level which requires no prior familiarity with statistics. Emphasized throughout this review paper the analysis of variance and the Dunnett multiple comparisons test.

Dilatometry has become a useful method for the study of proteins owing to its simplicity and accuracy. However, it has seldom been used in the study of immunoglobulins. Therefore possible applications of the method for that study are being discussed. A description is also given of the most common experimental set-up and procedure for dilatometric experiments. Finally, several papers describing the application of dilatometry to the study of immunoglobulins are reviewed.

The increased cost of maintaining and purchasing small laboratory animals used in the production of large quantities of antiserum resulted in a search for a more suitable animal. This paper describes the procedures used to raise antisera in Scottish mountain sheep.

Rosette formation between sheep erythrocytes (SRBC) and human thymus-derived lymphocytes (T cells) is used to monitor T cells in various human diseases. Rosettes are usually counted in a hemacytometer immediately after preparation. This paper reports a technique for permanently fixing and staining rosettes for sereial and comparative studies, a procedure which has probably been less well standardized, less reproducibly performed, and less widely used than many investigators appreciate. The technique described has the advantages of providing distinct morphological identification of the rosette-forming cell and it produces a permanent mount for future reference.

The addition of 2-mercaptoethanol to the medium used for the in vitro cultivation of mouse lymphoid cells has allowed the development of a sensitive and highly reproducible mixed leucocyte culture assay. In this system, the thymidine uptake by allogeneic cultures is 10 to 100-fold higher than that previously obtained with the same strain combination (CBA/J → C57B1/6J) and stimulation indices are 5 to 10-fold higher. This paper defines factors that affect the degree of stimulation in allogeneic cultures and those that contribute to the variability of the assay.

An immunonephelometric method for determination of fibrinogen and fibrin degradation products in plasma is described. Test plasms are diluted 1/600 in a solution containing 17 g NaCl and 3.8 g trisodium citrate/100 ml. To 3 cm 3 of this mixture 0.2 cm 3 of a concentrated antifibrin gamma globulin preparation is added. The reading of this sample minus the γ-globulin blank in a Thorp Nephelometer is directly proportional to the fibrinogen content of the plasma or to its FDP concentration in terms of intact fibrinogen. This paper examines the mechanism, accuracy and reproducibility of this method.