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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.

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.

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 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.