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In the present paper we describe in detail a simple peocedure for the preparation of monosopecific antisera to human and mouse serum fibronectin. A similar procedure could also be used to prepare antibodies to fibronectin from other species. The procedure, based on the recently reported affinity of fibronectin for gelatin, essentially consists of two steps. (1) Immunization of rabbits with fibronectin purified from serum by affinity chromatography using gelatin coupled to CNBr-activated Sepharose 4B. (2) Absorption of the antiserum obtained by an immunoabsorbent prepared using fibronectin-free serum proteins that remained after absorbing serum with gelatin-Sepharose. The antisera obtained were monospecific, as determined by immunoelectrophoresis and did not show any difference with respect to antisera prepared by different procedures.

Radioimmunoassay (RIA) provides a sensitive serological procedure for detecting rabies virus ribonucleoprotein (RNP) as well as its specific antibodies. RIA was carried out using highly purified RNPs labelled by the chloramine-T method. This paper describes optimal conditions for iodination of RNP with high specific activity. The optimal concentrations of 125I, RNP, chloramine-T, and reducing agent as well as the effect of pH on the reaction were investigated. RIA proved to be extremely sensitive for detection of homologous antibodies. In competition experiments the part-relationship of the group-specific RNPs of the three rabies virus serotypes (HEP, MOK and LBV) was confirmed.

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.

This paper examines the parameters necessary for the efficient measurement of anti-Theiler's murine encephalomyelitis virus (TMEV) antibodies in an affinity-dependent manner using a variation of a solid-phase particle concentration fluorescence immunoassay (PCFIA). By allowing antibody to react with fluorochrome-labelled virus in fluid phase and subsequently capturing the resulting virus-antibody complexes with anti-immunoglobulin coated polystyrene particles (fluid-phase PCFIA), the present assay allows for both greater sensitivity, specificity and preservation of conformational viral epitopes than do solid-phase immunoassays. Fluid-phase PCFIA proved to be a more rapid quantitative assay than ELISA and significantly diminished non-specific binding by both untreated and heat-inactivated normal mouse sera. This methodology also allowed us to perform competition assays and to determine the dissociation kinetics of anti-viral antibody preparations, investigations which cannot generally be performed as solid-phase immunoassays. Thus fluid-phase PCFIA is a rapid and efficient immunoassay with excellent reproducibility and great versatility.

This paper describes the use of Histopaque (Hp) density gradient medium and its advantage for separating mononuclear cells (mnc) from rabbit blood. Hp solutions of density (d) = 1.083 g/ml, 1.119 g/ml and 1:1 mixture of these solutions (final d = 1.103 g/ml) were compared to Ficoll-Paque (Fp, d = 1.077 g/ml) and Lymphopaque (d = 1.086 g/ml). The average leukocyte recovery was: Fp = 26% and Lp = 17%, while that with Hp was: Hp d = 1.083: 41%; Hp d = 1.119: 43%; Hp d = 1.103: 57%. Optimum mnc recovery (76%) was obtained with Hp d = 1.103. Average mnc purity for the various media was: Fp = 88% mnc; Lp = 91%mnc; Hp d = 1.083: 94% mnc; Hp d = 1.119: 93%; Hp d = 1.103: 94% mnc. Contamination was mainly from basophils. Viability was greater than 95% in all cases. Thus, Hp density gradient media provide increased recoveries of mnc from rabbit blood compared to Fp and Lp, while purity is not affected. Rabbit mnc appear to be denser than human mnc, which are recovered in greater numbers using Fp.

In the present paper possibilities for combination of autoradiography with other histochemical techniques in the same tissue section of cell smear are reviewed. Autoradiography may be combined with immunofluorescence and immunoenzyme techniques in order to detect two different compounds in the same tissue section or cell smear. Grains in autoradiographs may be stained by a colour coupling process in order to facilitate distinction between autoradiographic grains and the other labels.

This paper describes a method to enhance the sensitivity of enzyme immunoassays by the use of solid tagged latex beads. Various bead sizes (0.21 and 0.81 micron) were tested using particles which had been covalently modified (or coated) with biotinylated rabbit anti-mouse immunoglobulin antibodies. Detection limits of mouse monoclonal antibodies or antigens (which have been reacted with specific mouse antibodies) were compared to those obtained using the general homogeneous 'sandwich' ELISA assay. Compared to the homogeneous biotin-avidin ELISA an increase in sensitivity level of about 6-10 times was observed in antigen detection assays. The stability of the reagent was excellent. The method gave low backgrounds and was as simple to use as the standard procedure.

We have recently developed an immunoradiometric assay (IRMA) for specific measurement of immunoreactive kallikrein which allows a simultaneous determination of the enzymatic activity of kallikrein. This paper describes the application of this method for measurements of glandular kallikrein complexed with inhibitors. Interference by low molecular weight inhibitors such as benzamidine and Trasylol was easily overcome by increasing the amount of immobilized anti-kallikrein antibody added in the assay, and by prolonging the incubation time of the antigen-binding step. The recovery of kallikrein in complex with plasma inhibitors was complete only when the anti-kallikrein antibody was immunoadsorbed onto a solid-phase sheep anti-rabbit immunoglobulin. The dose-response curve of glandular kallikrein in plasma paralleled that of purified kallikrein in both the immunoradiometric and the immunoenzymometric assays. The concentration of immunoreactive glandular kallikrein in normal rat plasma was 12.8 +/- 4.3 nU/ml. The enzymatic activity of this immunoreactive kallikrein was 86% inhibited.

Immune lysis of liposomes has been utilized in immunoassays for detection of specific antibodies. Membrane immunoassays which have sensitivities comparable with those of radioimmnoassays are easy and rapid to perform. Information obtained so far has been limited by the fact that the method is based on empirical relationships. The present paper describes a simple theoretical model for analysing membrane immunoassay data. According to the model, relative dissociation constants for the antibody-antigen reaction and relative concentrations of high affinity antibodies for different antisera may be determined. Furthermore, the analysis yields the percentage of antibody bound to antigen under certain conditions. The method is also useful in the search for optimum experimental conditions in membrane immunoassay measurements.

An indirect anti-immunoglobulin rosetting reaction (IARR) can be successfully used to detect alloantibodies to human lymphocytes. In this paper we describe an adaptation of the IARR which allows detection of alloantibodies to human lymphocyte subpopulations. Fluorescein labelled sheep red cells, as a T cell marker, are incorporated into the rosetting reactions and by looking for rosettes with two indicator cell types, sheep and ox, one can determine if the alloantibodies are reacting with T cells or non-T cells. This type of assay is more sensitive than a standard cytotoxic test and can detect non-cytotoxic antibodies. The results show that this rosetting assay with two types of indicator cell can be useful in the study of pregnancy anisera, particularly those that are thought to contain reactivity directed solely against non-T cells. These antisera probably recognise the human DR antigens which are though to be equivalent to the rodent Ia antigens.

The augmentation of murine natural killer (NK) cell activity after culture is described. Increased NK cell activity occurred when spleen cells were cultured for 18 or 42 h at high cell density in macro culture plates at 37°C. Similar results were also achieved using the same cell density when micro culture plates were employed. The simple modifications of culture conditions described in this paper should provide an excellent tool to study murine NK activity after culture. Furthermore, the micro culture system has the added advantage of enabling one to test large numbers of samples.

Radionuclides or anti-cancer drugs may be coupled to antibodies for specific transport to target tissues. We have previously reported that several proteins could be rapidly and efficiently labeled with gallium (67Ga) by using deferoxamine (DFO) as a bifunctional chelating agent. In the present paper, we have described the use of hetero-bifunctional agents for the conjugation of DFO with antibodies and investigated the effect of coupling agents on in vitro properties and biodistribution of 67Ga-labeled antibodies. 67Ga-labeled monoclonal antibodies retained antigen-binding activity when prepared under optimum conditions. The use of hetero-bifunctional reagents, such as succinimidyl 6-maleimido-hexanoate (EMCS) or N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), which link thioether bonds and disulfide bridges prevented the formation of polymerized antibodies. Although high non-specific uptake in the liver was observed with radiolabels prepared by the homo-bifunctional agent glutaraldehyde, uptake in the liver was low with conjugates linked by hetero-bifunctional agents. 67Ga-labeled antibodies with thioether bonds showed in vivo stability, but the clearance from the circulation was the fastest with the radiolabel holding disulfide bonds. The coupling reagents used to link DFO and antibodies greatly influenced both in vitro properties and in vivo distribution of labeled antibodies and 67Ga-labeled antibodies provide a good model for the study of coupling methods and biodistribution of antibody conjugates.

This paper describes an immunoperoxidase technique for labelling cryostat tissue sections which is routinely used in the authors' laboratories both in the initial screening of hybridoma culture supernatants, and also during the subsequent cloning and growth of antibody-secreting cell lines. The technique can readily be performed on 100 samples in less than 3 h and is free of non-specific background labelling. The staining pattern of a monoclonal antibody on a single tissue section allows semiquantitative assessment of its reactivity against a wide variety of tissue constituents and is thus inherently much more informative than conventional screening techniques (such as binding assays) which yield only a single numerical value for each test performed. In consequence it is often possible to identify the probable specificity of a new monoclonal antibody at the primary screening stage. A further important advantage of immunohistological screening is that it detects antigens on cells or other tissue structures which do not readily enter suspension and also antibodies against nuclear and cytoplasmic antigens. Examples of monoclonal antibodies analysed by immunohistological screening include antibodies against C3b receptor, HLA-DR, factor VIII-related antigen, human syncytiotrophoblast, dendritic reticulum cells and a proliferation-associated cell surface glyco-protein.

This paper describes a new assay, based on the ELISA technique, for the quantification of antibodies to streptolysin-O (ASLO). We have compared its performances with that of a standard method (inhibition of hemolysis). Using a panel of 137 sera covering the whole range of ASLO titers, the results showed a good correlation between both methods but the ELISA method was more reproducible than the standard technique, thus represents a convenient alternative for the quantification of ASLO.

Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.

Isolating sufficient B cells for DR typing is often a problem, particularly in patients with low lymphocyte and/or B cell counts. This paper describes a simple method for preparing highly purified B cells using monoclonal antibodies and complement to kill the majority of non-B cells. Mononuclear cells are isolated by standard Ficoll-Hypaque techniques and incubated at 0 degrees C for 10 min with the following monoclonal antibodies: OKT3 (pan T cell), OKT11 (pan T and null cell), and Leu11b (NK cell). Rabbit complement is added and the mixture is incubated at 37 degrees C for 20 min. Dead cells are then removed by Percoll gradient centrifugation. This procedure results in cell preparations containing 90.1 +/- 1.3% B cells as measured by two-colow immunofluorescence with an Epics C flow cytometer. A yield of 52% can routinely be obtained. In contrast, nylon wool adherence results in preparations with only 44 +/- 9.1% B cells with a yield of 31%. B cells prepared by the monoclonal antibody procedure always show stronger and clearer reactions on DR typing trays. These results are consistent with the flow cytometric analyses of the B cell preparations. In summary, this procedure has the following advantages: increased accuracy of DR typing, decreased sample size (only 1.0 X 10(7) mononuclear cells are required), reduced time of cell preparation, and reduction in the number of repeat typings necessitated by poor yields and/or results.

This paper describes a system for determining the frequency and fine specificity of influenza A virus-immune memory cytotoxic T cell (CTL) clones from limiting dilution (LD) microcultures. We found that such experiments can only be performed (1) by analyzing the clonal response of CTL from wells of replica plates containing fractions of one original plate, (2) when it has been ascertained that splitting is possible at the clonal level, and that each fraction of a microculture well gives an identical response on target cells infected with the stimulating virus. With these requirements fulfilled we found that short term CTL clones from LD microcultures from influenza A virus (A/X-31)-immune mice (C57BL/6) occur at a frequency of f = 1:546 to f = 1:6303. The effector cells carry the Lyt-2.2 marker and are specific for target cells infected with the immunizing virus (influenza virus A/X-31). They do not lyse NDV (Newcastle disease virus) or HSV (herpes simplex virus)-infected or NK (YAC) target cells.

An isolation procedure for functionally active lamina propria lymphoid cells (LPL) from the murine intestine is described. The procedure involved EDTA-dithiothreitol incubation of intestinal tissue to remove epithelial and intraepithelial cells, followed by collagenase digestion of the basement membrane to liberate part of the LPL. The LPL were suspended by squeezing the remaining tissue strips through a nylon gauze filter. Functional activity was tested by enumeration of the immunoglobulin-secreting cells in the cell suspensions obtained by an isotype-specific protein A plaque-forming cell assay. On average 1-2 X 10(8) LPL were isolated from the intestine of C3H/He mice. 11% of these cells actively secreted Ig. From these Ig-secreting cells 99% produced IgA. The isolation procedure described in this paper permitted a higher recovery of viable cells than has previously been obtained with other methods.

This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromogen results in a stable colour reaction that can be semiquantitatively analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1:50, IgE could be detected in the range of 12.5-2500 U/ml. Using non-diluted serum samples IgE levels between 0.05-50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.

Rosetting techniques are widely used to quantify or purify various lymphocytic subpopulations; however, these techniques cannot discriminate between different receptors of similar specificities and different binding strengths, further, they do not provide any information concerning the molecular mechanisms involved in cell-cell adhesion. This paper describes a very simple technique of assaying rosette stability: cell suspensions are driven with known pressure through a calibrated needle with a syringe. Adhesion is quantified before and after this treatment. This procedure did not damage rat peritoneal cells used in a model system. Further, this method yielded fairly reproducible results and allowed a crude estimate of the force involved in the binding of glutaraldehyde-treated sheep red cells (GSRC) or immunoglobulin-coated sheep red cells (IGSRC) by rat macrophages (an average force of 0.8 x 10(-7) Newton was needed to separate 50% of bound IGSRC from macrophages). Binding and binding strength were found to be independent parameters. Last, this method possibly provided a way of separating two distinct subpopulations of rat macrophages. It is suggested that this technique might be routinely used to refine rosette studies.

This paper describes the analysis of both monoclonal and polyclonal murine insulin antibodies with time-resolved fluoroimmunoassay (TRFIA). This assay is based on the binding of antiserum by coated isotype-specific capture antibodies and the detection of insulin binding using europium-labeled human insulin (HI-Eu). This reagent was shown to have a detection limit of 10 −16 mol. Among various applications this technique permitted an isotype-specific determination of the affinity distribution of polyclonal antibodies from individual mice.

This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.

In this paper we compare a solid-phase enzyme immunoassay (EIA) with a neutralization-inhibition enzyme immunoassay (NI-EIA) for the determination of anti-idiotypic antibodies against Semliki Forest virus (SFV)-neutralizing monoclonal antibodies (MAs) UM 5.1 (IgG2a) and UM 1.4 (IgG2a). Against these MAs strong immune sera were induced in female BALB/c mice by two subcutaneous injections, 3 weeks apart, with keyhole limpet hemocyanin coupled MA mixed with the saponin Quil A. Rabbit immune sera were prepared by intracutaneous injections of purified MA mixed with either FCA (first immunization) or IFA (second and third immunization). In the NI-EIA serum is preincubated with neutralizing MA, in wells of 96-well plates, before SFV is added. Binding of anti-idiotypic antibodies to MA results in a diminished capacity of that MA to neutralize SFV. After 1 h incubation with SFV L cells are added and residual infectious virus is allowed to multiply for 6 h at 37 degrees C. Then the monolayers are fixed with glutaraldehyde and subsequently SFV is quantified with a horseradish peroxidase-labelled SFV-specific MA. Low absorbance values indicate that the neutralizing capacity of MA is intact and that blocking antibodies were not present in serum. In contrast high absorbance values indicate that blocking (anti-idiotypic) antibodies had abrogated the neutralizing capacity of MA. With the strongly neutralizing MA UM 5.1 as idiotypic antigen the NI-EIA proved to be at least as sensitive as the solid-phase EIA. Furthermore both normal mouse serum-absorbed rabbit immune sera and mouse immune sera were not cross-reactive in both solid-phase EIA and NI-EIA.

This paper describes the use of the immunoperoxidase technique for the screening of rat hybridoma culture supernatants on tissue sections. By combining the avidin-biotin system with mouse monoclonal antibodies specific to different rat immunoglobulin isotypes, it is possible to resolve the specificity patterns of complex mixtures of monoclonal antibodies from uncloned culture wells. This strategy is particularly useful in the derivation of monoclonal antibodies to cell surface antigens.

This paper describes a miniaturised method of cell cloning in Terasaki plates. It is rapid and technically simple enabling large numbers of putative clones to be tested. As Terasaki plates are designed for use in tissue typing procedures, the wells can be screened and the exact number of cells in any particular well can be seen. Colonies derived from wells containing only one cell can be then be chosen, eliminating the need for a second or third recloning usually necessary for statistical surety.

This paper describes a modification of the peroxidase technique by which immunophenotyping may be carried out on routinely air-dried blood and bone marrow (BM) smears. The method is simple and quick, requires no special equipment, can be performed on fresh or stored specimens and gives a standard of morphological detail equal to that of routine blood films. With a monoclonal anti-HLA-DR antibody as a prototype, it was possible to demonstrate reliably, the presence of positively and negatively stained cells of appropriate morphological types in the peripheral blood of leukemia patients. Although only about one-third of antibodies tested were effective with the technique, we identified monoclonal antibodies capable of demonstrating myelomonocyte, granulocyte, monocyte, pan-leukocyte, transferrin, platelet, pan-T, ‘cALLA plus B cell’ and other antigens. However, we have not yet found antibodies able to identify T cell subsets, nor to distinguish ‘common’ acute lymphoblastic leukemia from its rare B-cell counterpart. With these limitations the method is suitable for routine use alongside cytochemistry in the differential diagnosis of leukemias and lymphomas.

This paper describes a convenient method for the culture of sub-lines of the murine T cell cloned line, D10.G4.1, and the use of these lines in a highly sensitive and specific bioassay for interleukin-1 (IL-1). The cells are cultured with IL-1, interleukin-2 (IL-2), and concanavalian A (ConA), in the absence of feeder cells or antigen. Assays are routinely carried out in the presence of saturating IL-2, which enhances sensitivity and ensures that further IL-2 will not give false positives. Addition of interleukin-4 (IL-4) has a similar effect and can be used together with IL-2 where there is a potential for interference from either cytokine. The assay is not affected by high concentrations of human interleukin-6 or tumour necrosis factor-α (TNF-α) and only minimally affected by high concentrations of murine TNF-α.

A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

Testing hybridoma supernatants for antibodies of interest involves extensive screening, particularly when the immunogen comprises whole cells. The number of different screening procedures is often large and unmanageable and depends on whether one is interested in, for example, cell surface or intracellular binding. This paper describes an initial screening technique using whole tissue homogenate rather than the individual tissue components. The tissue is fixed to the surface of 96-well microtitre plates by microwaves using a conventional microwave oven. This technique provides a rapid and cost-effective means of screening large numbers of monoclonal antibodies.

This paper describes a simple methodology that allows reproducible generation of calibrated shear flows over a wide range (100-fold) of shear rates. This was illustrated by studying the dissociation by shear of alloimmune lymphocyte-target cell conjugates; mixtures of target tumor cells and immune lymphocytes of the same H-2 haplotype were used as controls. Also, current methods of cell agitation (vortexing and aspirating with a Pasteur pipette) were compared with calibrated shearing. Three conclusions are suggested: (i) there is a wide range of resistance of lymphocyte-target cell bonds, without any clearcut separation between weak and strong association. Hence any study made on this (and most probably other types of) intercellular adhesion is dependent on the arbitrary definition of ‘bound’ cells: such a definition should be as absolute as possible, especially when rosetting assays are performed to study different subsets of immunologically competent cells, and when results obtained in different laboratories are compared. (ii) There is no clearcut separation between the strength of specific and nonspecific interactions, there is thus no simple way of using shearing forces to selectively eliminate the latter. (iii) The methods described should help to study the mechanism of immunological processes involving intercellular contacts such as cell-mediated cytotoxicity or antigen-induced lymphocyte activation.

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the β- d -galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewis x ), which is directed against a tumor-associated antigen sialosylated Lewis x (S-Le x ). The antibody was first acylated with a heterobifunctional agent N -( γ -maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Le x levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.

This paper describes the development of a simple, sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the measurement of serum IgG, IgA and IgM anti-bodies to cow's milk protein. The specificity of this assay was determined by preparing immunosorbents of cow's milk and human serum albumin (HSA). Fractionation of the antiserum-enzyme label by gel filtration and affinity chromatography showed that the conjugate preparation contained moieties of antiserum-enzyme, and antiserum-enzyme polymers. The preparation also contained enzyme-enzyme and antiserum-antiserum conjugates. Purification of the labelled reagent did not improve the sensitivity. The reagents and conditions used have been analyzed for purposes of standardisation.

Immunoadsorption is a simple and effective method for purifying antigens and antibodies. The major interfering effect is non-biospecific binding, especially when crude preparations of antibodies or antigens are coupled to the matrix. In this paper various possibilities for eliminating these effects are compared and analysed. It was found that for antigen (AFP) purification pre-elution with a pH 10.0 buffer of high ionic strength (1.0 M) is effective. A major problem in purifying antibodies in the non-biospecific binding of IgG. This can be eliminated by using a non-biospecific immunoadasorbent column, where specific antibodies are not, or only weakly bound and IgG with irrelevant specificity but exhibiting high non-biospecific interaction is retained. This has been applied to preparation of sheep anti-pepsinogen II group protease antibodies.

It has generally been assumed that the binding of most proteins to nitrocellulose is stable in the non-ionic detergent Tween 20. However, the following paper demonstrates that in immunoassays where antibodies or other proteins are bound directly to the nitrocellulose, 0.05% Tween 20 may dissociate these proteins from the membrane. The degree of dissociation appears to be dependent on the individual protein studied. Some antibodies and other proteins bind tightly to nitrocellulose and dissociation of these proteins by Tween 20 is barely detectable. In contrast, other proteins are nearly completely stripped from the nitrocellulose by the same detergent. Therefore, unless it is known from control experiments what proteins will or will not be dissociated from nitrocellulose by Tween 20, the routine use of Tween 20 in the development of Western blots, native blots and dot-blots should be discontinued.

Existing methods of measuring IgE in in vitro peripheral blood lymphocyte (PBL) cultures are not sufficiently sensitive to detect IgE when it is present in small amounts. This paper describes a modification of a two-site ELISA which increases the sensitivity of the assay 10–20-fold. By using the Fab′ fragment of either rabbit or mouse monoclonal anti-IgE conjugated to alkaline phosphate (AP) as the detector, the background of the assay was reduced sufficiently to permit signal amplification, using a commercially available amplified AP substrate. With this assay as little as 10 pg/ml of IgE could be detected. The interassay coefficient of variation was 15–18% between 1200 and 100 pg/ml IgE ( n = 14) and there was a good correlation with a commercial IgE radioimmunoassay (RIA) ( r = 0.98, n = 38).

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.

A method for the quantitative determination of proteins by immunofixation-agarose gel electrophoresis is described. The proteins are separated electrophoretically and the resulting circular zone overlaid with specific antibody-impregnated filter paper (10.2 microliter/cm2). Following incubation for 20 h at room temperature the plates were processed, stained and the areas of the precipitin zones determined by planimetry. Intra-plate variation (coefficient of variation) for alpha1-antitrypsin (30 samples) and elastase (30 samples) at concentrations of 0.25, 0.5 and 1.0 mg/ml was 2.2--5.8% and inter-plate variation ranged from 2.9% to 5.2%. Overall, the analyses showed that the method is the statistical equivalent of rocket immunoelectrophoresis. The lowest concentration used was 0.25 mg/ml which corresponds to a sensitivity of 1.25 microgram (5 microliter per sample well). It is conceivable that lower amounts could be successfully determined. Application of the method to the determination of stoichiometry of the alpha1-antitrypsin--elastase interaction permitted the simultaneous quantitative determination of inactivated alpha1-antitrypsin (an acidic protein) and excess elastase (a basic protein) in alpha1-antitrypsin--elastase reaction mixtures (molar ratio elastase/alpha1-antitrypsin = 1.3) and thus by difference, the amount of elastase and and alpha1-antitrypsin in the alpha-antitrypsin--elastase complex. The results showed that the molar combining ratio of inhibitor to enzyme was 1.086 : 1 or 1 : 1. Conventional inhibition experiments (residual elastase activity as a function of increasing amounts of alpha 1-antitrypsin) showed this ratio to be 1.79 : 1 but 1.03 : 1 when the inactivation reaction, as determined by quantitative immunofixation, was taken into account. The quantitative immunofixation method should be applicable to interacting systems which, like elastase and alpha1-antitrypsin, result in complexes with electrophoretic mobilities intermediate to the parent proteins.

This paper describes the development of an enzyme immunoassay (EIA) for the detection of autoantibodies against thyroglobulin in an avian system. In this system EIA offers an efficient and alternative approach to already established methods such as double diffusion in gel, passive haemagglutination, indirect immunofluorescence and radioimmunoassay. The optimization of the different incubation steps is described and the expression of results of observed antibody activity is discussed.

A simple chemical method is described which creates cyanate groups on the saccharide core of bacterial lipopolysaccharide (LPS). The activated LPS molecule is stable at pH 3.5 and can be kept for months at -20 degrees C without loss of properties. The strong reactivity of the cyanate groups permits efficient covalent coupling of various molecules to LPS. This is done under non-denaturing conditions. This paper also describes the coupling of microquantities of antigen (5-10 micrograms) to LPS. The 'LPS-antigen' conjugates are easily purified upon centrifugation at 15,000 X g. These compounds are mitogenic for B cells and trigger in vivo or in vitro production of antibodies directed against the coupled antigen. In vitro, a concentration of conjugate as low as 10(-2) micrograms/ml triggers specific antibody synthesis. Injection of 5 micrograms of conjugate in mice induces a humoral response detectable 7 days after immunization. B cells cultured for 5 days with an adequate dose of the LPS-beta-galactosidase conjugate were fused with the Sp2-0-Ag cell line to give anti-beta-galactosidase hybridomas.

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.