Biospecific bimolecular binding reactions — a new ellipsometric method for their detection,quantification and characterization

A new ellipsometric method for detection, quantification and characterization of bimolecular, specific interactions on solid surfaces, e.g., binding between antigen and antibody and between ligand and receptor, is described. In the method, which we have called diffusion-in-gel (DIG) ellipsometry, one of the binding components is placed in a trough in a gel which has been poured over a solid surface coated with the other binding component. After diffusion, the gel is removed from the surface and ellipsometric measurement of thickness of adsorbed bimolecular layers is performed at different distances from the site of the diffusion trough. Bimolecular binding on the solid surfaces was also studied by wettability determinations with a water condensation technique.Three bimolecular binding systems were studied: bovine serum albumin (BSA)-anti-BSA, ganglioside GM1-cholera toxin, and C-polysaccharide-C-reactive protein. There was no tendency to saturation in the anti-BSA adsorption profile, which was steep with an endpoint thickness of about 16 nm. In contrast, the cholera toxin profile, within a narrow concentration range, rose to a plateau level of about 3 nm thickness of adsorbed cholera toxin. The C-reactive protein profile formed an intermediate pattern. Good agreement was observed between the thickness of the adsorbed ligand layers and wettability as determined by water condensation.Compared with other methods, the DIG ellipsometry technique has several theoretical and practical advantages for the detection and investigation of biospecific bimolecular binding.     

A paper radioimmunosorbent test (PRIST) for the detection of larva-specific antibodies to toxocara canis in human sera

A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 μg/ml, and could be stored for up to 3 weeks in vacuo at ?70°C. Antigen coated discs were incubated with test sera at 1 : 10 dilution for 3 h at room temperature (21°C), reacted with [¹²⁵I]anti-human IgG for 1 h and counts determined in a gamma counter. Sera from patients with fascioliasis, taeniasis, schistosomiasis, oxyuriasis, trichinellosis and ancyclostomiasis gave counts similar to cord serum controls. Sera from patients with ascariasis gave counts of up to twice as great as controls, but sera from patients with toxicariasis produced counts of 7,000–13,000, a 4–6-fold increase.     

Test systems for detection of human T associated Ig-related surface determinants using chicken antiglobulin reagents

In earlier experiments the mixed antiglobulin rosetting reaction (MARR) and the direct antiglobulin rosetting reaction (DARR) were found to be of similar sensitivity in demonstration of immunoglobulin (Ig)-bearing B lymphocytes. These test systems were used in the present study together with chicken anti-F(ab')2, anti-IgM and anti-IgA antibodies to detect Ig-related determinants on human peripheral blood T cells. Although for each antiglobulin reagent the reactivity of viable T cells in the MARR and the DARR appeared to be optimal after treatment of the lymphocytes with neuraminidase, the MARR was always more sensitive than the DARR. Further, when test conditions were established so as to avoid erroneous detection of Ig bound to T cell Fc receptors, the MARR was found to be more sensitive than indirect immunofluorescence. Under the most sensitive mixed antiglobulin rosetting conditions, the mean number of T cells expressing F(ab')2-, alpha- and Mu-related determinants was 99%, 50% and 39% respectively. The complete inhibition of the MARR by human IgM when anti-IgM antibodies are used and the more than 70% inhibition by IgA when anti-IgA antibodies are used, largely exclude false positive rosette formation due to contaminating specificities directed against non-Ig determinants on T cells. Trypsin treatment of T cells removed all determinants recognized by anti-IgM and anti-IgA antibodies and these cells reexpressed these determinants during in vitro culture; this indicates that these determinants are T cell products and do not represent adsorbed Ig molecules.     

Quantitation of helper factor activity. I. Utilization of the ELISA method to measure helper activity in the secondary antibody response

We describe here a simple culture system for measuring the effects of regulatory cells or factors on the secondary antibody response which uses the enzyme-linked immunosorbent assay (ELISA) to detect antibody production. This technique offers several advantages over methods which measure hemolytic plaque-forming cells. Helper factors purified from the supernatant of in vitro primed helper cells were shown to specifically enhance antibody production from primed spleen cells by from 2- to 4-fold in this system.     

Inter-alpha-trypsin-inhibitor (ITI): use of immunoadsorbents for preparation of anti-ITI antiserum, ITI-free human serum and purified ITI

Immunoaffinity chromatography was used to prepare various reagents required for studies of inter-alpha-trypsin-inhibitor (ITI), a human protease inhibitor. To absorb an initially polyspecific anti-ITI antiserum, a mixture of all serum proteins except ITI was prepared as follows: normal human serum was gel filtered (Sephacryl S-300) and the part of peak II containing normal ITI was removed; another aliquot of serum was heated, gel filtered, and peak I which contained all ITI molecules in aggregated form was discarded. The remaining fractions from both gel filtrations were immobilized on gel and used as an immunoadsorbent. The monospecific anti-ITI antiserum thus obtained was immobilized on gel and could bind ITI from human serum. Under conditions chosen to weaken non-specific adsorptions and desorb ITI without denaturation, this immunoadsorbent made it possible to prepare ITI-free serum and purified ITI with biological activity.     

P物质对小鼠腹腔巨噬细胞功能的调整作用

本文研究了神经肽P物质(Substance P,SP)对经硫代乙醇酸钠(TG)培基活化的小鼠腹腔巨噬细胞(Mφ)的免疫功能的影响。结果表明,经TG活化的Mφ在体外与SP共同培养18小时后,其吞噬中性红的能力明显提高,其有效浓度在10~(-6)～10~(-8)M之间(P<0.05)。实验中还发现SP在10~(-7)～10~(-9)M有显著增强Mφ产生IL-1的作用,SP与LPS(10μg/ml)联合应用可协同促进IL—1的产生。SP尚可增强Mφ对肿瘤细胞增殖的抑制作用,但这一作用较巨噬细胞活化因子(MAF)明显为弱。SP不能与Con A协同增强小鼠脾脏T细胞产生MAF的能力。     

Improved performance of a double antibody radioimmunoassay for carcinoembryonic antigen.

A new double antibody solid-phase radioimmunoassay (RIA) for carcinoembryonic antigen (CEA) is critically analyzed. The aim of the study was 4-fold: (a) to define the level of sensitivity (a comparison of 3 different assay procedures revealed that our sequential assay was more sensitive than most previously reported RIAs, while competitive and non-equilibrium assay had wider measuring ranges); (b) to analyze recoveries of CEA in either serum, plasma or urine (the recovery, even in urine, was very close to expected values, indicating that no CEA is lost or degraded during brief storage or in the extraction procedure); (c) to evaluate inter- and intra-assay variations, since most clinical management is dependent on serial assays rather than single determinations. The coefficients of variation were low both within and between assays. A change of 3 ng CEA is required for significant change (greater than 2 S.D.) at the normal serum level which is 16 ng CEA/ml in our assay. At levels above normal, a change of 4 ng is required; (d) the assay was also developed for determination of CEA levels in a large series of perchlorid acid treated serum, plasma or urine samples. This forms the basis for an assay suitable for serial assays with high sensitivity and accuracy in various neoplastic diseases.     

Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader

Two simple semiautomated microassays for the measurement of superoxide (O-2) and hydrogen peroxide (H2O2) production by cultured macrophages (MPs) are described. The measurement of O-2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H2O2 is based on the horseradish peroxidase (HRPO)-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 mul amounts per well of either a ferricytochrome c solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst (OB) and incubation of the plates at 37 degrees C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above test, when fitted with interference filters with wave lengths of 550 nm (for the assay of O-2) and 600 nm (for the assay of H2O2). The principal advantages of this techniques are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.     

The incorporation of high pH euglobulin precipitation in the isolation of C1q

A one-step polyvalent counterimmunoelectrophoresis (PIE) method for the typing of pneumococci is described. Only one antiserum is used (omniserum, containing antibodies to > 80 pneumococcal types) and it is not necessary to stock large numbers of monospecific typing sera. By observing reactions of identity between the precipitin lines produced by the pneumococcus under test, and the specific capsular polysaccharide precipitin lines in a reference pattern produced by the polyvalent pneumococcal vaccine ‘Pneumovax’, a cumulative percentage of 64.6% of pneumococcal types isolated could be typed.The method is simple, reproducible, inexpensive and provides a permanent stained record.     

Shedding and resynthesis of receptor for mouse red blood cells on lymphocytes from healthy subjects and chronic lymphatic leukaemia patients

The shedding and resynthesis of receptors binding mouse red blood cells was investigated in lymphocytes of normal peripheral blood and in that of chronic lymphatic leukaemia patients. In the first hours after incubation the release of the receptor from the membrane is observed and its resynthesis occurs after 5 h. Metabolic poisons inhibit the reappearance of receptor while specific inhibitors for protein synthesis do not influence these phenomena.Compared with normal lymphocytes, cells from CLL patients show different patterns of shedding and resynthesis of receptors suggesting an altered metabolism of leukaemic lymphocytes.     

A solid phase micro-radioimmunoassay to detect minute amounts of Ig class specific anti-viral antibody in a mouse model system.

A simple and rapid micro-radioimmunoassay was developed to detect and quantitate class specific mouse anti-Sendai virus antibodies. Two different 125I-labelled indicator systems were studied. After incubation of test serum with antigen one system used 125I-rabbit anti-mouse IgG (RIA 1) and the second employed rabbit anti-mouse IgG, IgA or IgM followed by 125I-sheep anti-rabbit immunoglobulin reagent (RIA 2). The RIA 2 method was adopted for routine use as it was more sensitive, gave better discrimination between sample and background counts and eliminated the need for several labelled rabbit anti-mouse Ig class specific antisera. The technique was found to be about 100 times more sensitive than conventional HI tests, specific, reliable and economical of reagents and time.     

Characterization of mouse allergens

The major allergens present in mouse skin, serum, and urine have been identified. Skin extracts, serum, and urine were chromatographed, and the activities of the fractions were monitored by histamine release from the leukocytes of individuals sensitive to mice. Fractionation of skin extracts revealed two major allergens. The large allergen has a molecular weight of approximately 67,000 daltons and by biochemical and immunochemical criteria appears to be identical to mouse albumin. The smaller molecular weight allergen is approximately 17,000 daltons. The same two allergens are also found in mouse serum and mouse urine. Histamine release by leukocytes of individuals allergic to mice demonstrated that some individuals react predominantly to the large allergen, some to the small allergen, and one group of patients reacts to both allergens.     

Immediate cytotoxicity of Chlamydia trachomatis for mouse peritoneal macrophages.

The toxicity of Chlamydia trachomatis was studied with mouse peritoneal macrophage culture. Inoculation of 30 inclusion-forming units of trachoma B/TW-5/OT organisms and 250 inclusion-forming units of lymphogranuloma venereum L2/434/Bu organisms per cell caused immediated toxicity, with the killing of 40 to 90% of the macrophages within 6 h after inoculation. Inhibition of phagocytosis by adsorption at 0 degrees C or by NaF pretreatment of macrophages prevented the toxicity, indicating that chlamydiae must be phagocytized to induce toxicity. Infectivity and toxicity could be dissociated, since ultraviolet-inactivated chlamydiae were still toxic. However, the toxicity was destroyed by heating the organisms at 56 degrees C for 10 min. Tetracycline, and antichlamydial drug, did not prevent toxicity, indicating that multiplication of the organisms was not required to induce toxicity. Toxicity was not prevented by treatment of macrophages with hydrocortisone. The toxicity of trachoma TW-5 was reduced by the rabbit immune serum of trachoma TW-5 but not by the rabbit immune serum of psittacosis meningopneumonitis.     

Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages.

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.     

Maturation of bone marrow lymphocytes. I. Quantitative rosetting methods of detecting Fc and complement receptors and surface immunoglobulin.

Cytocentrifuge rosetting procedures were developed for use with bone marrow cells to quantitate Fc and complement receptors (FcR, CR) and surface IgM on marrow small lymphocytes. Cell suspensions from 9–11-week-old C3H mice were mixed (1 : 30) with washed sheep red blood cells (SRBC) coated with either mouse anti-SRBC serum (for FcR), rabbit anti-SRBC stroma serum plus mouse serum (for CR) or goat anti-mouse IgM serum (for IgM). Centrifuged cell pellets were incubated for 30 min at either 37°C (FcR, CR) or 0°C (IgM) and examined in stained cytocentrifuge preparations. Small lymphocytes were defined as non-DNA-synthesizing lymphocytes smaller than 10 μm nuclear diameter. Many of these cells in the bone marrow formed specific rosettes for FcR (25%), CR (18%) and surface IgM (39%). The incidence of FcR- and CR-bearing small lymphocytes relative to IgM-bearing small lymphocytes was lower in the marrow than in the blood, spleen and lymph nodes. Rosette size ranged from 4 to > 20 SRBC per lymphocyte but tended to be smaller in the marrow and blood than in the spleen and lymph nodes. The methods and results are discussed as a basis for studies of B lymphocyte maturation and lymphocyte heterogeneity in the bone marrow.     

A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface

A quick and simple method for determining whether monoclonal antibodies react with the same or different antigens is described which utilises an indirect radioactive binding assay against cells. Six antibodies were selected, BK19.45, BK20.20, GenOx4.17, GenOx4.21, W6-34 and W6-46, which detected antigens present either only on leukocytes (BK19.45 and BK20.20) or on a wider range of cell types including fibroblasts, liver cells and neuroblastoma cells. The saturation binding levels for each antibody, in terms of the amount of ¹²⁵I-anti-mouse immunoglobulin bound, were determined with respect to a fixed number of cells. The addition of two antibodies with different specities (BK19.45 or BK20.20 to either W6-34 or W6-46) resulted in a higher plateau value of ¹²⁵I-anti-mouse immunoglobulin bound per fixed number of cells than for either antibody singly. The increase in the amount of antibody bound is equivalent to the sum of the two individual saturation binding levels. In contrast, the addition of BK20.20 to BK19.45 or W6-45 produced no detectable rise in the saturation level. From these data it is concluded that BK20.20 and BK19.45, and W6-34 and W6-46 are bound to either identical epitopes which are in very close spatial relationship. On the other hand 19.45 and W6-34, as expected, detect unrelated antigens. Observations from autoradiograph binding studies on the semi-quantitative distribution on bone marrow cells of the antigens recognised by 19.45 and 20.20 supported the conclusion that the antibodies were recognising identical antigens.A study of the antibodies to HLA (2A1) and β₂ microglobulin (M3) showed an increase in the saturation level, when both antibodies were added together, which was less than the sum of the two individual saturation binding levels. The close association of β₂ microglobulin and HLA on the cell surface may to some extent prevent independent antibody binding. These data suggest that the above approach will differentiate between monoclonal antibodies which detect antigenic determinants that are located on closely associated surface molecules.     

Appropriateness of drugs prescribed by primary care physicians for depressed outpatients.

In a sample of middle class individuals seeking martial and sexual counseling, 30% had diagnosable psychiatric illness, including 14% who had depressions at the time of interview. Those with psychiatric syndromes were significantly more likely to have prescribed psychoactive than those without these syndromes. Those with depression were more likely to have received diazepam and similar drugs than antidepressants. The same was true for those with other syndromes but in many of these cases, diazepam or other antianxiety agents seemed more appropriate. Thus, affective disorder might well be the psychiatric syndrome for which these drugs are most often inappropriately prescribed. Inappropriate treatment is a matter of concern in an illness which is potentially fatal.     

Determination of alternative pathway of complement activity in mouse serum using rabbit erythrocytes

Rabbit, mouse and sheep erythrocytes expressing different concentrations of membrane sialic acid were used to study possible modes of activation of the alternative complement (C) pathway in mouse, human and guinea pig serum. Mouse erythrocytes activated only human serum, whereas rabbit erythrocytes activated the sera of all three species. Based on the observation that rabbit erythrocytes activate the murine alternative C pathway a method for estimation of alternative C pathway activity (AP50 value) in mouse serum was devised analogous to that used for human AP50 determination. The method is not very sensitive to ageing or to batch variation of the indicator cells. The AP50 value of mouse serum measured by this method is of the same order as for human and guinea pig serum. Mouse serum AP50 activity is partly determined by natural antirabbit erythrocyte antibodies and is sensitive to heating (15′ at 48°C and 4′ at 56°C), and to the actions of cobra venom factor, zymosan and cysteine. Strain and sex differences with respect to AP50 activities of mouse sera were observed.