A general study of the binding and separation in partition affinity ligand assay. Immunoassay of beta 2-microglobulin

Partitioning in aqueous 2-phase systems was used to separate free and bound ligand in an immunoassay for beta 2-microglobulin. In order to get efficient separation in the phase system, the antibodies were modified to favour their partition in a different phase from that of antigen. However such modification of antibodies significantly decreased their binding capacity. This was overcome by using antibodies bound to previously modified staphylococci, which had proper partitioning behaviour. Alternatively, antibodies conjugated with biotin could be used in combination with modified avidin. This paper presents a method for the evaluation of data from immunoassays whereby 2-phase systems have been used to separate free and bound antigen.     

The effect of levamisole on E-rosette formation by trypsinized lymphocytes.

Normal lymphocytes treated with trypsin lost their ability to form normal numbers of rosettes with sheep red blood cells. The recovery in E-rosette forming capacity of trypsinized cells was considerably augmented when the cells were incubated in the presence of a wide range of concentrations of the immunostimulatory drug, levamisole. Substantial recovery of rosette-forming ability was seen 2 hr after incubation of trypsinized cells with levamisole.     

The influence of epitope density on the estimation of the affinity of antibody for complex antigens.

The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities.     

Determination of the binding affinity of different human papillomavirus E7 proteins for the tumour suppressor pRb by a plate-binding assay.

A plate-binding assay was developed to quantify the affinity of the E7 oncoprotein from different human papillomavirus (HPV) types for the tumour suppressor pRb. The method is highly reproducible, sensitive and easy to handle. It could be easily adapted for the quantitative study of other interacting proteins and for screenings of inhibitors of protein/protein interactions. The pRb-binding affinity of six different E7 proteins has been quantified. The K(D) values vary from approximately 4.5x10(-9) M for HPV16 E7 to more than 1x10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the high affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity reduction. The data indicate that the high affinity pRb-binding domain of E7, LXCXE, is essential for the association between the viral and cellular proteins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association.     

A modified E-rosette assay as a semi-empirical tool in search of new T lymphocyte stimulants

An "activity index" is defined for T cell modulators, which allows meaningful comparison of experimental results by eliminating the deviations due to differences in the immunological states of T lymphocytes.     

The influence of thymosin (TEX) on antigen-induced primary and secondary antibody responses of mice

Thymosin (TFX) administered in mice does not alter their primary IgM humoral responses, while it significantly increases the number of IgG plaque-forming cells and IgM PFC during the secondary response. In the thymectomized mice, thymosin partially restores primary and secondary IgM responses, but IgM production remains low.     

The influence of hapten density on the assay of penicilloylated proteins in fluids

The use of inhibition radioimmunoassays for the measurement of penicilloylated proteins in biological fluids is compromised by the dominant influence of hapten density. Precise quantitation, and therefore assessment of antigenicity and immunogenicity, cannot be achieved in the absence of knowledge of the number and distribution of haptenic groups on the protein carrier. These assays may not, therefore, be appropriate for the measurement of potential allergenic residues in food products.     

E-rosette inhibiting substance in Hodgkin''s disease spleen extracts.

Patients with Hodgkin's disease often manifest impairment of cell-mediated immune responses in both in vivo and in vitro tests, as well as a markedly decreased percentage of E rosette-forming (T) lymphocytes in the peripheral blood. This report describes the inhibition of E-rosette formation by normal peripheral blood lymphocytes after incubation with extracts prepared from the spleens of patients with Hodgkin's disease. Such extracts also depressed E-rosette formation by the peripheral blood lymphocytes of patients with Hodgkin's disease after those cells had been restored to normal function by prior incubation in foetal calf serum. Similarly prepared extracts from the spleens of normal donors had no immunodepressive effect. The E-rosette inhibitory substance in the Hodgkin's disease spleen extracts was found to be a complex containing beta-lipoprotein, C-reactive protein, and C1q.     

The influence of scoring method on variability in results obtained with the comet assay

As part of a project to develop high throughput versions of the comet assay (single cell gel electrophoresis), with a consequent need for more efficient scoring, we have compared the performance of visual scoring, automated and semi-automated image analysis when assessing comets in the same set of gels from dose-response experiments with typical DNA-damaging agents. Human lymphoblastoid TK-6 cells were treated with concentrations of methylmethanesulphonate between 0.04 and 0.6 mM, and peripheral human lymphocytes were incubated, after embedding in agarose, with H(2)O(2) concentrations from 2.5 to 160 μM. All three scoring methods proved capable of detecting a significant level of damage at the lowest concentration of each agent. Visual scoring systematically overestimates low levels of damage compared with computerised image analysis; on the other hand, heavily damaged comets are less efficiently detected with image analysis. Overall, the degree of agreement between the scoring methods is within acceptable limits according to a Bland-Altman analysis.     

The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

The acrosome reaction, sperm-zona pellucida binding, sperm-oolemma binding/fusion and subsequent fertilization are known to be influenced by homologous as well as heterologous follicular fluid and zona pellucida protein. In this study, the effect was investigated of different concentrations of solubilized porcine zona pellucida protein on the zona binding potential of human spermatozoa under hemizona assay conditions. Human spermatozoa incubated with 617 and 142 micrograms/ml porcine zona pellucida protein showed a statistically significant increase in zona binding when compared with control spermatozoa (106.5 +/- 18.0 versus 60.9 +/- 29.0, P less than 0.02 and 111.0 +/- 26.6 versus 63.0 +/- 25.5, P less than 0.0001, respectively). Concentrations of 67 micrograms/ml porcine zona pellucida protein did not show a significant increase in zona binding (78.7 +/- 21.7 versus 66.7 +/- 25.4, P greater than 0.05). Control zona binding values for different experiments did not differ significantly (60.9 +/- 29.0; 63.0 +/- 25.5; and 66.7 +/- 25.4, P greater than 0.6). In conclusion, it seems likely that a factor(s) present in the porcine zona pellucida might play a beneficial role during human sperm-oocyte binding. The results of the study might be used in future investigations to manipulate gamete interaction to such an extent that improved fertilization rates can be accomplished.     

Carbamylcholine modulation of E-rosette formation. Effect of plasmapheresis in myasthenia gravis.

Myasthenia gravis may result from a reduction of available acetylcholine receptors at the neuromuscular junction, likely secondary to the presence of anti-acetylcholine receptor antibodies. Since T lymphocytes appear to carry a similar nicotinic acetylcholine receptor, we investigated the capacity of T cells from patients with myasthenia gravis to bind sheep erythrocytes. In addition we determined the effect of carbachol, a cholinergic agonist, on E-rosette formation, and the role of myasthenic serum in modulating these responses. Two groups of patients were identified; one with normal numbers of E-rosettes forming cells (E-RFC) and the other with significantly reduced numbers. The majority of patients with myasthenia had a reduced number of carbachol-sensitive T cells. Incubation of their serum (or the IgG fraction) with normal T cells led to a reduction in numbers of E-RFC, particularly of the carbachol-sensitive subset. These effects were blocked by d-tubocurarine and not by atropine. Following plasmapheresis, normal numbers of E-RFC were detected in the patients and the serum inhibitory activity was no longer detected. The data suggest that in parallel to the achievement of some degree of clinical improvement, plasmapheresis may restore some aspects of lymphocyte function.     

Aging influence on the E-rosette forming cells, spontaneous cell mediated cytotoxicity, and antibody dependent cellular cytotoxicity in humans

In 131 adults and 107 aging healthy subjects of both sexes, the percentage of the T lymphocytes in peripheral blood and the cytotoxic activities of NK and K cells, were determined. No age-related differences have been found in the number of peripheral blood lymphocytes, the binding of NK cells to the K562 targets, the percentage of E rosette forming cells and the ADCC level. An increased proportion of T suppressor lymphocytes in elderly subjects and a large inter- and intra-individual variation of NK cells activity in both adult and aged subjects were found.     

The influence of penicillin on Lactobacillus leichmannii serum B12 assay

The influence of penicillin on vitamin B(12) assay using L. leichmannii as the test organism was investigated, and it was found that penicillin even in a low serum concentration invalidated the test.     

Vitamin D binding globulin levels and affinity in various clinical conditions.

The approximate association constants of the plasma vitamin D binding globulin (Gc-globulin) for 25-hydroxycholecalciferol (25(OH)D3) and the plasma 25(OH)D3 binding capacities were measured in samples from 123 patients with a variety of disorders. No gross differences in binding affinities were observed between different groups of patients and controls. Many patients, however, had moderately reduced, and several had grossly reduced, plasma binding capacities. The changes in Gc-globulin relative to some other proteins are also described in detail in three patients during the course of their illness. Gc-globulin concentration and hence plasma vitamin D binding capacity can undergo rapid and marked changes during illness.     

Mechanisms of E-rosette formation by mitogen-stimulated canine lymphocytes.

This study examined the mechanisms of E-rosette formation of mitogen-stimulated canine peripheral blood lymphocytes with human and guinea-pig erythrocytes (E). In vitro culture with phytohaemagglutinin-P (PHA-P) increased the number of E-rosette forming cells two- to three-fold within 1 h and ten-fold by 48 h. Mitogen-enhancement of 1 h rosettes occurred via cross-linking of E to lymphocytes by PHA-P. Forty-eight hour rosettes, mediated by natural cytophilic antibodies in serum, resulted from PHA-P-induced increases in the number of cellular Fc receptors for this immunoglobulin. The data obtained in this study indicate that E-rosette formation by resting or mitogen-stimulated canine lymphocytes is neither a reliable nor a specific T-cell marker in this species.     

E-rosette forming cell numbers in the blood of human renal allograft recipients.

The incidence of circulating T lymphocytes (ET-RFC) and a sub-population of T lymphocytes (EE-RFC) were monitored in the blood of seventy-one cadaver renal allograft recipients for the first 2 months after transplantation. In patients with uneventful post-operative courses, the incidence of both ET-RFC and EE-RFC fell promptly upon initiation of immunosuppression returning approximately to pre-operative levels 3-5 weeks after operation; the fall in cell numbers was greatest in those patients receiving adjunct ALG therapy. With the onset of an acute rejection episode, the EE-RFC level rose quickly eventually exceeding the pre-operative level; in 88% (thirty episodes) of cases this rise occurred 1-6 days before clinical diagnosis of rejection and in 12% of cases on the same day as clinical diagnosis. The incidence of ET-RFC rose in conjunction with some cases of acute rejection but remained unchanged in other cases. It is suggested that measurement of the incidence of EE-RFC in blood is valuable in predicting the onset of acute rejection and for the differential diagnosis of acute rejection and ischaemic renal damage.     

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.