Mean OD Values Research Articles

Fetal calf serum in growth medium obscures the detection of early anticardiolipin antibody secreting clones

We have examined the effect of fetal calf serum (FCS) on the detection of early anticardiolipin antibody (ACL) secreting clones, using two well-established human IgG clones, LJ1 and AH2. By plating cells at 50/well and growing both clones simultaneously in standard growth (SG) medium containing 10% FCS, and in serum-free (SF) medium, we were able to measure by ELISA the total IgG and ACL levels in the supernatants. The mean OD values (×1000) against cardiolipin for both LJ1 and AH2 were significantly higher for clones grown in SF than in SG medium: 331 OD units vs. 172, and 275 OD units vs. 166 respectively ( p < 0.001). Importantly, the number of wells in which the OD value was > 0.25 units above background was: for LJ1 in SG only 3 36 vs. 30 36 in SF; similarly, for AH2 1 36 in SG vs. 22 36 in SF. In comparison, the total IgG assay using an OD value > 0.7 units above background, detected immunoglobulin secretion in all but one of the wells. We conclude that in ELISA procedures FCS in SG medium competes with solid phase cardiolipin for antibody binding. We suggest that these antibodies are binding to phospholipid from microvesicles found in FCS. We recommend that minimally ‘positive’ clones on testing should be re-tested and, if necessary, switched to SF medium in order to prevent such clones from being discarded prematurely.

Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant

In order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (+/- SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 +/- 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.

ELISA of rat sera infected with Paragonimus iloktsuenensis

Enzyme-linked immunosorbent assay (ELISA) of paragonimiasis iloktsuenensis rat sera was performed using crude antigens of Paragonimus iloktsuenensis (PIA), P. westermani (PWA) and Clonorchis sinensis (CSA). Three crude antigens (PIA, PWA, CSA) were prepared to saline homogenated supernatants of whole adult worms. Infected rat sera were obtained biweekly from the albino rats fed 50-80 metacercariae of P. iloktsuenensis through gastric catheter. Experimental groups were divided into 4 groups: GI (controls), GII, GIII and GIV according to 1-7 worms as GII, 10-19 worms as GIII and 22-40 worms as GIV, respectively. In ELISA, the mean OD values of each group for the homologous antigen (PIA) were increased significantly compared to the control sera at the 4th week of infection. With the progress of duration of infection, the mean OD values of infected sera of GII & GIV continuously increased up to the 12th week (last week), but in GIII the mean OD value increased until the 10th week. No significance was noted among the infection dose groups (GII, GIII and GIV), after the 6th week of infection. Also, the OD values of all infected rats did not show any proportional relationships to the number of worms recovered. In brief, the antibody productivity of individual rats were strongly different. The rat sera infected with P. iloktsuenensis cross-reacted with those infected with P. westermani or C. sinensis, as identified by OD values.

IgG rheumatoid factor in human and rabbit transplantation sera.

Sera of human recipients of kidney allografts and rabbit recipients of skin allografts were tested by means of enzyme immunoassays (EIA) for IgG rheumatoid factor (RF) combining with human IgG (RF-H) or rabbit IgG (RF-R). In humans, the mean OD value in EIA for RF-H was significantly higher in 185 sera of allograft recipients than in 46 pretransplantation sera and 100 sera of normal subjects. Furthermore, the mean OD in patients who rejected their grafts was higher than in patients with satisfactory graft function. Interestingly, there was no difference in OD values for RF-R between sera of graft recipients and sera of normal subjects. Of 52 rabbit recipients of skin allografts, 24 were positive for both RF-R and RF-H, 4 were positive only for RF-R, another 4 only for RF-H, and 20 were negative for both. Renal allografts originating from the previous donors of skin grafts resulted, in most instances, in a significant decrease in the strength of the reaction for both RF-R and RF-H. The possible role of graft rejection in stimulation of RF formation as well as the possible role of RF in graft rejection should be considered.

Antibodies to the PF155 Antigen of Plasmodium falciparum: Measurement by Cell-ELISA and Correlation with Expected Immune Protection

Plasmodium falciparum polypeptide Pf155 is one of the main candidates for a vaccine against asexual blood stages of P. falciparum. Antibodies against Pf155 can be detected by a cell-ELISA technique with glutaraldehyde-fixed and air dried P. falciparum-infected erythrocytes as antigen. Using this assay, we measured the level of antibodies in sera from 230 subjects with various degrees of past exposure to P. falciparum. Significant levels of antibodies (OD492 greater than 0.5) were detected in 41/50 sera from Central African adults and in 34/50 sera from West African adults. All sera from 50 European adults suffering primary malarial attack and 28/30 sera from West African children (10 to 12 years old) were negative. Intermediate results were obtained with 50 sera from West African adults living in France for greater than or equal to 2 years (12 positive). Mean OD values of the sera of these five groups of subjects varied in the same direction as the positivity rates. These preliminary results suggest that the level of anti-Pf155 antibodies as detected by cell-ELISA might provide an assessment of protective immunity against P. falciparum which could complement clinical or epidemiological criteria.

Determination of vaccine-induced and naturally acquired class-specific mumps antibodies by two indirect enzyme-linked immunosorbent assays

Paired sera from 46 vaccinees and 22 patients with clinically typical or atypical parotitis were tested for class-specific mumps antibodies by two different indirect enzyme-linked immunosorbent assay (ELISA) procedures. Both ELISAs appeared suitable, specific and more sensitive than neutralization (NT) and complement-fixation (CF). However, the macro-ELISA (M-ELISA) method, using beads as antigen-coated solid phase, showed a higher sensitivity than micro-ELISA (m-ELISA), performed on microplates. Diagnostic rises in mumps IgG antibodies and mumps IgA antibodies were detected more frequently by M-ELISA, mostly in post-vaccination sera. In addition, higher mean OD values of mumps IgG, IgA and IgM antibodies were generally found by M-ELISA. Nevertheless, m-ELISA appeared more convenient for evaluating class-specific mumps antibodies in large-scale studies, since the procedure is simpler, more rapid and less expensive than that of M-ELISA. Conversely, M-ELISA may be considered the test of choice for detecting low class-specific antibody levels. However, the determination of class-specific mumps antibodies appeared as an essential tool for evaluating vaccine-induced or naturally acquired mumps immunity.

Partial characterization and clinical correlation of circulating human immunoglobulins directed against thyrotrophin binding sites in guinea pig fat cell membranes. Development of a direct enzyme immunoassay.

To obviate several problems inherent in indirect thyroid-stimulating hormone (TSH) receptor antibody assays, we developed an enzyme-linked immunosorbent assay (ELISA) that measures antibodies binding to guinea pig fat cell membrane, which contain high concentrations of TSH receptors. Solubilized guinea pig fat cell membranes were adsorbed to plastic microtiter plates and served as the solid-phase antigen. Test sera and affinity-purified alkaline phosphatase-conjugated anti-human IgG were co-incubated with membranes, after which p-nitrophenyl phosphate was added. Results were read when a positive control reached a standard color change (OD405nm). Specificity of this assay was demonstrated by the inability of albumin, insulin, TSH subunits, propranolol, or dexamethasone to block binding 30. normal subjects had a mean OD value of 0.080 +/- 0.050 (SD). 23 of 25 untreated Graves' patients had OD values at least 2 SD above the normal mean (Grave's mean +/- SD; 0.46 +/- 0.33, P less than 0.001) and in each case 10(-6) M TSH inhibited the binding by at least 60%, suggesting that the immunoglobulins were directed at the TSH receptor. Seven of 25 serum samples from patients with Hashimoto's disease, seven of 23 serum samples from patients with transient hyperthyroidism (subacute thyroiditis or painless thyrotoxic thyroiditis), and two of 10 samples from patients with thyroid carcinoma had significant elevations in the titers of membrane-directed immunoglobulins. Graves' patients who were treated with ablative therapy at least 6 mo earlier and who were euthyroid when restudied continued to have abnormally elevated membrane-directed immunoglobulins in six of eight samples studied. Further studies involved the substitution of affinity-purified alkaline phosphatase anti-IgM antisera for the anti-IgG antisera routinely used. Seven of 12 serum samples from patients with Graves' disease had significant elevations in binding which in every instance was inhibited by greater than 60% by 10(-6) M TSH. In sum, the present results indicate that (a) we have developed a sensitive, specific, reproducible, convenient ELISA for the measurement both of the total amount of circulating membrane-directed antibodies and of TSH-displaceable membrane-directed immunoglobulins. (b) This ELISA detected significant elevations in TSH-displaceable guinea pig membrane binding in 23 of 25 untreated Graves' patients as well as in approximately 30% of patients with Hashimoto's thyroiditis and subacute thyroiditis. (c) Elevated membrane directed antibodies may continue to be present many months or years after restoration of the euthyroid state. (d) Circulating membrane binding IgM immunoglobulins have been detected in patients with Graves' disease. Further studies using this ELISA should prove useful in a variety of investigative and clinical studies.