Anti-horse Antibodies Research Articles

Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics

We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

Pharmacokinetics in rabbits and anti-sphingomyelinase D neutralizing power of Fab, F(ab')2, IgG and IgG(T) fragments from hyper immune equine plasma

We describe the separation of whole IgG, IgG(T)-less IgG (called here merely IgG) and IgG(T) and the production of Fab and F(ab')(2) fragments. We studied the pharmacokinetics of these immunoglobulins and fragments in rabbits. Both, the isotypes and the whole IgG fragments were purified and/or produced from the same plasma lot from horses hyper immunized against sphingomyelinase D to produce anti-Loxosceles antivenom. The sphingomyelinase D neutralizing ability of the isotypes and their fragments was measured. Fab and F(ab')(2) PK was well described by a tri-exponential kinetics. IgG and IgG(T) PK, however, deviated from the tri-exponential kinetics 120h after injecting a bolus of the immunotherapeutics. The departure between tri-exponential PK and the experimental data, was shown to be due to a surge of anti-horse antibodies occurring after 120h, peaking at approximately 260h and decaying slowly afterward.

ATGAM™ LEVELS IN KIDNEY TRANSPLANT PATIENTS TREATED FOR ACUTE REJECTION

242 Purpose: To measure and model Atgam blood levels in patients receiving Atgam for treatment of acute rejection. Methods: A group of 81 patients undergoing acute rejection episodes were treated with Atgam™ (horse antithymocyte globulin), 15 mg/kg/day for 7 to 14 days as part of a double blinded trial comparing the efficacy of Thymoglobulin (rabbit antithymocyte globulin) and Atgam. Serial serum samples (n=526) (day 0 to day 90) from 75 of the patients were tested to determine the level of Atgam (i.e. horse IgG levels) using an ELISA. This assay was developed and validated at SangStat Medical Corporation. Results: The mean peak value was 1258.64 μg/ml (range 560 to 2690 μg/mL) at day 9 (n=46 patients).FigureThe peak, day 21 and day 30 concentration could be modeled using multiple regression. Independent variables were number of full doses (FD), number of partial doses (PD), and presence of anti-horse antibodies as measured by SangStat's Equistat (Estat) assay. Peak level = 436 + 81(FD) + 136(PD) + 45(EStat); p =.006; R = 0.61 Day 21 level = 30 + 71(FD)+56(PD)-108(EStat); p < 0.0001; R = 0.72 Day 30 level = 62(FD)+22(PD)-131(EStat); p < 0.0001; R=0.86 Of the 75 patients, 55 had an Atgam determination on days 7, 9 or 14. For each of these 55 patients, we chose the highest value at day 7, 9 or 14 as Cmax. Patients were then ranked according to their Cmax value. Treatment failure rate was 9 out of 55 (16.4%). Of those 9, 4 were in the 20th percentile for Atgam Cmax (44%). Treatment success rate was 46 out of 55 (83.6%). Of those 46, 7 had an Atgam Cmax in the 20th percentile (15%). There was a trend toward an association between a low Atgam Cmax and treatment failure: patients in the 20th percentile for Atgam peak level were 3 times as likely to fail to respond to therapy when compared to those with higher Atgam peak levels (p=0.067 by Fischer's Exact Test).Conclusions: Using Atgam and anti-Atgam assays we have developed a method to model Atgam concentrations. Additionally, results suggest a possible relationship between lower Atgam levels and treatment failure.

Characterization of anti-Thymoglobulin, anti-Atgam and anti-OKT3 IgG antibodies in human serum with an 11-min ELISA

Patients treated with OKT3 may become resistant to OKT3 therapy following induction of anti-idiotypic antibodies. Antirabbit or antihorse antibodies following Thymoglobulin (rabbit antihymocyte globulin (ATG)) or Atgam (hores ATG) theraphy may not similarly inhibit drug efficacy due to an insufficient anti-idiotypic response against the multiple idiotypic specificities of the polyclonal anti-T cell preparations. However, no standardized assay had been developed to monitor antihorse and antirabbit antibodies. To address this issue, we developed three rapid (11-min) and standardized semiquantitative plate enzyme-linked immunoassays (ELISAs) to monitor human serum immunoglobulin-G (IgG) antibodies to Orthoclone OKT3 IgG2a, Atgam and Thymoglobulin. The format was identical for the three assays, with the exception of the immunoglobulin antigen coated on the ELISA plates. As OKT3 is a monoclonal antibody, OKT3 itself is used as the capture antigen. However, for antibody responses against the polyclonal antibody preparations Atgam and Thymoglubulin, it was found tht horse IgG and rabbit IgG respectively were equivalent to Atgam and Thymoglobulin as capture antigens. Excellent correlation with a 3.5-h format was demonstrated ( r values between 0.986 and 0.845). Specificity was demonstrated by inhibition experiments. Correlation between endpoint titres calculated from serial dilutions and 1:50 dilution OD values were 0.821, 0.983 and 0.937 respectively for the mouse, horse and rabbit assay. High titre sera were selected to asess their ability to block in vitro the binding of OKT3, Thymoglobulin or Atgam to T cells, using flow cytometry. None of the sera containing antihorse ( n = 9) or antirabbit ( n = 8) antibodies blocked T cell binding of Atgam or Thymoglobulin. In contrast, OKT3 binding was blocked by the four highest titre sera of the 13 anti-OKT3 sera tested. Consequently, prospective monitoring of treated patients using standardized 11-min assays may allow a better assessment of the effect of presentization to OKT3, Atgam or Thymoglobulin.

Rapid serologic diagnosis of serum sickness from antithymocyte globulin therapy using enzyme immunoassay.

Although the use of antithymocyte globulin/antilymphocyte serum (ATG/ALS) has been shown to be beneficial in treating renal allograft rejection, the incidence and nature of serum sickness reactions following such treatment have received limited attention. In the setting of rejection or infection, the diagnosis of serum sickness is often difficult on clinical grounds, and biopsy may be an undesirable means of obtaining documentation. A sensitive enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against ATGAM (Upjohn) and was utilized in 35 patients treated for rejection with ATGAM following clinical unresponsiveness to bolus methylprednisolone therapy. Serum sickness was diagnosed clinically in 7 of these patients (20%) during or immediately following ATGAM therapy. Significant titers of anti-ATGAM antibody were found in these 7 cases, as well as 5 additional cases (14%). Upon retrospective review, the diagnosis of serum sickness was also made in each of these 5 additional cases based upon the immunopathologic detection of horse immunoglobulin deposits in vessels of tissue (lung or kidney) examined. In 2 other cases where serologic testing was negative, there was some clinical suggestion of serum sickness that could not be documented pathologically. ELISA for anti-ATGAM antibodies was negative in testing 46 control patients and pooled normal sera. In one case, retrospective testing of pre-ATGAM therapy serum samples showed significant antihorse antibodies. Despite a negative skin test prior to administration, this patient developed symptoms of serum sickness 15 days after the onset of ATGAM therapy. Extremely high titers of antibody were detected 5 days earlier (10 days posttherapy), and the presence of horse immunoglobulin immune complexes in the kidney was documented following graft loss that occurred within 4 weeks. These findings indicate (1) serologic testing for anti-ATGAM antibodies using a sensitive ELISA method provides a quick, inexpensive, noninvasive means for the presumptive diagnosis of serum sickness in complicated clinical situations, and (2) screening of patient sera prior to administration of ATGAM may be useful in avoiding potential serum sickness reactions.

Antigenicity of the Inhibitor of Influenza Virus Strain A/Asia/57

Summary The inhibitor of influenza virus strain A/Asia/57 possesses antigenic activity. By immunizing chickens, antisera can be obtained which specifically neutralize the inhibitor. The titer of the inhibitor neutralizing antibodies in the anti-inhibitory chicken serum shows no correlation with the titer of the antihorse antibodies in that serum. Within the range of dilutions in which the anti-inhibitory activity of hen serum is manifest, the intrinsic inhibitor of the chicken serum does not exhibit itself.