Antisperm antibody binding to human acrosin: a study of patients with unexplained infertility.

OBJECTIVE: Antisperm antibody binding to acrosin was investigated by Western Blotting. The clinical significance of this binding specificity was assessed in a 2-year clinical follow-up. DESIGN: Consecutive serum samples positive for antisperm antibodies by both enzyme-linked immunosorbent assay and immunobead testing were evaluated for acrosin-binding specificity. SETTING: The patients were followed in an outpatient setting by private infertility specialists. PATIENTS: Sixty-five consecutive infertile referral patients with positive antisperm antibody were evaluated. Clinical follow-up was obtained on 8 of 9 females with evidence of antibody binding to acrosin and 19 of 26 females with no specific binding to acrosin. INTERVENTIONS: Prednisone therapy was given during six courses of intrauterine insemination with husband's sperm. All treatment decisions were made by private physicians independent of the acrosin-binding result. MAIN OUTCOME MEASURES: Pregnancy status was obtained as part of a 2-year follow-up. RESULTS: Acrosin-binding specificity was demonstrated in 10 (15%) of the 65 patients. Two of the 8 women (25%) with antibody binding to acrosin and 6 of the 19 women (32%) with antisperm antibodies but no specific binding to acrosin delivered normal children. CONCLUSIONS: Although antibody-binding specificity to acrosin could be demonstrated, a 2-year clinical follow-up showed no difference in pregnancy rates when compared with women with antisperm antibodies showing no binding specificity to acrosin.

Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.

A direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The direct correlation between the results of these assays was 94%. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa.

Platelet antibody binding in systemic lupus erythematosus.

Using a combination of enzyme-linked immunosorbent assay and SDS-PAGE with protein blot (Western blotting), increased levels of serum platelet bindable immunoglobulin (SPBIg) were demonstrated in 10 of 10 thrombocytopenic patients with systemic lupus erythematosus (SLE) (7.0-60 fg Ig/platelet) with consistent binding to SDS-PAGE platelet fractions of approximate molecular weight (120 and 80 kDa). This pattern of Ig binding was characteristic of SLE and was not seen in 20 normal volunteers and infrequently seen in 20 patients with idiopathic thrombocytopenic purpura.

Antibody binding specificity to donor sperm in sera from infertile patients.

Indirect testing for elevated sera levels of antisperm antibody assumes that all relevant antigens are present on the target sperm utilized. In the present study, the heterogeneity of reactivity of positive sera with sperm from different donors was addressed. When 68 sera that previously tested positive by either sperm immobilization test (SIT) or ELISA or both were tested for antisperm antibody levels by a quantitative ELISA using nine different sperm donors, the frequency of positive reactions was 51% for men and 81% for women. A 50% correlation of SIT- and ELISA-positive results could be improved to 85% using the same sperm specimen. This would improve the overall correlation of functional and ELISA test results from 95% to 99% in the infertile population studied. The data suggest that individual sperm may vary in their antigenic expression and that comparison of methods between laboratories could be improved if equivalent target sperm were used.

A comparison of sperm agglutination and immobilization assays with a quantitative ELISA for anti-sperm antibody in serum.

An enzyme-linked immunosorbent assay (ELISA) that quantitates antisperm antibody in serum was compared with standard sperm agglutination and immobilization assays with the use of sera from 40 normal and 292 subfertile individuals. Quantitation of the assay was accomplished by standardizing assay parameters, including the incorporation of a standard reference curve, the number of whole target sperm, the optimal dilution of serum, the selection of microtiter plate, and the time and temperatures involved in the adsorption and incubation phases. With this method, the level of antisperm antibody binding to target sperm in 40 normal fertile individuals was found to be 2.3 (+/- 1.1 standard deviation [SD]) fg immunoglobulin (Ig)/sperm. An increased mean level of 7.4 +/- 3.7 fg Ig/sperm was determined in 84 infertile patients with positive agglutination and/or immobilization tests. In 208 individuals with negative agglutination and immobilization tests the mean concentration of antisperm antibody was 2.5 +/- 1.3 fg Ig/sperm. Postvasectomy patients assayed by this method had a mean Ig binding value of 7.1 +/- 2.4 fg Ig/sperm. The infertile group with positive agglutination and/or immobilization tests had a significantly higher mean antisperm antibody level than the normal fertile group, according to the Student's t-test for independent samples (P less than 0.001). This indirect serum-based assay reproducibly quantitates antisperm antibody binding to whole target sperm, suggests the normal and abnormal levels of antisperm antibody, and correlates with standard functional assays.

Antigenic determinants in idiopathic thrombocytopenic purpura.

Serum platelet bindable immunoglobulin (SPBIg) was determined in a group of 23 idiopathic thrombocytopenic purpura (ITP) patients and compared to 20 normal, healthy controls. The mean SPBIg of the ITP group was 16.1 (+/- 17.9 SD) fg/platelet, while the normals were substantially lower, 4.0 (+/- 1.2) fg/platelet. Sera from patients of both groups were then incubated with platelet fractions immobilized on nitrocellulose membrane strips (Western Blotting) to detect platelet antigen specificity using a peroxidase labelled indicator antibody. The normal patient sera did not react with platelet fractions on the nitrocellulose strips. However, 21 of 23 ITP sera bound to one or more platelet fractions with large variations in the number and molecular weights of the platelet fractions identified by ITP antibody. These observations suggest the presence of multiple antigenic binding sites for platelet specific immunoglobulin in ITP sera. This variation may reflect heterogeneous antibodies binding to diverse antigens or homogeneous antibodies to a limited number of antigenic determinants shared by several discrete platelet molecules.

Quantitation of sperm bindable IgA and IgG in seminal fluid.

Seminal fluid and serum from 95 infertile males were assayed for sperm bindable immunoglobulins using an indirect ELISA with whole target sperm. The ELISA method was compared to seminal fluid and serum immobilization and agglutination assays (functional assays). In this infertile group, the ELISA assay was positive in 22% of seminal fluids (greater than 1.2 fg IgA/sperm and greater than 0.3 fg IgG/sperm). The seminal fluid antibodies were IgA and had an accompanying elevated IgG component in 78% of patients. There was a 96% correlation between negative seminal fluid functional assays and negative ELISA, and a 95% correlation between positive seminal fluid functional assays and positive ELISA. Positive serum sperm antibody tests were found in 71% of the infertile males with positive seminal fluid sperm antibodies, but 29% of the infertile males with strongly positive IgA seminal fluid sperm antibodies showed normal levels of serum sperm antibodies by either ELISA or functional assays. The ELISA method gives reproducible quantitation of sperm antibodies in seminal fluid and correlates well with accepted functional assays. Comparisons with serum sperm antibody assays suggests that seminal fluid sperm antibody analysis complements the serum analysis of sperm antibodies.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

An enzyme-linked immunosorbent assay for the evaluation of thrombocytopenia induced by heparin.

Five patients with heparin-associated thrombocytopenia (HAT) were evaluated by platelet aggregation and quantitation of immunoglobulin binding to intact target platelets in both the presence and absence of heparin. These patients developed thrombocytopenia (12,000 to 70,000 platelets/microliter) 7 to 15 days and embolic and hemorrhagic complications 9 to 15 days after the initiation of heparin therapy. Platelet aggregation after the addition of heparin was demonstrated in two of four HAT serum samples, whereas normal serum samples showed no significant platelet aggregation. The five HAT serum samples showed normal to elevated baseline serum platelet-bindable immunoglobulin (SPBIg) with a range of 4.3 to 11.4 fg/platelet (normal less than or equal to 1.0 to 6.5 fg/platelet). When HAT sera were incubated with target platelets and heparin (5 U/ml), the SPBIg increased to 8.5 to 37.5 fg/platelet, a mean increase of 148% in the presence of heparin. Normal and control serum samples (from 10 normal laboratory volunteers, nine patients without thrombocytopenia receiving heparin, nine patients with autoimmune thrombocytopenic purpura, and nine patients with nonimmune thrombocytopenia not receiving heparin) showed only a slight increase in SPBIg of 0 to 2.8 fg/platelet above baseline, a mean increase of 15% after heparin incubation with the serum samples. The measurement of SPBIg of washed platelets incubated with test serum samples in the presence and absence of heparin is potentially a specific and sensitive in vitro test for the diagnosis of HAT and may prove more sensitive than platelet aggregation studies with heparin.

A quantitative ELISA procedure for the measurement of membrane-bound platelet-associated IgG (PAIgG).

A quantitative ELISA assay for the measurement of in vivo bound platelet-associated IgG (PAIgG) using intact patient platelets is presented. The assay requires quantitation and standardization of the number of platelets bound to microtiter plate wells and an absorbance curve using quantitated IgG standards. Platelet-bound IgG was measured using an F(ab')2 peroxidase labeled anti-human IgG and o-phenylenediamine dihydrochloride (OPD) as the substrate. Using this assay, PAIgG for normal individuals was 2.8 +/- 1.6 fg/platelet (mean +/- 1 SD; n = 30). Increased levels were found in 28 of 30 patients with clinical autoimmune thrombocytopenia (ATP) with a range of 7.0-80 fg/platelet. Normal PAIgG levels were found in 26 of 30 patients with nonimmune thrombocytopenia. In the sample population studied, the PAIgG assay showed a sensitivity of 93%, specificity of 90%, a positive predictive value of 0.90, and a negative predictive value of 0.93. The procedure is highly reproducible (CV = 6.8%) and useful in evaluating patients with suspected immune mediated thrombocytopenia.

Preoperative AT-III values and clinical postoperative thrombosis: a comparison of three antithrombin-III assays.

Three antithrombin-III assays (Sigma functional plasma, von Kaulla functional serum, and Calbiochem-Behring radial immunodiffusion) are compared using preoperative serum and plasma from 48 patients admitted for cardiovascular or other major vascular surgery. Medical records were reviewed for evidence of thrombotic complications. Eight patients (17%) in this selected population had clinical evidence of postoperative thrombotic complications. The sensitivity and specificity for each AT-III assay were calculated, and the positive and negative predictive values in this population were determined. Sigma's plasma AT-III had the highest positive predictive value (67%) and negative predictive value (100%). The functional serum and RID assays had significantly lower positive predictive values (23% and 38% respectively) and negative predictive values of 86%. Using the Two Sample t-Test to evaluate differences in AT-III values between the two patient groups, i.e., those who experienced thrombotic complications and those who did not, only the functional plasma AT-III method was statistically significant at a 95% confidence interval.

An enzyme-linked immunosorbent assay for the quantification of serum platelet-bindable IgG.

An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.

Early detection of oxacillin-resistant staphylococcal strains with hypertonic broth diluent for microdilution panels.

A total of 292 coagulase-positive and 111 coagulase-negative staphylococcal strains were tested in microdilution MIC panels containing 16 to 0.13 microgram of oxacillin per ml diluted in cation-supplemented Mueller-Hinton broth with and without an additional 2% NaCl. All strains were tested using the stationary-phase inoculum procedure with an incubation temperature of 35 degrees C. Test results were recorded after 16 to 20 h of incubation; staphylococcal strains susceptible to oxacillin (less than or equal to 2 micrograms/ml) were reincubated for 20 to 24 h, and endpoints were determined again. Oxacillin resistance was found in 27 (9%) of the 292 coagulase-positive strains and 39 (35%) of the 111 coagulase-negative strains. Of these resistant strains, 5 (19%) of the 27 coagulase-positive strains and 13 (33%) of the 39 coagulase-negative strains were detected 24 h earlier in cation-supplemented Mueller-Hinton broth with 2% NaCl than in cation-supplemented Mueller-Hinton broth without the additional NaCl. However, 9 (33%) of the 27 resistant coagulase-positive strains and 10 (26%) of the 39 resistant coagulase-negative strains were detected only after an additional 24 h of incubation. Oxacillin MICs for the 265 coagulase-positive susceptible strains and 72 coagulase-negative susceptible strains were not affected by the additional 2% NaCl. These results support the utility of adding 2% NaCl to the broth diluent for the early detection of oxacillin-resistant staphylococcal strains and the necessity of extended incubation for those strains which initially appear to be susceptible to oxacillin after only 16 to 20 h of incubation.

The role of carboxylic acids in EDTA-dependent panagglutination.

The investigation of a patient blood sample showing a discrepancy between cell grouping and serum confirmation demonstrated a serum agglutinin which reacted with all red blood cells tested when exposed to EDTA. This reaction was 4+ macroscopic at room temperature, 2+ macroscopic with hemolysis at 37 degrees C in albumin, and 1+ macroscopic in the anti-human globulin phase. Agglutination was abolished following dithiothreitol treatment of the patient's serum or following saline washing of the EDTA-exposed test cells. The agglutination reaction was not limited to EDTA, but could be produced with polycarboxylic acids (citrate, L-tartrate, succinate) and monocarboxylic acids (acetate, lactate, propionate, valerate, butyrate). Non-carboxylic acids and low molecular weight ketones or alcohols failed in the agglutination reaction. This study reports an additional example of an IgM "EDTA dependent agglutinin" and demonstrates the dependence on carboxyl groups for its agglutinating activity.