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At least 19 recordsLinked to original sources
PubMed1993

Detection of hepatitis C virus infection with recombinant immunoblot assay, synthetic immunoblot assay, and polymerase chain reaction.

The newly developed immunoblot assay, RIBA SIA (recombinant and synthetic polypeptide immunoblot assay), Chiron, Calif., was compared with the commercially available second generation recombinant immunoblot assay (RIBA-2) for the detection of antibody to hepatitis C virus (anti-HCV). The two immunoblot tests were also compared with the polymerase chain reaction (PCR) for the detection of HCV RNA. Ninety-one percent of samples reactive by RIBA-2 were positive for anti-HCV by RIBA SIA. A total of 31% of RIBA-2 indeterminate samples became reactive by RIBA SIA, 24% became non-reactive, and 45% remained the same. Samples reactive by RIBA-2 or SIA from different risk groups, were mostly positive (67-100%) by PCR for HCV RNA. All indeterminate samples from hemophiliacs and intravenous drug users were PCR positive. RIBA SIA is more sensitive and specific than RIBA-2 and correlates well with PCR results.

Female↗
PubMed1988

Immunoblotting of pollen proteins: Tween 20 mediates nonspecific binding of gammaglobulins to immunoblots of oak-pollen proteins.

Oak-pollen extracts, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose, demonstrated extensive banding in the absence of human sera or with nonatopic sera with both a horseradish peroxidase-conjugated goat antihuman IgE and 125I-labeled rabbit antihuman IgE. When the nonionic detergent Tween 20 was removed from all incubations and washes, negative banding appeared, corresponding to bands that were clearly visible when Tween 20 had been used as a blocking agent. When Tween 20 was reintroduced, the bands reappeared. These results demonstrate that, although Tween 20 is effective in blocking unbound sites on nitrocellulose membranes, this and other nonionic detergents may cause nonspecific binding to immunoblots of oak-pollen proteins. Thus, care must be used in the interpretation of immunoblots to detect allergens when Tween 20 is used as the blocking agent.

Animals↗
PubMedDate not supplied

[The enhancement of the diagnostic reliability of immunoblot for HIV-1 antibodies by enriching the preparation for immunoblot with the HIV-1 gp120 protein].

The diagnostic value of original immunoblot system depends on the availability of enveloped protein GP120 because it is the antibodies to this polypeptide that frequently indicate the running virus infection. This polypeptide is lost during purification of viral material but remains free in culture medium. The extraction of GP120 from culture fluid with immunosorbent based on sepharose 4B with ligated immunoglobulins from HIV-1-infected persons enriched the preparation for immunoblot with proteins increasing its diagnostic value.

Carrier State↗
PubMed2004

Western immunoblotting in avian shell gland sample immunoblotting methods.

Avian shell gland tissue was subjected to Western blot analysis using anti-human estrogen receptor-alpha antibody H222. Initial attempts to obtain consistent, high-quality blots were unsuccessful because, as it turned out, excessive lipid in tissue preparations interfered with protein separation. Incremental additions of acetone eventually proved to be the critical step in solubilizing lipids and allowing consistent separation of bands on gels. A detailed description of the methodology is presented.

Animals↗
PubMed1999

An improved recombinant IgG immunoblot for serodiagnosis of Lyme borreliosis.

We have previously described the use of the following recombinant antigens for serodiagnostic immunoblots: p83/100, p39, OspC and p41 (flagellin) internal fragment [Wilske et al. (1993) Med Microbiol Immunol 182:255-270; Rossler et al. (1997) J Clin Microbiol 35:2752-2758]. In our currently used immunoblot p83/100 is derived from strain PKo (Borrelia afzelii), p39 (BmpA) and OspC from strains PKa2 (B. hurgdorferi sensu stricto), PKo and PBi (B. garinii), respectively; the p41 (flagellin) internal fragments were cloned from strains PKo and PBi. In this study we describe the use of two additional recombinantly expressed highly immunogenic proteins Osp 7 (derived from PKo) and p58 (derived from PBi). A clinically well-defined panel of sera from 147 Lyme borreliosis patients and 139 controls previously tested by a standardized whole cell lysate immunoblot [Hauser et al. (1997) J Clin Microbiol 35:1433-1444] was investigated in the recombinant immunoblot without (old recombinant immunoblot) and with Ospl7 and p58 (new recombinant immunoblot) for IgG antibodies. The sensitivity of the recombinant IgG immunoblot for diagnosis of stage II and stage III could be significantly improved by addition of Osp17 and p58 without loss of specificity. With the exception of sera from patients with erythema migrans the diagnostic sensitivity is comparable to the whole cell lysate IgG immunoblot. The main advantage of the recombinant immunoblot is the easy identification of diagnostic bands, whereas the identification of bands in the whole cell lysate immunoblot is difficult. The recombinant immunoblot is especially suitable where large series of sera need to be investigated.

Animals↗
PubMed2004

[Immunoblot in the serological diagnosis of hepatitis C virus infection].

The objective of the study was to assess three immunoblot assays, the Deciscan HCV Plus, the Riba and the Inno-Lia, on 44 discordant samples with three EIA kits. These immunoblots were considered as confirmation reagents. A result was considered as a false positive by anti-HCV antibody assay if the three immunoblots were negative or if two immunoblots were negative with the third being indeterminate and a negative virological genomic diagnosis observed on all the samples. The result was positive if at least two immunoblots out of three were positive. Thus, 34 samples were considered as false positive and ten samples were excluded because it was impossible to conclude between true or false positive result. The 44 discordant results were never confirmed as positive by the use immunoblot or PCR. The three immunoblots were negative for half of the samples and two immunoblots and one indeterminate were observed for 77% of the samples. The false positive results by the Monolisa assay were more often found indeterminate with the Deciscan assay than with the other immunoblots. That was also checked for Vitros/Riba pair. One of the explanations could be the use of common antigens for the reagents from the same manufacturer. The Inno-Lia test is the most specific immunoblot according to the results obtained in our study.

Hepacivirus↗
PubMed1995

Immunoblot interpretation criteria for serodiagnosis of early Lyme disease.

We monitored the antibody responses of 55 treated patients with early Lyme disease and physician-documented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [> 19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P = 0.006).

Antibodies, Bacterial↗
PubMed1997

Immunoblot assay for serodiagnosis of Helicobacter pylori infections.

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.

Adult↗
PubMed2006

Neospora caninum immunoblotting improves serodiagnosis of bovine neosporosis.

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.

Abortion, Veterinary↗
PubMed1996

Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease.

In October 1994, the Second National Conference on the Serologic Diagnosis of Lyme Disease recommended a two-step approach to serological testing. The first step was the performance of an enzyme-linked immunosorbent assay (ELISA); the second step was a confirmatory immunoblot. New criteria for the interpretation of a positive immunoblot were also recommended. The committee decided to omit the 31- and 34-kDa bands (OspA and OspB, respectively) from the choice of bands considered diagnostic for a positive immunoblot. Since we had previously included these in our diagnostic criteria for Lyme disease-positive immunoblots, we reviewed data for all patients attending a Lyme disease center with positive ELISAs and immunoblot assays for Lyme disease from 1 September 1992 to 31 December 1993. The criteria for a positive Western blot (immunoblot) were the presence of 5 or 12 bands, including the 10 recommended by the conference, and the presence of the 31- and 34-kDa protein bands. Of the 136 patients evaluated, 50 were considered to have Lyme disease. Of these 50, 4 (8%) would not have met immunoblot criteria for the diagnosis if the new recommendations were used. Had the 31- and 34-kDa bands been included as part of the diagnostic requirements for immunoblot, these patients would have been included. Although overdiagnosis of Lyme disease appears to be the more frequent problem, our concern is that the exclusion of the 31- and 34-kDa protein bands from the diagnostic criteria may result in the underdiagnosis of Lyme disease by those who would rely too heavily on serological confirmation. The addition of the 31- and 34-kDa bands to those recommended for confirmatory immunoblot should be reconsidered.

Adult↗
PubMed1992

Detection of chronic hepatitis C virus infection by four diagnostic systems: first-generation and second-generation enzyme-linked immunosorbent assay, second-generation recombinant immunoblot assay and nested polymerase chain reaction analysis.

Serum samples from 100 patients with non-A, non-B hepatitis-related chronic liver disease and 100 patients with hepatitis B-related chronic liver disease were tested by first-generation and second-generation enzyme-linked immunosorbent assays, a second-generation recombinant immunoblot assay and the nested polymerase chain reaction. In non-A, non-B hepatitis-related chronic liver disease, second-generation enzyme-linked immunosorbent assay (anti-c22 and/or c200) and second-generation recombinant immunoblot assay showed 98% positivity, whereas first-generation enzyme-linked immunosorbent assay (anti-c100-3) showed 89% positivity. The two second-generation recombinant immunoblot assay-negative samples were positive by nested polymerase chain reaction, but one second-generation recombinant immunoblot assay-positive sample was polymerase chain reaction negative. However, when this second-generation recombinant immunoblot assay-positive sample was tested by polymerase chain reaction using another set of primers, it was polymerase chain reaction positive. Therefore, 100% of the non-A, non-B hepatitis-related chronic liver disease serum samples were hepatitis C virus RNA positive by polymerase chain reaction. Nine hepatitis B-related chronic liver disease samples were first-generation enzyme-linked immunosorbent assay positive. Of the eight second-generation enzyme-linked immunosorbent assay-positive hepatitis B-related chronic liver disease samples, six were first-generation enzyme-linked immunosorbent assay positive and five were second-generation recombinant immunoblot assay positive and polymerase chain reaction positive. One indeterminate second-generation recombinant immunoblot assay sample was polymerase chain reaction negative. Therefore, second-generation recombinant immunoblot assay appears to be as useful as polymerase chain reaction for detecting a chronic hepatitis C virus infection, although some discrepancies were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2000

Is there a role for immunoblots in the diagnosis of latex allergy? Intermethod comparison of in vitro and in vivo IgE assays in spina bifida patients.

BACKGROUND: The best diagnostic method for latex allergy is still controversial. This investigation was designed to evaluate the diagnostic efficiency of immunoblotting in comparison with established in vitro and in vivo test systems. METHODS: A total of 108 spina bifida patients were investigated by questionnaire and skin prick test (SPT). Specific serum IgE to latex was analyzed by the Pharmacia CAP FEIA immunoassay, DPC AlaSTAT microplate immunoassay, and DPC AlaBLOT immunoblot. Patients were regarded as latex allergic if they reacted positively to challenge by the latex glove wearing test. RESULTS: Thirty-four patients reacted positively to challenge. The sensitivity rates were 97% (SPT), 94% (immunoblot, CAP), 74% (AlaSTAT), and 35% (clinical history). The specificity rates were 92% (clinical history), 88% (AlaSTAT), 77% (SPT), 76% (CAP), and 69% (immunoblot). If two methods were combined, efficiency rates were highest for SPT combined with CAP (sensitivity 94%, specificity 82%), with AlaSTAT (sensitivity 74%, specificity 92%), or with immunoblot (sensitivity 91%, specificity 84%). The sera of challenge-positive patients recognized more immunoblot bands than challenge-negative patients, and the severity of symptoms correlated with the number of recognized bands. CONCLUSIONS The diagnostic efficiency of immunoblotting is not superior to that of SPT. However, immunoblotting may serve as an additional tool to increase slightly the specificity of SPT and specific serum IgE tests.

Adolescent↗
PubMed1998

Validation of a new immunoblot assay (LiaTek HIV III) for confirmation of human immunodeficiency virus infection.

BACKGROUND: Used as a supplemental assay, new anti-human immunodeficiency virus (HIV) immunoblots, employing recombinant and synthetic antigens, appeared to resolve the majority of samples with false-reactive Western blot results. Would it be possible to completely replace the Western blot by an immunoblot for confirmation and exclusion of HIV infection? STUDY DESIGN AND METHODS: The sensitivity of the new LiaTek HIV III immunoblot assay (Organon Teknika, Turnhout, Belgium) was tested on 416 Western-blot positive samples (386 HIV-1, 22 HIV-2, 1 HIV-1/2, and 7 HIV-O) and on 45 HIV-1 seroconversion samples. The specificity was tested on 146 samples from noninfected donors with false-positive results on a HIV screening test. RESULTS: All Western-blot-positive samples tested positive in the immunoblot (sensitivity: 100%). The immunoblot could not discriminate between HIV-1 and HIV-2 infection in 22 of 416 (5%) samples. The LiaTek assay showed reactivity in 28 of 45 seroconversion samples, whereas the Western blot reacted in 30 of 45 seroconversion samples. With false-positive donor samples, the immunoblot was indeterminate in 10 of 146 samples (specificity: 93%), and the Western blot was indeterminate in 44 of 146 samples (specificity: 70%). CONCLUSION: Like the Western blot, the immunoblot runs the risk of missing samples that are reactive by enzyme immunoassay during the early stage of HIV infection. Nevertheless, considering its superior specificity on false-positive donor samples, it appears that the immunoblot offers a cost-effective alternative to the Western blot assay for confirmation and exclusion of HIV infection.

AIDS Serodiagnosis↗
PubMed2001

Diagnosis of babesiosis using an immunoblot serologic test.

Although the current indirect immunofluorescent assay (IFA) diagnostic antibody test for human babesiosis is sensitive and specific, an immunoblot antibody test may be easier to standardize and to perform. Our objective, therefore, was to determine the efficacy of and develop interpretive criteria for an immunoblot antibody test for diagnosing acute human babesiosis using a Babesia microti whole-cell lysate as the antigen. We compared the reactivity of sera to a B. microti immunoblot assay in 24 human subjects experiencing symptoms and expressing laboratory evidence of babesiosis, 28 subjects who experienced Lyme disease, 12 subjects who experienced human granulocytic ehrlichiosis, and 51 subjects who reported no history of any of these diseases and whose sera did not react against B. microti antigen in an IFA test. Immunoblot strips were impregnated with proteins derived from the GI strain of B. microti that had been electrophoresed in an acrylamide sodium dodecyl sulfate gel, followed by electroblotting onto nitrocellulose membranes. The sera of all subjects who experienced babesiosis reacted against the B. microti antigen in the IFA and against at least one of nine immunoblot protein bands specific to B. microti. In contrast, none of the sera from people who appeared not to have experienced this infection reacted against the B. microti antigen in the IFA (compared to 4% in the immunoblot assay). When two reactive bands were considered as definitive, immunoblot test sensitivity was 96%, while specificity was 99% and predictive positivity and predictive negativity were 96 and 99%, respectively. Our B. microti immunoblot procedure shows promise as a sensitive, specific, and reproducible assay for routine clinical diagnosis of acute babesiosis.

Antibodies, Protozoan↗
PubMed2000

Immunoglobulin M and G immunoblots in the diagnosis of parvovirus B19 infection.

BACKGROUND AND PURPOSE: To identify parvovirus B19 infection by means of immunoglobulin (Ig) G and IgM immunoblots among immunocompetent patients who tested negative or had low-titer B19 IgM antibodies in enzyme-linked immunosorbent assays (ELISA). METHODS: Serum samples were obtained from 20 patients with parvovirus B19 infection. Another 130 study subjects presumed to be without B19 infection (40 medical personnel and 90 prisoners) were also included. All sera from the patient and study groups tested positive for IgG or IgM with ELISA and were further evaluated using the immunoblot method. Detection of B19 DNA by nested polymerase chain reaction (PCR) was also performed on IgG and IgM positive sera. RESULTS: IgM immunoblots disclosed one false positive IgM ELISA result in the patient group and three false positive results in the study group. In the patient group, four patients were in the latter stage of antibody response to B19 infection as suggested by the low titer of anti-B19 IgM, incomplete IgM immunoblots, with only a weak viral capsid protein VP-N reaction band, and fading but still strong reaction bands on IgG immunoblots. Strong reaction bands on IgG immunoblots comparable to these four patients were found in three of the 130 study group sera. Furthermore, B19 DNA was detected in three of the four patients and one of the three study subjects by means of nested-PCR. A serum sample from one study subject showed strong IgG but no IgM reactivity to viral capsid protein VP2; nested PCR identified B19 DNA in this serum sample. CONCLUSIONS: Immunoblots and nested PCR should be applied in the diagnosis of B19 infection for patients with low-titer anti-B19 IgM tested by means of ELISA. For diagnosis of B19 infections in certain clinical entities such as chronic arthritis of recent onset and hydrops fetalis, B19 IgM antibodies may have disappeared but B19 infection can still be recognized by the intensity of the reaction bands on IgG immunoblots. The correlation between chronic B19 infection and persistence of antilinear VP2 epitopes requires further study.

Adult↗
PubMed1990

Immunoblotting of plasma in a pregnant patient with hereditary angioedema.

Hereditary angioedema (HAE), an autosomal disorder caused by a deficiency of C1 inhibitor, is characterized by attacks of localized swelling, laryngeal edema, or abdominal pain. Plasma samples from one pregnant patient were studied serially by functional and quantitative immunochemical assays as well as immunoblot assays for high molecular weight kininogen (HMWK) and/or prekallikrein/kallikrein (PK/K). An immunoblot of this patient's HMWK from plasma obtained before she became pregnant and when she was well revealed that it was mostly an intact protein of 120 kd, similar to immunoblot results of normal plasma HMWK. In plasma samples taken throughout her pregnancy, before, during, and after clinical attacks of angioedema, all of her plasma HMWK was shown to be cleaved into the 45 kd light chain form. After delivery of the infant the 120 kd form of intact plasma HMWK returned to her plasma. In comparison, immunoblot studies on 21 normal and abnormal pregnancies revealed that plasma HMWK was an intact protein at 120 kd. That this patient's plasma during her pregnancy was contact activated was determined by additional immunoblot studies for PK/K. Immunoblot assay for plasma PK/K revealed kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form seen only in activated plasma samples. The findings of kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form disappeared after delivery. These combined studies on this patient show that the structures of HMWK and prekallikrein as indicated by immunoblot assays were altered during pregnancy. Immunoblot assays for detection of changes in the structure of HMWK and prekallikrein may be objective laboratory studies for documenting clinical attacks of hereditary angioedema, their onset, and their resolution.

Adult↗
PubMed1995

Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII.

Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.

Animals↗