Identification of antigenic and allergenic natural rubber latex proteins by immunoblotting

Quantitation of proteins in finished natural rubber latex (NRL) products is essential in predicting their allergenic potential. The ASTM standard Modified Lowry method for measuring total protein content has been used for several years. Most recently, ASTM published a standard for more sensitive and more specific enzyme immunoassay for quantitation of antigenic NRL proteins. It is an ELISA inhibition assay, using rabbit anti NRL sera. Since the measurement of proteins in this method depends on recognition capacity of rabbit antibodies, the selection of an appropriate protein source for rabbit immunization is crucial for the accuracy of such test. In this study, we evaluated the composition of NRL proteins from ammoniated (AL) and nonammoniated (NAL) raw latex and from finished NRL products, and compared the effectiveness of sera from rabbits immunized with NRL proteins, to react with those extracts. Immune rabbit sera were analyzed by immunoblotting against extracts of several samples of AL, NAL, and glove proteins. In the NAL extracts, we identified 26-28 protein bands by SDS-PAGE. AL samples had between 6 and 9 bands with a great variation in the band positions among the samples. The Western blot analysis showed that anti-AL rabbit serum reacted with 4-9 protein bands in various AL extracts. The highest intensity of reaction was observed with the extract used to immunize the rabbits. Similar reaction was observed with anti-NAL serum. However, when the antisera were blotted against NAL extracts, anti-NAL serum reacted more strongly and with a larger number of proteins than anti-AL serum. In summary, anti-NAL serum recognized an equal number of proteins in AL extract as anti-AL serum. However, anti-AL serum recognized fewer protein molecules in NAL extract than anti-NAL serum. Our findings suggest that NAL extract contains more individual proteins than other extracts, and sera from rabbits immunized with this antigen have a greater capacity to react with a wide spectrum of NRL proteins. This finding may be helpful in selecting the representative reference antigen and antiserum for further efforts in NRL protein quantitation.     

Correlation of bioluminescent assay of gentamicin in serum with agar diffusion assay, latex agglutination inhibition card test, enzyme immunoassay, and fluorescence immunoassay.

An improved bioluminescent assay of gentamicin in serum, based on the dose-dependent effect of the agent on the accumulation of extracellular ATP in Escherichia coli LU 14 cultures, is presented. The accuracy of the bioluminescent assay of gentamicin, expressed as the mean coefficient of variation over the therapeutic range, was 2.8%. Corresponding figures for an agar diffusion assay, a latex agglutination inhibition card test, an enzyme immunoassay, and a fluorescence immunoassay were 6.4, 17.5, 4.2, and 9.4%, respectively. All of the methods correlated well (r = 0.926 to 0.976), with the possible exception of the card test (r = 0.777 to 0.841). The bioluminescent assay requires only 1 microliter of serum, which allows for capillary sampling, and results are available within 75 min.     

Detection of antimitochondrial antibodies: characterization by enzyme immunoassay and immunoblotting

Mitochondrial antigens were purified from rat liver and characterized by immunoblotting. Sera from 19 well defined patients with primary biliary cirrhosis (PBC) reacted with two mitochondrial polypeptides of 68 Kd and 45 Kd. Antibodies to these antigens were not detected in any of the sera of patients with cirrhosis of the liver, chronic active hepatitis or other autoimmune diseases. The two polypeptides were derived from the soluble fraction of the mitochondrial matrix. An enzyme-linked immunosorbent assay (ELISA) employing these rat liver mitochondrial antigens is described. Positive results were obtained with all except one PBC sera (95%), five out of 47 patients with cirrhosis (11%), one out of 20 patients with chronic active hepatitis (5%), and two out of 19 patients with various autoimmune disorders (11%). The titers detected in PBC were markedly higher than those recorded in patients with other liver and autoimmune diseases. Strong correlation was found between immunoblotting and the ELISA in determining antimitochondrial antibodies. The ELISA presented is easily performed and seems to be a useful diagnostic tool for antimitochondrial antibodies in patients with PBC.     

Falsely low MMP-3 by latex turbidimetric immunoassay

We experienced a case with a falsely low value by a blood MMP-3 measuring reagent employing a recently structured new latex immunoturbidity. The case involved duplicate orders for one patient in a single day, and the blood collection amounts and measured values were approximately 6.0 mL and 206.6 ng/mL and approximately 1.0 mL and 107.5 ng/mL. The latter MMP-3 concentration was 48% of the former, showing a low tendency. Therefore, an experiment was conducted by adding serum to the blood collection tubes with or without a serum-separating agent of four different manufacturers (Terumo, Sekisui Medical, Nipro, and Becton Dickinson), and similar results as our experienced case were obtained with the Terumo tube with serum-separating agent, which had been used in this case. The amount of whole blood was obtained by conversion assuming a hematocrit value of 40%, and the addition ratio was calculated relative to the predetermined amount of the tube showing a falsely low value. Falsely low values were observed at < or = 56%, < or = 21%, < or = 20%, and , or = 33% for Terumo, Sekisui Medical, Nipro, and Becton Dickinson, with tubes containing a serum-separating agent, and at < or =10%, < or =8%, < or =19%, and < or =14% for Terumo, Sekisui Medical, Nipro, and Becton Dickinson, respectively, with plain tubes. Falsely low values were not observed with the 10-ml plain tube of Terumo and the 9-ml plain tube of Nipro (untreated tube). Based on these results, care should be taken if samples are below the predetermined amount of the blood collection tube to determine the serum MMP-3 by this method.     

A sensitive kinetic latex agglutination immunoassay adapted to centrifugal analysis

Latex reagents for HCG obtained from commercially available pregnancy test kits were adapted for use on Instrumentation Laboratory's Multistat III Plus centrifugal analyzer. The clearance rate of an agglutinating reaction mixture can be measured by absorbance over a time period of 15 min. The automated system has a sensitivity of less than 5 mIU HCG/ml in buffer or urine with a range of up to 100 mIU HCG/ml. Statistical analyses on urine samples indicated CVs of between 7.6% and 10.7% for within-run precision and between 6.8% and 10.8% for between-run precision. The correlation between the centrifugal latex agglutination method and two commercially available RIAs was found to be about 87%.     

Surgical latex glove allergy: characterization of rubber protein allergens by immunoblotting.

Immunoblot analysis employing IgE antibodies derived from sera of 3 physicians and 2 nurses allergic to surgical latex gloves, disclosed 10 allergens in natural rubber sap. Nine of the 10 allergens were detected in ammoniated natural rubber latex, but only 4 allergens in a latex glove extract. The allergenic proteins had apparent molecular weights ranging from 14 to 70 kD. Allergens with molecular weights of 14 and 21 kD showed the most intense immunoblot reactions suggesting that these proteins could be the major allergens in the natural rubber. An 11-kD protein and a 26-kD protein were only seen in the glove extract, indicating that they could be modified rubber proteins formed during glove manufacture.     

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.     

Effect of potential interference factors on performance of enzyme immunoassay and latex agglutination assay for cryptococcal antigen.

The PREMIER Cryptococcal Antigen Enzyme Immunoassay (Meridian Diagnostics) did not give discrepant results with rheumatoid factor, syneresis fluid, or serum macroglobulins from systemic lupus erythematosis patients. The Cryptococcal Antigen Latex Agglutination System (Meridian Diagnostics) did cross-react with syneresis fluid but not with the other serum factors tested.     

Humoral immune response associated with lyme borreliosis in nonhuman primates: analysis by immunoblotting and enzyme-linked immunosorbent assay with sonicates or recombinant proteins

The immune response to Borrelia burgdorferi, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of B. burgdorferi. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.     

Meningitis antigen detection: interpretation of agglutination by ultrasound-enhanced latex immunoassay

Detailed instructions for performance and interpretation of ultrasound-enhanced latex agglutination tests for the rapid identification of bacteria causing meningitis are described. This recently developed technique, which enhances the sensitivity of most latex immunoagglutination assays, has been studied mainly in the context of detection of antigens of meningitis-causing bacteria. The test concentrates on the Wellcogen bacterial antigen kit (Murex Diagnostics Ltd) that contains five latex suspensions specific for Haemophilus influenzae type b, Neisseria meningitidis ACYW135, N. meningitidis B/Escherichia coli K1, Streptococcus group B and Streptococcus pneumoniae. Light photomicrographs of positive agglutination are shown. Particular attention is paid to the appearance of the latex in negative control samples following exposure to ultrasound. Guidance is given on interpretation and assessment in clinical samples.     

Serum antibodies in rickettsia patients as determined by immunoblotting technique.

The Western-blot technique (WB) was used to determine which polypeptides of Israeli spotted fever (ISF) isolates and other spotted fever group rickettssia (SFGR) reference isolates (G212, S484, A828) and two reference strains. R. Rickettsii (Sheila Smith strain) and R. conorii (Boutonneuse fever), were used as antigen sources for the WB. Immunoperoxidase assay (IPA) seropositive (titer greater than 80) and seronegative (titer less than) sera were examined with the separated polypeptides of the above strains. WB analysis of the rickettsial polypeptide-serum reactions showed that R. conorii and the three isolates of ISF reacted identically with the sera, except that in the three ISF strains a 175 kD protein was present. It was also observed that all of the IPA seropositive sera examined reacted with the following polypeptides: 18kD, 20kD, 22kD (28kD to 37 kD LPS group), while each seropositive and seronegative serum reacted differently with polypeptides 23kD, 42kD, 45kD, 46kD, 52kD, 55kD, 70kD, 82kD, 105kD, 125kD, 155kD and 175kD. Using this technique, no heat labile polypeptides (preelectrophoretic treatment: 100 degrees C for 2 min vs 37 degrees C for 20 min) were observed in SFGR strains used in this study. Our results indicate that the immunoblot technique shows no difference between R. conorii and ISF antigens except the existence of 175kD protein antigen in the latter.