A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin

We have developed an avidin-biotin assay for the detection of lactoferrin in human seminal plasma. We compared 5 modifications of this assay, and found the non-competitive avidin-biotin assay (NABA) to be the most sensitive. The detection limit of 3-step NABA was 0.2 or 0.1 ng lactoferrin/ml, depending whether avidin-biotin-peroxidase complex (ABC) or avidin-peroxidase was used. The intra-assay and inter-assay coefficients of variation for 3-step NABA were 6.2 and 8.5%, respectively. The lactoferrin levels of human seminal plasma samples measured by 3-step NABA and radioimmunoassay (RIA) showed good correlation (r = 0.96). The total performance time of 3-step NABA is flexible and the method can be modified for rapid (less than 1 h) or overnight assay according to need.     

A rapid and sensitive non-competitive avidin-biotin immuno-enzymatic assay for lysozyme

A non-competitive avidin-biotin immuno-enzymatic assay (NABA) for lysozyme is described. The assay was found to be more sensitive than a competitive assay with biotinylated lysozyme. The lower detection limit of NABA was 0.1 ng lysozyme/ml compared to 1 ng/ml for the competitive assay. The intra-assay and inter-assay coefficients of variation for NABA were 5.9 and 9.1%, respectively. The total time for NABA can be decreased (to less than 1 h) without influencing the detection limit or the analytical range. Serum lysozyme levels measured by NABA and the enzymatic assay in 32 samples showed a correlation coefficient of r = 0.97.     

Method of analysis of non-competitive enzyme immunoassays for antibody quantification

A computerized analysis of a quantitative enzyme-linked immunoadsorbent assay (EIA) using a non-specific immunoglobulin (IgG) of known concentration as the standard has been developed for measuring specific antibody levels in serum without the need for affinity purification of the positive control antibody. The computer program utilized logit-log linear regression analysis of sigmoid serial dilution curves plus a weighted least-squares best curve fit analysis and an iterative manipulation to eliminate errant data points. The EIA was performed using serial dilutions of standard and unknown antibodies, and a double sandwich technique. A comparison of antibody levels determined by EIA using non-specific IgG as a standard relative to antibody levels determined using affinity-purified specific antibody as a standard were 1.04, 0.53, 0.48, and 0.97 for four different polyclonal antibody systems. Five monoclonal antibodies to carcinoembryonic antigen gave ratios as described above of 1.07, 1.59, 1.73, 2.32, and 2.42. The corresponding antibody affinity constants (1/mol) were 1.0 × 10⁸, 3.8 × 10⁸, 5.5 × 10⁹, 1.8 × 10¹⁰, and 2.6 × 10¹⁰ respectively. This method permits accurate quantification of serum antibody levels when affinity-purified antibodies are not readily available and avoids errors due to loss of antibody activity during affinity purification.     

Murine monoclonal anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining

To improve the sensitivity of conventional immunoassay methods using avidin-biotin-enzyme complex reagents, we have developed several murine monoclonal antibodies to the egg white avidin glycoprotein. These anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays by selectively enlarging the avidin-biotin-enzyme complex through bridging avidin to a second layer of avidin-biotin-enzyme complex, thereby increasing the signal from substrate conversion. Six hybridomas producing murine monoclonal antibodies which bind avidin-biotin-enzyme complexes were isolated and cloned. Two of these anti-avidin antibodies, WC19.10 and WC19.7, have been shown to produce a four-fold enhancement of signal obtained in avidin-biotin-enzyme ELISAs and qualitative enhancement of immunohistologic staining procedures.     

Nonvalue of antigen detection immunoassays for diagnosis of candidemia.

We evaluated the Cand-Tec (Ramco Laboratories Inc., Houston, Tex.) and LA-Candida antigen detection system (Immuno-Mycologics Inc., Norman, Okla.) tests as possible rapid alternatives to blood cultures for the identification of patients with candidemia. Tests were performed on sera from (i) 33 patients with candidemia, (ii) 82 patients with fever and risk factors for invasive candidiasis, and (iii) 13 healthy controls. A total of 21 patients had no evidence of invasive candidiasis, as determined by clinical course, blood culture, and/or autopsy; results for 61 patients were indeterminate regarding the presence of invasive candidiasis, or else the patients had invasive candidiasis with organ involvement. By using a threshold positive Cand-Tec titer of greater than or equal to 1:4, the sensitivity in candidemic patients was 49%; the specificity was 43% (patients with true-negative results had neither candidemia nor other evidence of invasive candidiasis). Coexistent disseminated candidiasis in some candidemic patients may have accounted for some positive Cand-Tec tests and possible overestimation of the sensitivity of the test for candidemia. Cand-Tec test results were negative for healthy controls. All test results obtained by the LA-Candida antigen detection system assay were negative. Our findings indicate that neither of these assays reliably identifies patients with candidemia.     

Unsatisfactory specificities and sensitivities of six enzyme immunoassays for antibodies to hepatitis B core antigen.

To examine the consistency and comparability of anti-hepatitis B core antigen (anti-HBcAg) assays, four blood donation centers of the Red Cross in the Federal Republic of Germany tested 4,080 unselected blood donors with six different tests in parallel. Confirmation testing of reactive samples was done in the National Reference Center for Viral Hepatitis. Depending on the test kit used, 4.1 to 9.9% of serum samples were initially positive and 2.9 to 7.5% were repeatedly positive. Sixteen percent of serum samples were positive in at least one test but only three percent were positive in all six tests. Statistical analysis of frequency distribution of optical densities for each test suggested that there should be a correction of the cutoff values. This reduced the number of false-positive results by half, but a significant proportion of discrepant results could not be resolved. The lack of specificity and consistency requires cautious interpretation of isolated anti-HBcAg results in clinical specimens. Screening of predominantly anti-HBcAg-negative populations (e.g., blood donors) by the current anti-HBcAg test kits will almost necessarily give unsatisfactory results.     

Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.     

Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin

We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were > or = 99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by approximately 75 to 80% and two-step testing was complete in < or = 3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US dollar 143,000 less than if CCNA alone had been performed on all 5,887 specimens.     

Evaluation of two enzyme immunoassays for detection of human immunodeficiency virus antigen in African and European sera

The performance of two HIV antigen enzyme immunoassays (EIAs), the HTLV-III antigen EIA (Abbott) and the Innotest HIV antigen EIA (Innogenetics), was assessed using 276 HIV-1 antibody positive and 42 HIV antibody negative sera from European and African individuals. On the initial screening 16 % (51/318) of the sera were reactive in the Abbott EIA and 21.4 % (68/318) in the Innotest EIA. The Innotest detected significantly more confirmed HIV antigen positive sera (55/58, 94.6 %) than the Abbott EIA (41/58, 70.7 %). The presence of free detectable HIV antigen in African and European sera was strongly associated with the clinical classification CDC IV and with low p24 antibody levels.     

An avidin-biotin ELISA for the measurement of serum and secretory IgD

A lipid vesicle-mediated immunoassay for small haptens (digoxin) is described. Using a detergent removal procedure for vesicle formation, a water-soluble marker system like alkaline phosphatase is stably entrapped within 150–200 nm unilamellar lipid vesicles composed of egg yolk lecithin and cholesterol. Specific lysis of the lipid vesicles is achieved upon addition of a hapten-melittin conjugate. Inhibition or modulation of this lysis by the hapten-melittin conjugate can then be achieved by adding stoichiometric amounts of high affinity antibody. Finally, the antibody inhibition of hapten-melittin lysis can be modulated by the addition of competing amounts of free hapten. This assay approach is simple, fast, highly sensitive, and versatile such that it can be carried out in either a homogeneous or heterogeneous mode. Furthermore, unlike all other liposome-mediated immunoassays, complement is not required for lysis.     

Comparison of a chemiluminescent immunoassay with two microparticle enzyme immunoassays for detection of hepatitis B virus surface antigen

A comparative evaluation of the following commercial immunoassays for the detection of hepatitis B virus surface antigen (HBsAg) was performed: the Abbott AxSYM, Abbott IMx, and DPC IMMULITE assays. The specificity was 100% for all assays. Twelve samples were identified and were confirmed to be positive for HBsAg by all three methods. One additional sample was identified as reactive and was confirmed to be positive by the Abbott AxSYM assay only. Prior to confirmation testing the DPC IMMULITE assay produced significantly fewer false-positive results than the Abbott AxSYM assay (P < 0.05).     

Application of sandwich immunoassays for the determination of sample-specific background signals and its use for calibration of immunoassays

Sample-related background signals in immunoassays can be measured by a variation of the double antibody sandwich principle, in which the unlabelled specific antibody is substituted by a similar unrelated non-specific antibody. This permits differentiation between the analyte-specific and the background signal components for each sample. The method permits selection of sera with no or low analyte content for use as analyte diluent and for defining the zero point of the calibration curve. The method also permits control of analyte content during production processes which may change the background signal as well as identification of samples with atypical background signals. The procedure has been used for the calibration of enzyme immunoassays for alpha-fetoprotein (AFP) and human thyroid-stimulating hormone (TSH).     

Characterisation of the polymerised and monomeric human serum albumin binding sites on hepatitis B surface antigen

Receptors for polymerised human albumin are present on the pre-S sequence of the envelope protein of HBV and on the hepatocyte membrane and are thought to be involved in uptake of the virus by hepatocytes. Using a solid phase radioimmunoassay we demonstrate binding of HBsAg to polymerised human serum albumin (pHSA) in both HBe antigen-positive and -negative patients, and this binding is linearly related to the HBsAg titre in both groups. There are probably several modes of interaction between HBsAg and pHSA. Here we show that pHSA binds to the 22,000-dalton polypeptide of HBsAg, which does not contain the pre-S sequence. This pHSA-HBsAg interaction is inhibited by physiological concentrations of human serum albumin, suggesting that the albumin known to be present in the envelop of HBsAg plays a role in this binding. The inhibition of pHSA/HBsAg interaction by native albumin suggests that this interaction is probably not an important mechanism of virus uptake during infection of hepatocytes.     

An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B

The avidin-biotin system was incorporated into the enzyme-linked immunosorbent assay (ELISA) to establish a new detection method for staphylococcal enterotoxins A and B in culture supernatants. Staphylococcal protein A (SPA) does not interfere significantly with the procedure. The test shows good sensitivity for the toxins and the ease and rapidity with which the assay can be performed make it a valuable tool for the routine detection of enterotoxins.     

Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies.

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.