Antigen-specific detection of soluble immune complexes by a solid phase specific antibody system.

An antigen-specific immune complex assay based on the following principle has been developed: xenogeneic, antigen-specific antibodies are attached to a solid phase. During first incubation with patient's serum, immune complexes in antigen excess are bound to the xenogeneic antibody by their free antigenic determinants. In a second incubation a labeled antibody, specific for human immunoglobulins, is combined with the antibody part of the immune complex. Quantitation of the label allows the determination of the immune complexes. The principle of the method has been varied using artificial soluble immune complexes of tetanus toxoid and human anti-tetanus toxoid antibody, and immune complexes prepared by mixing patient sera containing either HBsAg or anti-HBsAg antibodies. The reliability of the results is shown by their coefficient of variation (2.5%). With the method described soluble immune complexes predominantly in slight antigen excess, which are thought to be responsible for development of immune complex disease, have been detected.     

Evaluation of human immunodeficiency virus seroprevalence in population surveys using pooled sera.

The pooling of individual serum samples to determine human immunodeficiency virus (HIV) seropositivity was examined to assess whether testing pooled sera was technically feasible, cost-effective, and accurate for estimating seroprevalence in large population surveys. The sensitivities and specificities of three commercially available HIV enzyme-linked immunosorbent assay (ELISA) kits were tested using 65 serum pools of 15 individual serum samples each (975 total serum samples) at two different dilutions. With pooled sera, the Organon Teknika Bio-EnzaBead ELISA at half the dilution recommended by the manufacturer showed the best agreement with ELISA and Western blot results of individual sera. In subsequently testing 92 pools, each containing 15 individual serum samples from a population of American patients attending a sexually transmitted diseases clinic, the estimated seroprevalence was 5.27 compared with 4.93% in a test of 1,380 individual serum samples and 5.19% in a test of 4,028 individual serum samples from the same population. In an evaluation of 1,380 African patients using 10 serum samples per pool, the estimated seroprevalence was 5.79 compared with 6.16% in a test of individual sera. These results indicate that ELISA testing with pooled sera is highly sensitive and specific and appears to be a cost-effective means for estimating HIV seroprevalence in large population-based surveys.     

Mutation detection by solid phase primer extension

A mutation analysis method based upon a wild-type DNA sequence is presented. Oligonucleotides were utilized for primer extension by T7 DNA polymerase to discriminate between wild-type and mutant sequences in two solid phase approaches. 1. Oligonucleotides were annealed to an immobilized template, extended with fluorescent dideoxynucleotides (ddNTPs), and analyzed on an automated fluorescent DNA sequencer. The oligonucleotide length identified the known mutation site, and the fluorescence emission of the ddNTP identified the mutation. 2. Template DNA was annealed to an oligonucleotide array, extended with α-³²P dNTPs, and analyzed with a Phosphor Imager. The grid position of the oligonucleotide identified the mutation site and the extended base identified the mutation. © 1996 Wiley-Liss, Inc.     

The detection and specificity of class specific antibodies to whole bacterial cells using a solid phase radioimmunoassay.

A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml.     

Sensitive and fast mutation detection by solid phase chemical cleavage

We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA sequencing system to analyze the cleaved fragments. © 1996 Wiley-Liss, Inc.     

Routine antenatal screening for hepatitis B using pooled sera: validation and review of 10 years experience.

AIMS: To validate the sensitivity of universal antenatal screening for hepatitis B surface antigen (HBsAg) by testing pools of 10 sera, and to review 10 years' experience using this method. METHODS: 66,945 antenatal patients were tested between 1986 and 1996 using the pooled method. All sera from 1996 (n = 6050) were retrieved and retrospectively tested individually. An in vitro determination of the effect of pooling on sensitivity was performed by checkerboard neutralisation assay. RESULTS: 26 HBsAg positive women were detected by universal screening over 10 years; 12 had non-European surnames and five had known risk factors for hepatitis B infection. High titre anti-HBs sera in the pool reduced the sensitivity of the HBsAg assay, though the effect was only significant at low levels of HBsAg carriage. CONCLUSIONS: The prevalence of hepatitis B is extremely low in the antenatal population served by Plymouth PHL. Pooling is unlikely to reduce sensitivity enough to lead to significant preventable vertical transmission, and is a cost-effective and valid strategy in areas of low seroprevalence.     

A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favorably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants.     

A comparison of two solid phase systems for antibody detection

Two solid phase methods of antibody detection, Capture-R (CR) and Capture-R Ready-Screen (RS), were compared to determine their acceptability for use in prenatal antibody screening. Ninety-six serum samples, screened using a saline antiglobulin test, were coded and tested by CR and RS at two laboratory sites using a blinded study design. Thirty of the samples were free of antibody, and 66 samples contained antibody. Parallel testing was also performed in both laboratories on 648 prenatal samples. The sensitivity and specificity of CR, based on the 96 previously screened samples, was 95 percent and 90 percent, respectively, and the sensitivity and specificity of RS was 90 percent and 89 percent, respectively. Antibodies detected only by CR included anti-K(1), -Ch(2), -Jka(1), -Lea(1), -Fya(1), -Mca(1), and -e(1). Antibodies detected only by RS included anti-Jka(5) and -E(1). The sensitivity and specificity of CR for the 648 prenatal samples was 100 percent for each, while the sensitivity and specificity of RS was 97 percent and 100 percent, respectively. Both CR and RS are acceptable techniques for prenatal antibody screening.     

Diagnosis of pancreatic lesions using fine needle aspiration cytology: detection of K-ras point mutations using solid phase minisequencing.

AIMS--To improve the diagnostic value of fine needle aspiration biopsy of pancreatic lesions using a simple mutation detection method based on the polymerase chain reaction (PCR). METHODS--Fine needle aspirates from 21 suspected pancreatic lesions were analysed for K-ras codon 12 point mutations using solid phase minisequencing. RESULTS--A point mutation in codon 12 of the K-ras gene was detected in 14 of 17 cases of pancreatic carcinoma. No false positive results were recorded. The concordance of the result with routine cytology was 78%. All patients diagnosed as having malignant disease on cytology also had a K-ras point mutation. Additional information on the presence of malignancy was obtained using molecular genetic analysis in two cases. CONCLUSIONS--PCR based minisequencing is a promising method for the analysis of cytological material. K-ras point mutation analysis was modified to enable it to be carried out in a clinical laboratory. Advantages of the method include its simplicity and speed. Adequate sampling guidance is important but analysis can be performed even with small amounts of cellular material.     

The sensitivity of antibody detection testing using pooled versus unpooled reagent red cells

Because the sensitivity of antibody detection testing may be reduced when pooled reagent red blood cells (RBCs) are used, the American Association of Blood Banks (AABB) prohibits the use of pooled reagent RBCs when performing pretransfusion antibody detection testing. This restriction imposed upon the use of pooled reagent RBCs is based, at least in part, on the belief that pooled reagent RBCs are less likely to detect clinically significant antibodies than are sets of unpooled reagent RBCs. Little data, however, have been published to support this contention. In the present study, the data show a decreased sensitivity for antibody detection when pooled reagent RBCs are used. This reduced sensitivity could result in failure to detect some clinically significant RBC alloantibodies, which might result in the occurrence of overt hemolytic transfusion reactions, especially if an indirect antiglobulin test is not performed at the time blood is crossmatched.     

A bioluminescent solid phase for immunoassays and the detection of nucleic probes

A luminescent adsorbent constituted of bacterial luciferase, FMN oxidoreductase and a protein, such as an antibody or an oligonucleotide coimmobilized on Sepharose, has been used to detect a label enzyme (Glucose 6 phosphate dehydrogenase). The label enzyme, bound to the solid phase, produces NADH and start an enzymatic chain reaction leading to light emission. The dehydrogenase, which is not bound to the solid phase, produces NADH in solution which is rapidly oxidized by a scavenger system (lactate dehydrogenase plus pyruvate) and thus does not participate in light emission. Using this solid phase, binding assays do not require separation of the excess of label, and the assay protocol is limited to the addition of sample, and luminescent reagents. The authors have used this solid phase for rapid immunoassays of haptens and proteins but also for the rapid quantitation of DNA sequences obtained by enzymatic amplification catalysed by a thermostable DNA polymerase.     

The sensitivity of antibody detection testing using pooled versus unpooled reagent red cells

Evaluation of paternity (alleged father, mother, and child) can range from a straightforward resolution to a complex problem that cannot be resolved without family studies. We present a case of disputed paternity in which tests for crossreactive groups (CREcS) and antigen subtypes (splits) within the human leukocyte antigen (HLA) system could not be used confidently to prove or disprove paternity. Further analysis, red cell enzyme tests, enabled a final verdict and confirmed the current reliability of HLA antisera defining splits.     

高度敏感与特异的微量固相放射免疫分析法检测人IgE

应用两个针对不同抗原决定簇的抗人IgE单克隆抗体分别作为固相抗体和标记~(125)I,建立了一步法微量固相放射免疫分析系统。该系统灵敏度为12.5-15.0pg/ml,特异性强,与100μg/ml人IgG,IgA,IgM和IgD均无反应;标准曲线的线性范围宽,为25-51200pg/ml。批内变异系数为2.4-17.1%。全部材料均可采用国产品,可用于定量测定人淋巴细胞体外培养上清及外分泌液中的微量IgE。     

Genetic mapping using fluorescent quantification of allele frequencies in pooled DNA loaded by solid support

An efficient and labour-saving method for fragment analysis in linkage studies using biotinylated primers and streptavidin-coated combs is presented. The level of streptavidin attached to the combs was used to control the amount of immobilised material. Thus, the need for titration of PCR products to fit the dynamic range of the sequencer was reduced. The method was used to investigate the possibility of quantitating allele frequencies in pools of DNA from family members with the autosomal dominant eye disorder Best's macular dystrophy. The method allowed the detection of one unique allele in a background of 39 other alleles. Using independent datasets, it was further found that the method was able to detect distorted allele frequencies in affected individuals of one family as compared to reference individuals, for markers located more than 30 cM from the disease locus. It was found that this procedure is a powerful alternative to conventional linkage analysis and the method may prove useful in a genome scan for genes involved in complex disorders.     

Sensitivity and specificity of pooled versus individual sera in a human immunodeficiency virus antibody prevalence study.

We evaluated the efficacy of testing pooled versus individual sera for the detection of human immunodeficiency virus antibody. A total of 5,000 individual specimens and 500 pools of 10 specimens each were assayed by an enzyme-linked immunosorbent assay. There was complete agreement in human immunodeficiency virus enzyme-linked immunosorbent assay reactivity for pooled versus individual specimens. An estimated savings of 60 to 80% (labor and supplies) can be realized dependent upon pooling and assay format.     

Antibody detection and identification in a hospital blood bank.

A six-year serological survey has demonstrated that an average of 2.2% per annum of individuals requiring compatibility testing possessed antibodies de novo and that the frequency for individuals forming antibodies following transfusion is 4.1%. The increased detection rate from 1.5% (1969) to 4.1% (1971-5) was almost entirely due to the sensitivity of the two-stage papain technique; a total of 124 antibodies were detected only in this test. Three of these enzyme-active antibodies were found to be the cause of mild haemolytic transfusion reactions. Since 60% of the listed 332 alloantibodies were Rh-specific, the transfusion of appropriately Rh phenotyped blood is strongly recommended.     

A rapid solid phase assay for the detection of circulating immune complexes

A simplified process, which we have termed Enzyme Immune Complex Assay (EICATM) for the detection of circulating immune complexes (CICs), is described herein. The method utilizes readily available reagents, small quantities of serum, and can be performed quickly with a minimal amount of equipment. Serum from 38 normal controls, 98 burn patients, 36 frostbite injury patients, and 21 patients with elevated rheumatoid factor (RF) were tested for CICs in an immune function study. Elevated immune complex levels were found in the group of patients with frostbite injury, and in the group with elevated RF. Serum from thermally injured patients had slightly depressed yet normal CIC levels. The detection of elevated CICs by the EICATM method compared favorably with the more cumbersome Raji cell method, with the added advantage of simplicity, speed, and the ability to detect non-IgG immune complexes.