Immunoassay of pesticides: an update.

Measurement of levels of pesticides residues in foods and crops most often requires extensive cleanup and instrumental techniques such as gas chromatography. Immunoassay measurement techniques, on the other hand, may be used directly on the test portion or require only minimal cleanup. Further refinements of the common antibody-enzyme-based solid-phase assays, such as use of coated magnetic particles, antibody-coated crystals, and continuous-flow devices, have extended the measurement range and applicability of these assays. Likewise, new immunoassays for pesticides have been developed, and existing assays have been refined, optimized, and more completely characterized and validated. In addition to their ability to accurately and reliably measure amounts of residues present in food and crops, immunoassays can be readily used as rapid screening methods for contaminants in field samples. We have previously reviewed much of the work in the area of pesticide immunoassay; this report updates previous information and discusses some new immunoassay techniques.

The prevalence of depression in geriatric medical outpatients.

One hundred and fifty-nine patients attending a geriatric medical outpatient clinic were screened for depression using the geriatric depression scale (GDS) and brief assessment schedule depression cards (BASDEC). Twenty-two (13.8%) patients screened positive with one or other test, 13 (8.2%) being positive with both screening tests. There was a highly significant correlation between the two screening tests but the BASDEC instrument was quicker and easier to administer. Eighteen of 22 patients who screened as positive proved to have clinical depression. On subsequent re-testing of 17 of these patients 4-6 weeks later, eight were still positive, but nine patients were now negative and subjectively felt better. These findings suggest the possibility of a transient mood disturbance occurring in elderly patients akin to recurrent brief depression seen in younger subjects, and have implications for starting therapy in apparently depressed elderly people.

Do acute diseases transiently impair anterior pituitary function in patients over the age of 75? A longitudinal study of the TRH test and basal gonadotrophin levels.

Several studies have shown that anterior pituitary function is affected by the ageing process. Little is known, however, about the effect acute diseases have on the anterior pituitary. Should they be associated with a disturbance of anterior pituitary hormone production, they might make anterior pituitary function tests difficult to interpret when they are most needed. To test this hypothesis, we studied the thyrotrophin (TSH) releasing hormone (TRH) test and basal plasma gonadotrophins, taken as indicators of anterior pituitary function and reserve, in 74 consecutive patients (49 women) mean age 82 +/- 5.1 suffering from acute diseases soon after admission to hospital. A total of 44 (30 women) were followed up and retested after 3 disease-free months following recovery and discharge home. Seventy-one age- and sex-matched healthy controls (47 women), mean age 82 +/- 5.0 living in the community were also studied. The mean peak TSH increment after TRH (mean delta max TSH) increased from 5.7 soon after admission, to 7.7 U/l at follow-up (P = 0.01). The mean plasma LH increased from 25 to 35 U/l in women (P = 0.0004) and from 9.7 to 14 U/l in men (P = 0.03). The mean plasma FSH increased from 21 to 25 U/l in women (P = 0.04) and from 7.5 to 9.4 U/l in men (P = 0.01). Controls had greater TSH responses to TRH and higher plasma gonadotrophins levels when compared with acute patients (LH women P = 0.17, for all other tests P < 0.05). We conclude that a transient reduction of anterior pituitary function is a common occurrence in patients over the age of 75 suffering from acute diseases. This has diagnostic relevance and therapeutic implications.

Immunoassay of pesticides.

Determination of the presence and levels of pesticide residues in food is fundamental in monitoring and regulatory programs. Residues are separated from the food matrix by solvent extraction, followed by cleanup steps. The residues are most often identified and quantitated by instrumental analysis, usually liquid or gas chromatography. Extraction and cleanup are often laborious and time-consuming; determination requires expensive, sophisticated instrumentation. There is a need for rapid, easily performed tests, such as immunoassays, that could be used for screening under field conditions or for quantitation of residues in foods in the laboratory. Although a large number of immunoassays have been developed for pesticide chemicals, very few have been specifically applied to foods, and only a very small number are currently available commercially. The agencies charged with monitoring and regulatory responsibilities--Environmental Protection Agency, Food and Drug Administration, and U.S. Department of Agriculture--as well as professional societies such as AOAC and the International Union of Pure and Applied Chemistry, are investigating and developing guidelines for test kit evaluation and standards to be met before a kit can be accepted as a practical and useful method of analysis for use in their programs.

Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection.

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.

Single-chain antigen-binding proteins.

Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

Monoclonal antibodies against dengue 2 virus E-glycoprotein protect mice against lethal dengue infection.

A panel of 11 murine monoclonal antibodies directed against dengue type 2 was evaluated for antigen specificity by dot immunobinding assay and Western blot analysis and for in vitro and in vivo biological activities. Nine of the 11 monoclonal antibodies reacted with viral E-glycoprotein based on the Western blot analysis; one reacted with a 36 Kd protein present in dengue-infected C6/36 mosquito cells. The nine E-glycoprotein-reactive monoclonal antibodies also neutralized dengue 2 virus in a plaque reduction assay. Of the neutralizing monoclonal antibodies, five passively protected mice in vivo against lethal intracerebral dengue 2 challenge. The protective monoclonal antibodies were directed against viral determinants that fell into at least three spatially separate families of epitopes on E-glycoprotein, the antigenicities of which were preserved after heat/detergent denaturation.

Factors influencing invasion of erythrocytes by Plasmodium falciparum parasites: the effects of an N-acetyl glucosamine neoglycoprotein and an anti-glycophorin A antibody.

When schizont-infected erythrocytes were incubated with N-acetyl glucosamine coupled to bovine serum albumin (GluNAc-BSA), the number of new ring forms which appeared several hours later was reduced and the number of abnormal and unruptured schizont-infected erythrocytes was increased compared with controls, indicating that GluNAc-BSA prevents invasion by a toxic effect on schizonts rather than by receptor blockade. Invasion of erythrocytes by Plasmodium falciparum was inhibited by a monoclonal antibody against glycophorin A, but inhibition also occurred with P. knowlesi, a parasite that is known to invade independently of glycophorin A. Inhibition of invasion with anti-glycophorin A is unlikely to be related to receptor blockade and is probably related to decreased deformability of the erythrocyte membrane caused by the binding of this antibody. Previous studies suggesting that GluNAc-BSA and anti-glycophorin A antibodies inhibit invasion by receptor blockade should be reevaluated. Erythrocytes deficient in glycophorin C and band 4.1 were also resistant to invasion by both P. falciparum and P. knowlesi.

Monoclonal antibodies reactive with K1-encapsulated Escherichia coli lipopolysaccharide are opsonic and protect mice against lethal challenge.

Seven murine monoclonal antibodies (MAbs) directed against O-side-chain determinants of the K1-encapsulated Bortolussi strain of Escherichia coli (O18:K1:H7) were evaluated for their in vitro and in vivo activities. All the MAbs reacted well in Western blots against E. coli O18 lipopolysaccharide antigens. Two MAbs of the immunoglobulin G (IgG) class promoted in vitro opsonophagocytosis and protected mice lethally challenged with bacteria. Two IgM MAbs showed partial protection, although they had no in vitro opsonic activity, and the remaining three IgM MAbs showed no apparent functional activities. Monoclonal IgG antibodies against bacterial lipopolysaccharide can be opsonic and protective in spite of the presence of the K1 capsule on the bacterium.

Cervical diastematomyelia: a case report with operative management.

A case of midcervical diastematomyelia presenting as acute traumatic hemiplegia in an adult is discussed, with emphasis upon features reflecting this particular location. Advantages of metrizamide computed tomographic imaging in the diagnosis of this entity are presented, and a method of operative management is outlined.

Epitope ratio analysis (ERA): a simple radioimmunological method using monoclonal antibodies for the simultaneous analysis of several antigens.

Monoclonal antibodies specific for human cell surface antigens were used to develop a technique for the simultaneous analysis of several antigenic determinants. The procedure was used to measure the relative expression of specific cell antigens during cultural growth. Epitope ratio analysis (ERA) is applicable to many systems requiring measurement of such differential antigen expression.

Persistence of hapten-antibody complexes in the circulation of immunized animals after a single intravenous injection of hapten.

To study the fate of a low molecular weight antigen (hapten) in the circulation of animals whose sera contain antibodies specific for that low molecular weight antigen, a single injection of digoxin-(3)H (0.4 mg/kg) was administered intravenously to 18 rabbits. Thirteen animals (nine nonimmunized and four immunized with bovine serum albumin) served as control animals. In five rabbits which had been immunized with a digoxin-bovine serum albumin conjugate and whose sera contained digoxin-specific antibodies, the mean 12-h serum digoxin concentration was 8,300 ng/ml (control: 92 ng/ml) and the mean serum concentration 12 mo after the single injection of digoxin-(3)H was 85 ng/ml. In digoxin-immunized rabbits, less than 10% of the digoxin-(3)H was excreted in the first 10 days (control: 77% recovered in urine and feces) and the mean biological half-life of digoxin, as calculated from serum digoxin-(3)H disappearance curves, was 72 days (control: 3.4 days). In sera of digoxin-immunized rabbits, more than 90% of the circulating digoxin-(3)H was immunoglobulin bound, as determined by the double-antibody and dextran-coated charcoal methods. The serum disappearance rate of (125)I-antidigoxin antibodies was similar in nonimmunized and in immunized animals and in the presence or absence of digoxin. It is concluded that the biological half-life of a hapten may be markedly prolonged when the hapten is bound to specific antibody. The persistence of antibody-hapten complexes in the circulation suggests that these complexes may not be deposited in tissues and raises the possibility that low molecular weight determinants may be capable of preventing or reversing the deposition of immune complexes, containing macromolecular antigens, in the tissues of experimental animals and man.