Journal of immunoassay最新文献

We have developed two bromocriptine enzyme immunoassays with different specificities for applications in human and animal pharmacokinetic studies. The first assay uses antibodies directed against the cyclopeptide structure of bromocriptine, and is specific for untransformed bromocriptine. The second assay uses antibodies directed against the bromolysergic part of the molecule and allows the measurement of both bromocriptine and its metabolites. Enzymatic tracers were obtained by covalent coupling of bromocriptine analogs to acetylcholinesterase from the electric eel Electrophorus electricus. Both assays have a limit of detection of 10 pg/ml and a limit of quantification of 50 pg/ml. The specificity of the assays was determined following fractionation by high-performance liquid chromatography of rat samples obtained after administration of bromocriptine.

A secondary monoclonal antibody (mAb2) was generated by immunization with immune complexes of human IL-1 beta and a primary monoclonal (mAb1). mAb2 bound to a neoepitope on the IL-1 beta/mAb1-complex with a dissociation constant (Kd) of 26 pM but not to uncomplexed IL-1 beta. As assessed by the binding of labeled IL-1 beta and neutralization of bioactivity, mAb2 enhanced the affinity of IL-1 beta to mAb1; Kd-values were 108 pM in absence and 5.4 pM in presence of mAb2. By analyzing a series of mutants of IL-1 where surface loops had been exchanged with the corresponding loops of human IL-1 receptor antagonist protein, a critical region responsible for mAb2 binding was localized to the C-terminal region. In addition to mAb1/IL-1 beta-complexes, mAb2 bound pro-IL-1 beta/mAb1 complexes as well as pro-IL-1 beta suggesting that mAb2 recognized a conformation of IL-1 beta resembling that of pro-IL-1 beta. Using this pair of mAbs, chemiluminescent and enzyme linked assays with detection limits of 2 pg/ml hIL-1 beta have been established.

Plasma immunoreactive vasopressin (iAVP) was studied by RIA in a patient suffering from polyuria during chronic treatment with lithium. The combined use of two antisera specific for different regions of the AVP molecule allowed us to detect circulating forms which are modified in the acyclic tripeptide portion. In this lithium-treated patient, iAVP was abnormally low with respect to plasma osmolality. However, iAVP increased during hypertonic saline infusion, probably through an osmosensitive mechanism. A remarkable finding was that contrary to the observations made in healthy subjects and in another patient with diabetes insipidus, iAVP measured with the antiserum specific for the acyclic portion of the AVP molecule was below the values measured with the antiserum specific for the hexapeptide ring. This unusual immunoreactivity profile suggests that the plasma of this polyuric lithium-treated patient contains vasopressin-like peptides which differ from arginine vasopressin in the structure of the C-terminal tripeptide tail.

The existence of soluble forms of HLA class I and class II antigens in human serum is well established and altered concentrations of these serum proteins have been described to be associated with various diseases. Since soluble HLA antigens (sHLA) can be measured both in serum and plasma samples, we investigated whether anticoagulant treatment influences the determined levels of soluble HLA class I (sHLA-I) or soluble HLA-DR (sHLA-DR). Analyzing paired samples of serum and plasma of 40 healthy individuals we found significantly lower serum levels of sHLA-DR (0.31 +/- 0.15 ng/ml) compared to EDTA plasma levels (0.58 +/- 0.20 ng/ml). By contrast, serum levels of sHLA-I (0.89 +/- 0.74 micrograms/ml) were only slightly lower than EDTA plasma values (0.95 +/- 0.86 micrograms/ml), a situation similar to that of sIL-2R and sCD4 levels. Further experiments intended to clarify the reasons of the reduced sHLA-DR serum levels revealed that (i) the blood storage time before centrifugation did not influence the sHLA-DR level, (ii) treatment of serum with anticoagulant did not augment the measured sHLA-DR concentration, and (iii) the recovery of spiked sHLA-DR was significantly lower when added to native blood than to serum or anticoagulant-treated blood. These results suggest that sHLA-DR is partly removed by the process of blood clotting thus resulting in diminished sHLA-DR serum levels.

A procedure for the radioimmunoassay (RIA) of Antarelix (teverelix) in plasma has been developed for the pharmacokinetic study of this potent LHRH antagonist in dogs. Antiserum was produced by coupling the deamidated Antarelix analog to bovine serum albumin by a carbodiimide reaction and immunizing rabbits with the conjugate. The crossreactivity of the antiserum with LHRH, LHRH agonist Metereline and LHRH antagonists tested was negligible, except for Antide which displayed a crossreactivity of 33%. No crossreactivity with Antarelix metabolites could be detected. The RIA is suitable for the direct determination of Antarelix in plasma, with a minimum detectable level of 1.12 fmol/assay. The accuracy and precision of the method were assessed with plasma samples spiked with Antarelix at concentrations ranging from 0.4 to 6.4 pmol/ml. The recovery was 104.8% with intra- and interassay CV between 1 and 3.7%. Pharmacokinetic profiles of Antarelix in dogs were established following an i.v. or a s.c. dose of 10 micrograms/kg.

Naturally occurring antibodies to alpha-linked galactose (anti- gal) has been reported to be present in large quantities in normal human sera and they seem to play an important role in a variety of infectious as well as autoimmune diseases. A cell-ELISA using glutaraldehyde fixed normal rabbit erythrocytes was developed for quantification of anti-gal in human sera. This assay was compared with three other(commonly used) immunoassays viz. a) agglutination b) enhanced agglutination and c) lipid ELISA-assays for detection of anti-gal in human sera. The cell-ELISA was found to be the most sensitive assay followed by lipid-ELISA, enhanced agglutination and agglutination assay in decreasing order. Anti-gal affinity purified through a column of melibiose-agarose was tested by cell-ELISA. Monolayers of RRBC pre-treated with alpha-galactosidase was not reactive while in monolayers treated with beta-galactosidase, the anti-gal reactivity was comparable to those in untreated RRBC monolayer, thus indicating the high specificity of cell-ELISA for detection of antibodies to alpha-linked galactose.

Streptavidin coated microtitre wells can be used to provide attachment to biotinylated peptides, regardless of charge and size, for use in an enzyme immunoassay. Considerable non-specific human serum binding to streptavidin at the recommended concentration was noted. The level of binding varied considerably with serum samples and was unrelated to previous streptococcal disease. Strategies used to reduce this phenomenon included pre-absorption of serum with streptavidin and reducing the streptavidin concentration in the wells. Significant reduction in binding was found with a reduction in streptavidin concentration from the recommended concentration of 0.5 ug/well to 0.01 ug/well.

A method of quantifying and visualizing herpes simplex virus type 1 antigen by indirect enzyme-linked immunosorbent assay (ELISA) is described. This assay is simplified by the use of polyclonal serum and can be applied to the quantification of free antigen as well cell-bound. Moreover, cell viral antigen can be visualized. Antigen sources were viral suspensions, infected cells and proteins extracted from infected cells. The assay was specific and its sensitivity was dependent on the antigen source. The technique was regarded as specific within a range showing a direct correlation (r > 0.8) between the concentration of the antigen and the net absorbance value (the difference of the absorbance obtained with the viral antigen minus the control antigen). The technique has advantages over other ELISA procedures: does not require monoclonal antibodies, or labelled antiviral immunoglobulins or antiviral serum from two different species. In addition total free antigen can be measured.

A direct enzyme-linked immunosorbent assay (ELISA) employing 2/215 mouse monoclonal hybridoma antibody is described. The assay is capable of detecting activated factor XII in human plasma and can be used to assess early detection of the intrinsic blood coagulation pathway. No cross reactivity with human factor XII zymogen has been found. The assay was used to assess activation of factor XII in patients with renal failure, pregnancy and diabetes compared to a control group.

An IgM monoclonal antibody (7C5B71) which reacted with the blood stages of Plasmodium vivax, but not with those of Plasmodium falciparum was used in a cell-ELISA to detect parasites in samples of peripheral blood. Blood thin smears were probed with monoclonal antibody 7C5B71 and then reacted with a peroxidase conjugate of the appropriate specificity and the insoluble chromogen amino-ethyl-carbazole. Infected cells which appeared dark red coloured were rapidly identified under a light microscope using a low magnification. The conventional microscopic examination of thin films coloured with Giemsa was used as reference test. Under laboratory conditions the test showed a positive result in samples with a level of parasitaemia of < or = 500 parasites/microliter of blood. In a preliminary field trial the test showed 100 % specificity for the diagnosis of P. vivax malaria.