SECOND ANTIBODY CHEMICALLY LINKED TO CELLULOSE FOR THE SEPARATION OF BOUND AND FREE HORMONE: AN IMPROVEMENT OVER SOLUBLE SECOND ANTIBODY IN GONADOTROPHIN RADIOIMMUNOASSAY

1976 ◽  
Vol 81 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Ph. Koninckx ◽  
R. Bouillon ◽  
P. De Moor
Keyword(s):  
Detection Limit ◽  
Protein Concentration ◽  
Cyanogen Bromide ◽  
Solid Phase ◽  
Phase System ◽  
Cellulose Matrix ◽  
Classical Technique ◽  
Definite Improvement ◽  
Cyanogen Bromide Activation

ABSTRACT Coupling the second antibody to a solid pase (DASP) was found to be a definite improvement over the classical technique of soluble second antibody (DA) separation of bound and free hormone in the radioimmunoassays of gonadotrophins in plasma or in serum. The duration (2.5 to 7.5 min) and the pH (10.5 to 12.5) of the cyanogen bromide activation of microcristalline cellulose did not affect the coupling capacity or the immunoreactivity of the second antibody, nor did it augment aspecific adsorption of free gonadotrophin on the cellulose matrix. Precision of duplicates was comparable for both methods but the accuracy and the detection limit of the solid phase system were higher due to a better separation of bound and free hormone, to the lower blank value and to the absence of aspecific interference of protein concentration. Cost was lower since much less second antibody was needed in the solid phase system than in the soluble system and since the experimental procedures were simplified by the absence of prozoning effect and the solid nature of the cellulose matrix.

RADIOIMMUNOASSAYS EMPLOYING IMMUNOSORBENTS

1969 ◽  
Vol 62 (1_Suppl) ◽  
pp. S207-S221 ◽  
Author(s):  
Leif Wide
Keyword(s):  
Liquid Phase ◽  
Cyanogen Bromide ◽  
Solid Phase ◽  
Binding Capacity ◽  
Phase System ◽  
Two Phase ◽  
System A ◽  
Technical Procedure ◽  
Particle Form ◽  
Insoluble Polymers

ABSTRACT The use of a reaction between compounds in a two phase system – a solid phase and a liquid phase – has simplified the technical procedure of the radioimmunoassays. Antigens or antibodies can be coupled covalently to insoluble polymers to form stable conjugates with retention of immunological binding capacity. Such immunosorbents can be used in radioimmunoassays in many different ways and with the solid phase in different forms. Some of these techniques seem to be universally applicable for the assay of antigens or antibodies. In one of these, antigens or antibodies are chemically coupled to cyanogen bromide activated insoluble polysaccharides in a particle form. This method which has several advantages when compared with most other radioimmunological methods is discussed in particular. Finally, a detailed description of the preparation of an immunosorbent and its use in such a radioimmunoassay system is given.

Modified solid-phase (tube) radioimmunoassay of human growth hormone

1969 ◽  
Vol 47 (9) ◽  
pp. 803-814
Author(s):  
R. M. Bala ◽  
K. A. Ferguson ◽  
J. C. Beck
Keyword(s):  
Growth Hormone ◽  
Human Growth Hormone ◽  
Protein Concentration ◽  
Solid Phase ◽  
Ionic Composition ◽  
Rabbit Antiserum ◽  
Human Growth ◽  
Phase System ◽  
Nonspecific Binding ◽  
Free Antibody

Polystyrene tubes were coated with rabbit antiserum to human growth hormone (HGH). Bovine serum albumin (BSA) in buffer, HGH standard or unknown samples, and 125I-HGH were added to the antibodycoated tubes. After incubation the radioactivity in each tube was counted, the incubate discarded, the tubes were washed, and radioactivity was recounted. The sensitivity of the assay was consistently 0.05 mμg HGH or less. Nonspecific binding by uncoated tubes was less than 1%. It was preferable to control the surface area exposed to excess antiserum rather than limit the concentration of the antiserum. Polypropylene and polystyrene tubes gave similar results. Variation of ionic composition or pH of the incubate had no significant effects. Maximum precision and sensitivity occurred with incubation at 4 °C for 40 to 60 h. Addition of posthypophysectomy human plasma or varying the protein concentration had similar effects. The immobilized antibody may be more selective in binding with antigen than is free antibody in solution. Our modifications combine favorable features of the solid-phase system with an increase in sensitivity and greater economy of materials.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA <1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

Determination of Ochratoxin A in Feed by Immunoaffinity Cleanup and Liquid Chromatography

2013 ◽  
Vol 96 (3) ◽  
pp. 599-602 ◽  
Author(s):  
Ping Ding ◽  
Ziyou Mi ◽  
Yali Hou ◽  
Yigang He ◽  
Jianhua Xie
Keyword(s):  
Liquid Chromatography ◽  
Detection Limit ◽  
Ochratoxin A ◽  
Cyanogen Bromide ◽  
Polyclonal Antibodies ◽  
Immunoaffinity Column ◽  
Low Levels ◽  
Sepharose 4B ◽  
Feed Samples

Abstract A method using LC was developed for determination of ochratoxin A (OTA) in feeds. The extracted samples were cleaned up by an immunoaffinity column prepared by covalently coupling polyclonal antibodies against OTA to cyanogen bromide-activated Sepharose 4B. The eluates were determined by LC with fluorescence detection. Recoveries of OTA from fortified samples of 1–10 μg/kg levels ranged from 84.3 to 90.0%, with CVs of 3.3–7.8%. The detection limit was 0.045 μg/kg based on an S/N of 3:1. A total of 65 feed samples were screened for OTA with the proposed method. The results showed that only nine samples were contaminated with OTAs at low levels. The presented method was successfully applied to quantify OTAs in real feed samples.

scholarly journals An Alternative Coupling Procedure for Preparing Activated Sepharose for Affinity Chromatography of Penicillinase

1976 ◽  
Vol 29 (4) ◽  
pp. 305 ◽  
Author(s):  
RG Coombe ◽  
AM George
Keyword(s):  
Affinity Chromatography ◽  
Room Temperature ◽  
Cyanogen Bromide ◽  
Dry Weight ◽  
Enzymic Activity ◽  
Covalent Attachment ◽  
Affinity Column ◽  
Coupling Procedure ◽  
Coupling Techniques ◽  
Cyanogen Bromide Activation

Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axtm et al. (1967). Porath and Sundberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. a-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8� 0-8� 6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41 % of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.

Influence of biomass on hydrodynamics of an internal loop airlift reactor

2006 ◽  
Vol 60 (6) ◽  
Author(s):  
M. Juraščík ◽  
M. Hucík ◽  
I. Sikula ◽  
J. Annus ◽  
J. Markoš
Keyword(s):  
Aspergillus Niger ◽  
Gluconic Acid ◽  
Solid Phase ◽  
Biomass Concentration ◽  
Airlift Reactor ◽  
Phase System ◽  
Two Phase ◽  
Experimental Conditions ◽  
Internal Loop ◽  
Three Phase

AbstractThe effect of the biomass presence on the overall circulation velocity, the linear velocities both in the riser and the downcomer and the overall gas hold-up was studied in a three-phase internal loop airlift reactor (ILALR). The measured data were compared with those obtained using a two-phase system (air—water). All experiments were carried out in a 40 dm3 ILALR at six different biomass concentrations (ranging from 0 g dm−3 to 7.5 g dm−3), at a temperature of 30°C, under atmospheric pressure. Air and water were used as the gas and liquid model media, respectively. Pellets of Aspergillus niger produced during the fermentation of glucose to gluconic acid in the ILALR were considered solid phase. In addition, liquid velocities were measured during the fermentation of glucose to gluconic acid using Aspergillus niger. All measurements were performed in a bubble circulation regime. At given experimental conditions the effect of the biomass on the circulation velocities in the ILALR was negligible. However, increasing of the biomass concentration led to lower values of the total gas hold-up.

Direct solid-phase enzymoimmunoassay of testosterone in saliva.

1989 ◽  
Vol 35 (10) ◽  
pp. 2044-2047 ◽  
Author(s):  
K Howard ◽  
M Kane ◽  
A Madden ◽  
J P Gosling ◽  
P F Fottrell
Keyword(s):  
Detection Limit ◽  
Analytical Procedure ◽  
Solid Phase ◽  
Medical Applications ◽  
Microtiter Plates ◽  
Large Numbers ◽  
Coefficients Of Variation ◽  
Serial Sampling ◽  
Direct Solid

Abstract This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

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