Solid-phase Radioimmunoassay Technique Research Articles

PHYTOESTROGEN IN SEVERAL FRUITS AND LEAVES

Phytoestrogen is molecularly almost similar with and acts the same as estrogen and is found a lot in several fruits and leaves grown either in tropical or subtropical countries. However, the quantity of molecular contents are not yet known exactly. In menopause and andropause, people need substitution of estrogens as a replacement therapy of sex hormone, due to the significant hormone decline and impacted disturbance of several organ functions and thus progressively causing severe organ dysfunctions. The objective of this study was to know the estrogen content by analyzing extractions of pegaga, green clover leaves, tomato and papaya fruit of which certain communites have overviewed contents of the leaves and fruit The samples which were collected for this purpose used 10 times replication in four different groups: the pegaga, green clover leaves, tomato and papaya fruit. All these groups were divided into two (2) subgroups based on the process or subspecies. All samples were made as an infusion 1:4) w/v), and then extracted after centrifugation 1000xg for 15mnts, with 1:5 petroleum ether) v/v). After it was evaporated, each extraction then was kept dry-frozen at –20° C until the analysiswas performed. Solid phase Radioimmunoassay technique was used to identify the estrogen contents, up to a total of 80 samples The binding percentage of each sample was then interpolated on a logit-log paper to find out the real concentrations.14 The lowest estrogen level was found in fresh pegaga leaves extract (Mean+SD) was 47.9+5.5 pg/g, but in dry leaves extract the level was increased twice, about 96.1+11.2 pg/g. Meanwhile the estrogen level in fresh green clover leaves extract was 538.0+30.5 pg/g, more than ten times higher compared to fresh pegaga level, but twice lower than the estrogen level compared to dry green clover leaves extract, which was 1068.0+97.2 pg/g. In the fruit group, the fibrin part of tomatoes had more or less the same estrogen content compared with Thai papaya subspecies, 1037.0+37.7 pg/g and 1175.0+67.7 pg/g, respectively. On the other hand, it was noted that the inner part/fibrin part of tomato had a higher estrogen level of four (4) times compared to the outer part which was 315.0+18.4 pg/g. While it was noted that local Java papaya besides being not so sweet, the estrogen level was also not as high or the same as that found in fresh green clover which is 530.1+50.7 pg/g and 538.0+30.5 pg/g. Based on this study so far, it can be concluded that semanggi/green clover, tomato and papaya could be suggested as a replacement therapy for certain people who are considered to have reduced estrogen content, except that pegaga leaves are not recommended. The last mentioned plant besides that it is difficult to obtain, its estrogen content is also very low.

Blood and seminal plasma concentrations of selenium, zinc and testosterone and their relationship to sperm quality and testicular biometry in domestic cats

The aim of this study was to assess seminal plasma (SP) and serum concentrations of zinc (Zn), selenium (Se) and testosterone (T) in domestic cats and determine whether these are related to sperm quality and testicular biometry. Six tomcats were collected using an artificial vagina and sperm analysis included motility by CASA, morphology, plasma membrane integrity, and sperm count. Serum and SP were submitted to total T concentration determination using a solid-phase radioimmunoassay technique while Zn and Se were measured by atomic absorption spectroscopy. Serum T concentrations were greater compared to SP concentrations, but both values were significantly correlated. Se concentrations were higher in serum, whereas SP had greater Zn values. Concentrations of Se, Zn and T were not correlated with each other either in serum or SP. Negative correlations were detected between Se concentrations in SP and total sperm head defects, and between Se concentrations in serum and VAP, VSL, STR, and LIN. Serum concentrations of Zn were negatively correlated with total abnormal sperm and midpiece defects and positively related to progressive motility. Both serum and SP concentrations of T had no relationship with sperm quality. Concentrations of Se exhibited a negative correlation with total testicular weight, whereas T concentrations in SP and serum were correlated with total testicular volume and weight. In conclusion, both Se and Zn concentrations in serum were correlated to sperm quality variables in the domestic cat, thus, making these potential candidates for fertility markers.

Natural Phytoestrogen Contents In Severals Fruit And Leafs; The Future Replacement Hormone Therapy In Menopause Women

Fitoestrogen secara melekul hampir sama dan mempunyai aktifitas sama seperti estrogen dan ditemukan pada jenis tanaman dan buah buahan terutama di negara tropis. Tetapi jumlah kandungan melekulnya hormone belum diketahui. Pada orang-orang menopause dan andropause diperlukan sulih hormone seks karena mereka umumnya mengalami penurunan hormone tersebut secara berarti yang bisa berdampak pada gangguan fungsi organ. Tujuan dari penelitian ini untuk menganalisis kandungan estrogen ekstrak daun pegaga, semanggi, buah tomat dan papaya sehingga dampak positifnya pada komunitas masyarakat yang memerlukan.

Quantification of Myotrophin From Spontaneously Hypertensive and Normal Rat Hearts

A novel peptide, myotrophin, has been isolated, purified, cloned, and sequenced from the hearts of spontaneously hypertensive rats (SHR) and from dilated cardiomyopathic human heart tissue. Myotrophin accelerates myocyte growth by stimulating protein synthesis (not by altering myocardial cell division). Our successive studies were conducted to evaluate the pathophysiological significance of myotrophin; a solid-phase radioimmunoassay technique was developed for quantifying the protein in hypertrophied and normal hearts. Specific antipeptide antibody was raised in rabbits against a peptide that represents a selected amino acid sequence of a 17-amino acid myotrophin segment by using the multiple antigenic peptide technique. The specificity of the antibody was evaluated by determining the affinity constant after constructing the Scatchard plot obtained from the ratio of bound to free myotrophin against bound myotrophin. The value obtained was 2.61 x 10(7) L/mol. The specificity was further demonstrated by Western blot analysis, in which a single protein band was obtained in the region of 12 kD. Pretreatment of the antibody with myotrophin completely blocked the binding sites, because no protein band was detected on the immunoblot. The antibody prevented the myocardial protein synthesis induced by myotrophin as revealed by the blockage of the stimulation of [3H]leucine incorporation into myocyte protein. Quantification of myotrophin from different heart tissues was achieved by Western blot and dot blot analyses. Amounts of myotrophin present in different dots were determined by using a video image analyzer. The level of myotrophin in the embryonic tissue was found to be similar in male normal and SHR hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis.

To determine the prevalence and specificity of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis and assess whether increased levels of antibodies to B. burgdorferi were attributable to rheumatoid factor. Retrospective case-control study. Urban referral center in an area devoid of infected ticks as a source of endocarditis sera. Sera from 30 consecutive patients with culture-proven subacute endocarditis between 1979 and 1981 were compared with 30 control sera collected between 1989 and 1990. In addition, sera from 20 consecutive patients with rheumatoid arthritis who were positive for rheumatoid factor were collected between 1991 and 1992. Sera were compared with a convenience sample from 15 patients who met the criteria for Lyme disease. Antibodies to B. burgdorferi were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. IgM rheumatoid factor was quantified using solid-phase radioimmunoassay or latex agglutination techniques. Thirteen of 30 patients with endocarditis (43%) compared with 3 of 30 normal controls (10%) had increased levels of antibodies to B. burgdorferi (P < 0.01). Of these 13 patients, only 1 had an immunoblot consistent with previous infection. The others had nonspecific immunoblots: 5 showed isolated 60-kd reactivity; 1 patient had isolated 41-kd reactivity; and 6 had no bands of reactivity. Immunoblots of the 3 controls with increased antibodies showed only isolated 41-kd reactivity. Thus, the specificity of the B. burgdorferi antibody test in patients with endocarditis was only 60% (95% CI, 42% to 78%), compared with 90% (CI, 79% to 100%) in controls. No correlation was noted between IgM rheumatoid factor and antibodies to B. burgdorferi in patients with endocarditis (r = 0.2; P > 0.2). Only 1 of 20 patients with rheumatoid arthritis without known bacterial infections had antibodies to B. burgdorferi. Although a positive ELISA test for B. burgdorferi may be a "true positive," a positive serologic test alone does not ensure that the clinical problem is due to Lyme borreliosis. Cross-reactive antibodies to shared epitopes between B. burgdorferi and the endocarditis organism may account for the high false-positive results.

Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination

Antibody levels in 41 sets of human acute- and convalescent-phase meningococcal sera were compared with those in 23 sets of human prevaccination and 2-week postvaccination sera. We used a modification of a solid-phase radioimmunoassay (SPRIA) technique to test each of the human serum samples as inhibitors of monoclonal antibodies (MAbs) that bind (HIMSPRIA) to the outer membrane complex from a 2a:P1.2:P5.1 strain. We used three murine MAbs specific for the 2a, P1.2, and P5.1 epitopes on meningococcal class 1, 2, and 5 proteins, respectively, to detect antibodies with similar specificities in human sera. Each of 40 available matching strains from patients were also screened with the three MAbs in a nitrocellulose spot blot assay. A total of 37 (92%) were positive for the 2a epitope, 36 (90%) were positive for the P1.2 epitope, and 16 (40%) were positive for the P5.1 epitope. Of 38 available convalescent-phase sera, 27 (71%) matched with these strains and had detectable inhibiting antibody for each of the MAb-defined protein epitopes of the infecting strain. Three convalescent-phase sera had no HIMSPRIA activity for MAb-defined epitopes that were present on the infecting strain; others had activity for one or two of the epitopes. The results were similar for pre- and postvaccination sera. The average level of HIMSPRIA activity for the P1.2 epitope was greater than fivefold higher in postvaccination sera compared with that in convalescent-phase sera. Sera with distinct patterns of HIMSPRIA activity also were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis and showed a correlation between the HIMSPRIA activity for particular epitopes and the level of antibody binding to the immunoblotted proteins possessing those epitopes. A comparison of the HIMSPRIA and the bactericidal activity of selected postvaccination sera indicated a possible correlation between HIMSPRIA and bactericidal activity, but it also suggested the presence of bactericidal antibodies with specificities other than those defined by the MAbs.

Immune responses in mice to different noncovalent complexes of meningococcal B polysaccharide and outer membrane proteins.

Noncovalent complexes of meningococcal group B polysaccharide and outer membrane proteins (OMPs) from serotypes 2a, 2b, 6, 8, and 15 were made either by coextraction of the two components from culture supernatants or by separate preparation of both OMPs and B polysaccharide with subsequent mixing of the two components. The former method showed a markedly greater degree of binding of B polysaccharide to OMPs, a less heterogeneous composition of OMPs, and a lower lipopolysaccharide content. Immunization of mice with these complexes showed that a higher degree of binding of B polysaccharide to OMPs resulted in a higher anti-B response. Western blotting (immunoblotting) and solid-phase radioimmunoassay techniques confirmed that antibody cross-reactions occur among OMP serotypes. The occurrence of common T-cell determinants between OMP serotypes was suggested when mice primed with a type 6 OMP complex and challenged with a homologous or heterologous serotype complex responded with significantly higher anti-B titers than unprimed animals. These memory T cells persisted for at least 12 months in mice.

Bradykinin in carcinoid syndrome.

Bradykinin concentrations in peripheral venous blood were measured in seven patients with carcinoid syndrome. The diagnosis was based on typical symptoms and raised urinary excretion of 5-hydroxy-3-indole acetic acid; the carcinoid tumour was verified histologically. Two patients were flushing constantly and the other patients had flushing attacks two to 10 times daily. Several blood samples were taken at weekly intervals from six of seven patients. During 30 sampling procedures the patients were flushing during sampling in 12 instances. Bradykinin was measured by a sensitive solid phase radioimmunoassay technique. Blood bradykinin concentration was normal in all patients. Bradykinin is unlikely to be the vasoactive mediator of flushing.

Serum antibody against Tamm-Horsfall protein in patients with renal cell carcinoma.

We assayed sera from 24 patients with metastatic renal cell carcinoma for class specific antibody against urinary Tamm-Horsfall protein (THP) using indirect solid-phase radioimmunoassay techniques. Overall, 16 patients had elevated antibody levels (greater than 2 SD above the mean levels of control subjects). IgG against THP was elevated in eight of the patients, IgA in 12, and IgM in five. However, THP was not identifiable by immunohistochemical techniques in the sections of 15 of 24 tumors available for study. Nine of the 15 tumors were from patients with elevated antibody levels. Serum antibody against THP in patients with renal cell carcinoma appears to result from antigenic stimulation by THP derived from the urine rather than from the neoplasm.

Purification of amatoxin-specific antibodies from rabbit sera by affinity chromatography, their characterization and use in toxicological studies

Rabbits were immunized with fetuin - β-amanitin. They produced amatoxinspecific immunoglobulins of various classes, predominantly IgG. The crude IgG fraction was isolated by gel filtration on Sephacryl S-300. By polyethylene glycol precipitation in the presence of tritiated amatoxin, as well as by a solid phase radioimmunoassay technique, the portion of amatoxin-specific antibodies in the IgG fraction was determined to be 5 – 13%. The affinity of the amatoxin-specific IgG for a tritiated amatoxin derivative was measured by equilibrium dialysis and calculated according to the method of Scatchard. The dissociation constant was 2.6 × 10 −9 M. The amatoxin-specific immunoglobulins were extracted by their affinity to α-amanitin - Sepharose 4B and eluted with excess α-amanitin. The complex was isolated in 95% yield with a ratio immunoglobulin: toxin of c. 2:1. Alternatively, the uncomplexed IgG could be eluted from the α-amanitin - Sepharose 4B with 5 M guanidine hydrochloride; this treatment, however, decreased the binding capacity for amatoxin by 30% (ratio 1.4:1). The purified amatoxin-specific IgG was assayed for its therapeutic efficiency in mice poisoned with α-amanitin, but was unable to neutralize the toxic effects of the mushroom toxin. On the contrary, equimolar amounts of the amatoxin-specific immunoglobulins enhanced the toxicity of α-amanitin in the mouse by a factor 2.

Development of a sensitive solid-phase radioimmunoassay technique for quantification of class specific Escherichia coli antibodies

A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions. Preparation of affinity-purified anti- E. coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 μl of sample. This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection. RIA also compares favourably with ELISA in sensitivity and sample size required. Affinity-purified standards may also be used to quantify the ELISA test.

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4–23, 31–46, and 56–77; these studies suggested that an antigenic site might be restricted to residues 60–77 in the 56–77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61–77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu 60, thus confirming our suggestion that a major antigenic site is located in residues 60–77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60–77 indicating that the antibodies were specifically bound to the resin.

Detection of varicella-zoster virus-specific IgA antibodies in varicella and zoster patients and in healthy adults of various ages by solid-phase radioimmunoassay.

The varicella-zoster virus(VZV)-specific serum immunoglobulin A(IgA) response was studied in varicella and zoster patients and in healthy adults of various ages by a sensitive solid-phase radioimmunoassay technique. Significant rises in VZV-specific IgA titer were found for both varicella and zoster patients, while VZV-specific IgA was also detected in greater than 90% of healthy adults but at lower titers. No significant decrease in VZV-specific IgA titer with increasing age was found. The role of humoral versus cell-mediated immunity in VZV reactivation is discussed.

An epidemic outbreak of hepatitis A among homosexual men in Stockholm. Hepatitis A, a special hazard for the male homosexual subpopulation in Sweden.

In 1979-1980, a distinct outbreak of hepatitis A occurred among homosexual men in Stockholm, Sweden, city and county area. The epidemic comprised 145 known cases. It began in December 1979 and progressed in waves during the following 10 months, with three distinct peaks separated by about six-week intervals. Actually, the incidence of hepatitis A in the Stockholm area showed a fivefold increase during 1980 as compared to the previous year. Clinical serologic, and social characteristics were studied more closely in 98 of the 145 homosexual men. Verification of hepatitis A was made by a solid-phase radioimmunoassay technique for detection of antibody to hepatitis A virus of the immunoglobulin M class. In addition, 64% of the men showed findings consistent with a prior hepatitis B (antibody to hepatitis B core antigen and/or antibody to hepatitis B surface antigen) and 34% were Treponema pallidum immobilization-positive from a prior or concomitant syphilis. Employment in risk professionals was common; thus, 19% worked in restaurants or otherwise handled food and 20% were engaged in medical care as compared to the 1% occupied in either branch of work among the general population in Sweden. Sexual habits with multiple partners and oral-anal sexual contacts were judged to be of major importance in the spread of this epidemic. Some spread of hepatitis A to the general population probably occurred due to the risk occupations of many homosexual men.

A rapid miniaturized solid-phase radioimmunoassay technique for secreted immunoglobulins, employing microtiter plates, antibody bound to polyacrylamide beads, and filter strip harvesting

We have developed a solid-phase radioimmunoassay technique, which allows a large number of samples to be assayed, while minimizing amounts of reagents and technician time. The assay is run in microtiter plates, which results in improved organization, shorter assay set-up time and advantageous miniaturization. On the first day only 3 reagents are required: sample or standard, a 125I-labeled antigen, and an antibody attached to a polyacrylamide bead. After an overnight incubation period, the assay is harvested using a commercial microharvesting apparatus (24 samples/2 min) and placed directly into a gamma counter for counting. In the present study, we have developed the assay to measure secreted IgG, IgM and IgA, and we have compared our results with those of a standard radioimmunoassay technique. This rapid, simple, economical approach should be applicable to the radioimmunoassay of other substances as well.

A simple technique for enhancing the sensitivity of a commercial solid-phase radioimmunoassay for hepatitis B surface antigen (HBsAg).

A simple procedural modification of a commercial solid-phase radioimmunoassay technique resulted in increased sensitivity for detecting HBsAg. The main feature of the modification consisted of agitation of the sample and solid-phase antibody during the first incubation. This was achieved by dispensing the bead and sample into a vial and rotating the vial gently during the incubation period. The modified technique resulted in the detection of HBsAg in the range of 1.0 to 1.2 ng/ml (ay subtype) and 0.16 to 0.38 ng/ml (ad subtype). Samples that were reactive by the rotation technique could be shown to be specific by using an immunological neutralization test. Plasma as well as serum could be tested by the rotation technique and gave comparable results when samples were incubated at 45 C or at room temperature. Three (1.3%) of 227 donor samples containing anti-HBc as the sole hepatitis B marker were specifically reactive for HBsAg by the modified procedure, although no sample was reactive by the standard technique.

Antitreponemal IgE in early syphilis.

Using a solid-phase radioimmunoassay technique, mean serum IgE concentrations were found to be raised in patients with early syphilis. Antitreponemal specificity of the IgE response was investigated by the fluorescent treponemal antibody absorption test using a fluorescein-isothiocyanate-labelled antiserum against the Fc-fragment of human IgE. Validity of this test procedure was assessed by blocking experiments. The results provide evidence of the antitreponemal specificity of the IgE response in syphilis and indicate a possible role for antitreponemal IgE in the pathogenesis of the Jarisch-Herxheimer reaction and in the immune-complex origin of some of the lesions of secondary syphilis.

Clinical significance of total serum IgE determination in screening of Iranian Russian thistle pollen hypersensitive individuals.

Total serum IgE concentrations of normal and Russian thistle (Salsola pestifer) pollen hypersensitive individuals were determined in an Iranian population, using the solid phase radioimmunoassay technique. The mean values were 963.7 and 885.3 ng/ml, respectively, which are much higher than normal values given for non-Iranian populations. In addition, no significant differences were found between observed values (P = 0.1) for normal and allergic individuals. The effect of common parasitic infestation in the Iranian population in causing high serum IgE levels is discussed.

Radioimmunoassay for Gram-negative bacterial lipopolysaccharide O antigens: influence of antigen solubility

We have developed a solid-phase radioimmunoassay technique for specific gram-negative bacterial lipopolysaccharide (LPS) O antigens. The method exploits the high-titer, specific immunoglobulin M response of the rabbit to LPS immunization to measure as little as 5 ng of homologous LPS per ml with less than 0.5% cross-reactivity toward heterologous LPS or culture supernatants. We found that O antigen in complete LPS was less available for antibody binding than O antigen in the soluble polysaccharide derived by mild acid hydrolysis of LPS and that triethylamine-induced disaggregation of complete LPS increased its activity in the assay. Quantitation of O antigen with the assay was thus influenced by the physical state of LPS or "free" O antigen.

Persistence of antibodies to rotavirus in human milk

Human milk obtained from 21 American nursing mothers was studied for the presence of secretory immunoglobulin A antibody to rotavirus, the most common etiological agent of infantile gastroenteritis. Antibody was quantitated by adaptation of a recently described solid-phase radioimmunoassay technique that employs simian rotavirus as a convenient substitute antigen for human rotavirus. Of the mothers tested, 80% (12 of 15) possessed milk antibody within a week of parturition, whereas 56% of those tested (5 of 9) secreted milk antibody as late as 6 or 9 months postpartum. Specificity of the radioimmunoassay was demonstrated by absorption of antibody with purified rotavirus. Our detection by radioimmunoassay of antibody to rotavirus in milk samples collected past the colostrum stage is in contrast to other studies that have failed to detect antibody in human milk by immunofluorescence or neutralization tests. The present study also suggested that the appearance of secretory immunoglobulin A antibody in the milk of mothers previously lacking milk antibody may be correlated with subclinical infection of the mother with rotavirus.