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Biomedical subjects

C Wadsworth

Publications and source records attributed to C Wadsworth.

At least 19 recordsLinked to original sources
PubMed1989

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates.

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.

Blood Proteins↗
PubMed1985

A critical analysis of commercially available latex particle reagents for C-reactive protein (CRP) slide agglutination tests.

C-reactive protein (CRP) was assessed in pediatric serum samples using different commercial latex reagents, which were analyzed for species origin of the coating antibodies, homogeneity and density of the latex particles, and prozone agglutinating capacity. All reagents correctly agglutinated the positive and negative control sera. The antibodies coating the particles differed with regard to species origin: one was coated with rabbit, one with horse and goat, one with horse, goat, rabbit and swine, while the reference reagent had horse, goat and rabbit antibodies. Only the monospecies specific antibody-coated latex showed obvious prozoning; this reagent also had the smallest and most homogenous latex particles and showed the most clear-cut reactions. False agglutination was observed at 7-26% according to quantitation with the spot immunoprecipitate assay, which compared favorably with radial immunodiffusion measurements. The lowest percentage of false readings was noted for the rabbit antibody-coated particles; the highest for the reagent with particles coated using antibodies from 4 different species. No reagent had satisfactory precision for the low positive sera between 10 and 40 mg CRP/l.

C-Reactive Protein↗
PubMed1985

Complexes of IgG and plasma proteins in factor VIII preparations--a possible cause of adverse reactions.

IgG in factor VIII preparations was studied as a possible cause of adverse reactions following infusion of hemophiliacs. Thin-layer immuno-gel filtration analysis of nine products from seven firms demonstrated aggregated and monomeric IgG at highly variable amounts and proportions. These factor VIII products contained from 20 to 700 mg IgG per 1000 IU of factor VIII; IgG-fibrinogen complexes were demonstrated by double-antibody testing. A relationship is suggested between aggregated IgG and/or IgG complexes in factor VIII concentrates and adverse clinical reactions. They may also partially explain abnormalities and aberrations of the immune system previously reported in hemophiliacs.

Blood Proteins↗
PubMed1984

Efficacy of latex agglutination and quantification methods for determination of C-reactive protein (CRP) in pediatric sera.

Described is a procedure for serum C-reactive protein (CRP) determination, consisting of a semiquantitative rapid CRP latex agglutination test, using dilutions of the serum, and the quantitating spot immunoprecipitate assay (SIA). These methods are performed with standard laboratory equipment using no more than 30 microliters of serum for both assays. With visual inspection, the SIA results are available one to two hours after blood sampling. CRP levels obtained by agglutination testing with five batches of latex reagents coated with rabbit anti-CRP agree well with the quantification, i.e. essentially 100% for the negative sera and about 80% for positive sera containing greater than 40 mg CRP/l. The remaining 20% of the samples are classified as low positive at 10 to 40 mg/l on agglutination. False positive or negative agglutination findings are below 2.3% with concordance at 88% between SIA and the CRP agglutination with rabbit antibodies. Two lots of CRP-latex reagents coated with sheep antibodies, however, gave 15.3% and 10.1% false positive findings and poor concordance with SIA ratings, particularly for low positive sera at only 20 and 29%. SIA is suggested for CRP quantification because it compares well with radial immunodiffusion in accuracy (less than 91%) and provides results in 2 h rather than 1-2 days. Rocket electroimmunoassay is less reliable with lower ratings than found in SIA, probably due to the electrophoretic heterogeneity of CRP. This is demonstrated for two of three purified CRP preparations, for which varying agglutination is seen. The combination of methods is especially recommended for diagnosis and monitoring of CRP in infectious processes in neonates and infants because of the required small sample volume--0.5 ml heel-prick blood--the rapidity of reliable (greater than 80%) reporting and the possibility of rating sera with moderate levels of CRP.

Animals↗
PubMed1983

Spot immunoprecipitate assay in gel compared with immune reactions in solution and applied to antibody titration.

Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable (r = 0.9979) working range for quantification between 0.1 and 0.83 microgram protein in 3 microliters samples (greater than or equal to 0.03 g/l). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8-9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 microliters sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.

Albumins↗
PubMed1983

Mucosal immunity.

Mucosal defense is provided by a number of host factors countering the specific virulence factors of the many microorganisms infecting the mucous membranes. Secretory IgA antibodies presumably play an important role. Increase of the sIgA antibodies may most advantageously be attained by parenteral immunization, following mucosal priming. This was demonstrated in a rat model, where it was also noted that antigen injection into PP induced high milk IgA antibody levels. In man, parenteral vaccination against polio increased the sIgA antibody levels in the milk of mothers previously exposed naturally to the poliovirus. The response was relatively short-lived. In the previously unexposed, there was little or no response. By contrast peroral immunization with live poliovirus vaccine did not increase, or even decrease, the milk sIgA poliovirus antibody levels. Although salivary sIgA antibodies against antigens of colonizing E. coli appear during the first days of life, they are slow to increase. This deficiency is richly compensated for by all the sIgA antibodies that are provided the baby through the milk. No transfer of dimeric IgA into the milk could be shown in lactating rats, in contrast to what has been reported in mice. There is no evidence for a contribution to milk sIgA from serum in man. Close to parturition, human milk often contains some 7S IgA and various sizes of free SC, in addition to the dominating 11S sIgA. A few days later there is almost exclusively monomeric SC and 11S sIgA. IgG antibodies also play a role at the mucosal level. IgG2 antibodies against the bacterial polysaccharide capsule are as slow to appear as sIgA in ontogeny, possibly explaining the prevalence of infections with encapsulated bacteria and the poor response to polysaccharide vaccines in early childhood. Other defense factors preventing infections by way of mucous membranes may be important. Thus, oligosaccharides present in human milk seem to specifically prevent pneumococcal attachment to retropharyngeal cells. This anti-attachment capacity, in addition to that provided by milk and salivary IgA antibodies, may explain why breast-fed babies have less otitis media than formula-fed ones.

Adult↗
PubMed1983

Intramuscular and intravenous administration of immunoglobulin to patients with hypogammaglobulinemia.

Hypogammaglobulinemia patients, who seem to be more numerous than previously known, have an unacceptable diagnostic delay of 12 years. They often get insufficient Ig prophylaxis, frequently due to the side effects caused by the im administration. A new Ig preparation for IV use has been utilized, which is much more acceptable to the patients, causing very few side effects. In contrast to im preparations, IV immunoglobulin can be used at high doses, quickly normalizing the patient's serum IgG level, and seems to permit longer intervals between infusions during long-term prophylaxis.

Agammaglobulinemia↗
PubMed1979

Immunoglobulins G, A and M in normal and pathologic human sera determined with the spot immunoprecipitate assay (SIA) in multi-assay plates.

A simple rapid spot immunoprecipitate assay (SIA) is presented. The antigen--antibody reaction takes place in a field of alternating current; small amounts of specific antisera are used, and the influence of diffusion or electromobility of the antigen is avoided. Determinations of IgG, IgA and IgM in sera from healthy individuals correlated highly significantly with radial immunodiffusion (RID) results. The reaction conditions in SIA, particularly the availability of a sufficiency of specific antibodies, may permit more accurate estimates of monoclonal forms of Ig than in RID, where the combination depends on diffusion of one reactant. The precision of SIA quantitation of IgG and IgA was within +/- 10% and +/- 13% for IgM. Working ranges of the standard curves were from about 25--150 ng for G and A and 75--300 ng for IgM; least squares regression coefficients for four sets of standards on three SIA plates were 0.995 for IgG, 0.969 for IgA, and 0.986 for IgM. IgA at 7.5 ng in a 2.5 mg/l dilution was at the lower limit of demonstrability. Photometric registrations and estimates made by visual inspection compared highly significantly (P less than 0.001), differing by +/- 3%.

Antigen-Antibody Reactions↗
PubMed1979

C-reactive protein (CRP) in early diagnosis of neonatal septicemia.

The usefulness of CRP in early detection of neonatal septicemia/meningitis and urinary tract infection was studied in a neonatal unit using a semiquantitative latex-agglutination as a rapid screening method, and electroimmuno assay as reference method for CRP determination. In 94% of non-infected infants CRP was less than or equal to 15 mg/l and 82% had CRP less than 10 mg/l up to 3 days of age. After 3 days of age 96% had CRP less than 10 mg/l. The initial CRP level was increased in 16 out of 18 patients (89%) with bacterial septicemia. Low CRP was seen in one patient with total agranulocytosis and septicemia from Streptococcus type B and in one patient with Staphylococcus albus sepsis. A rise in CRP was also seen in very pre-term infants with septicemia. Increased initial CRP was uncommon in neonatal urinary tract infection (2 of 9), but a rise was seen in 3 additional patients. A comparison between CRP, total neutrophil blood cell count and band neutrophil count as diagnostic parameters was in favour of CRP at this early stage of infection. CRP is of definite value as an aid in early diagnosis of neonatal septicemia and bacterial meningitis.

C-Reactive Protein↗
PubMed1977

A new specific quantitation-in-gel method differentiating commercial human serum standards intended for RID analyses.

A rapid, sensitive technique is described which measures small amounts of protein applied on agarose gel containing specific antibody. Alternating current through the gel reduces the influence of diffusion, and specific immunoprecipitate spots are formed. Their density after staining is proportional to applied protein. Quantitation of IgG and IgA in human serum standards was highly reproducible (P less than 0.001, Spearman coefficient of rank correlation test), and as little as about 35 ng Ig could be detected. Results are available within hours. Human sera and some purified preparations of 7S IgG and secretory IgA were grouped according to immuno-gel filtration findings. The group of samples containing fragments, aggregates, or unusually small amounts of monomeric IgG or IgA was also differentiated by specific immunoprecipitate spot (SIS) analysis from those mainly monomeric in character (Wilcoxon rank test, P less than 0.02). A World Health Organization reference serum (67/97) was used as standard. The study indicates that a human serum pool stored in aliquots at -20 degrees C is a good standard for quantitating serum IgG and IgA and that purified preparations are no better. It is suggested that the SIS assay could be advantageously applied to screening biologic fluids for unusual amounts or types of IgG and IgA.

Chemical Precipitation↗
PubMed1977

A rapid spot immunoprecipitate assay method applied to quantitating C-reactive protein in pediatric sera.

A simple, rapid spot immunoprecipitate assay (SIA) technique is compared with the rocket electroimmunoassay and a latex agglutination method for determination of C-reactive protein (CRP) in pediatric sera. The SIA reaction conditions are special, since the antigen is made passively available in a gel for reaction with an abundance of antibody. In the present study the antigen-antibody interaction occurs in the presence of alternating electric current. The reaction is visualized by protein staining and quantitated by comparison with simultaneously run standards. Highly significant (P less than 0.001) Spearman's coefficients of correlation between SIA and rocket results were found along with evidence of discrepancies between the methods which could be related to the heterogeneity in electric charge of the CRP molecules. In this respect some sera with negative (greater than or equal to 10 mg CRP/l) rocket results had two- to ten-fold higher SIA ratings. In contrast to the rocket method, SIA could differentiate sera that were negative and low positive in agglutination. Some discrepancies between an agglutination technique and SIA estimates were noted in low-positive sera differing in electromobility from the cathodic traveling CRP standard. Reproducibility of photometrically registered SIA was +/-16.2%; visually evaluated it was +/-9.7%. As little as 0.5 ng CRP was demonstrable in a human serum pool dilution at 0.17 mg/ml.

C-Reactive Protein↗
PubMed1976

A simple stained protein assay (SPA) for rapid total protein determination applied to spinal fluids.

A rapid, simple technique measures small amounts of total protein applied on an agarose gel slab. Alternating current through the gel may reduce diffusion of the protein, which is chemically fixed. The intensity of the Coomassie-Blue-stained spots is a function of the amount of protein determined by comparison with simultaneously run standards. Densitometric and visual estimates of protein in spinal fluids by the stained protein assay correlated with Lowry values highly significantly (n = 28; P less than 0.001, Spearman test of rank correlation). As little as 0.2 mug protein was measured; this figure should be compared with 10 mug for the Lowry method. The reproducibility of the method was +/- 5%.

Adolescent↗
PubMed1976

Suppressor cell activity in a male infant with T-and B-lymphocyte dysfunction treated with thymosin.

A male infant with bilateral iris coloboma who had had repeated infections and malabsorption was studied. The levels of total lymphocytes and of T and B cells were normal or high, but IgA became undectable and IgG low, whereas IgM was normal. His lymphocytes did not respond to phytohemagglutinin (PHA), concanavalin A, pokeweed mitogen (PWM) or in mixed lymphocyte reactions (MLR), nor did they respond in vitro when thymosin was included in the test systems. He was skin-test-negative, even to dinitrochlorobenzene. His crudely isolated T lymphocytes and the supernatant of his PHA-stimulated lymphocytes inhibit the response of normal lymphocytes to PHA, PWM, and in MLR. During thymosin treatment skin test and lymphocyte reactivity to mitogen remained negative. He became faintly positive in MLR, and the suppressor activity in the supernatant of his PHA-stimulated lymphocytes no longer inhibited the response of normal lymphocytes to PHA, PWM, or in MLR. In parallel with thymosin treatment he showed quite marked clinical improvement.

B-Lymphocytes↗
PubMed1976

A Clq immunosorbent assay compared with thin-layer gel filtration for measuring IgG aggregates.

A new sensitive technique measures C1q-binding of human IgG aggregates. The method is based on the principle of the enzyme-linked immunosorbent assay with C1q-coated tubes. The IgG aggregates attaching to this C1q are shown by enzyme-linked anti-human IgG. Less than 0.01 mug of aggregates per milliliter of sample can be detected. The results with this new method showed significant correlation (P less than 0.01; Spearman rank correlation test) with the estimates of IgG aggregates of 13S or more and 10S in size obtained by thin-layer gel filtration. Both of these methods showed significant correlation with the classic hemolysis inhibition method for measuring complement fixation.

Chromatography, Gel↗
PubMed1976

IgA in commercial gamma-globulin preparations.

The presence of anti-IgA has been related to adverse reactions to injection or infusion of IgA-containing material. In this study IgA was demonstrated in all of the investigated commercial gamma-globulin preparations. In the material from a few producers it was, however, very low. By immuno-gel filtration it could be shown that the IgA consisted of aggregates as well as a 7S component and fragments. By immunoelectrophoresis-immunofluorescence IgG-IgA complexes could also be detected. The IgA aggregates could be almost completely degraded by reduction-alkylation. It is implied that such changed IgA in gamma-globulin preparations for injection may increase the risk of immunization against IgA.

Fluorescent Antibody Technique↗
PubMed1976

Estimation of aggregates of IgG and their effect on quantitation of IgG. I. A comparison of four quantitating methods.

Four quantitating techniques - radial immunodiffusion (RID),electroimmunoassay (EIA), thin-layer Sephadex gel filtration (GF), and GF with an added, specific immunoprecipitation step, immuno-gel filtration (IGF) - are compared for their accuracy in quantitating IgG of various molecular sizes - that is, pure 7S IgG and aggregated IgG, from large polymers to dimeric 10S molecular size. The aggregate fractions differed and were stable with regard to their migration in thin-layer gel filtration, their electromobility in agar and agarose, and their diffusibility in agarose. All four methods quantitated the largest aggregates four to five-fold lower and the next largest at no better than half the Lowry ratings. Only GF and IGF quantitated the two fractions of smaller aggregates within 10% of the expected value. The presence of about 50% of the two larger aggregates in prepared mixes with 7S IgG reduced quantitation results by about half to two thirds with all four methods; the smaller aggregates at 50% concentration were estimated within 16% of the correct values. Twenty percent aggragated IgG about the mean amount found in commercial gamma-globulin preparations, influenced measurments with RID, GF and IGF by less than 10% and the results with EIA by less than 16%.

Chromatography, Gel↗