Immunoelectrophoretic Techniques Research Articles

False spurs in quantitative immunoelectrophoresis.

Antigens which show reactions of identity in double-diffusion tests in gel may cause spur formation in crossed immunoelectrophoretic techniques. To differentiate these misleading spurs from spurs of double-diffusion tests which show the true immunochemical relationship between antigens, we have denoted the former type "false spurs". False spurs are often formed in tandemcrossed immunoelectrophoresis and fused rocket immunoelectrophoresis when antigens with different electrophoretic mobility are compared. On the basis of model experiments, criteria are set up for accepting a spur as a true spur, and procedures are described for avoiding formation of false spurs in quantitative immunoelectrophoresis.

Factor IX in warfarin treated patients.

Abstract The presence of inactive factor IX molecules (acarboxy-IX) was demonstrated in plasma from warfarin treated patients by immunoelectrophoretic techniques using a precipitating rabbit antiserum against human factor IX. The level of factor IX activity (mean 0.42 units/ml) was lower than the level of factor IX antigen determined by an electroimmunoassay technique (mean 0.70 units/ml) in 34 of the 35 patients studied. The percentage Thrombotest values correlated positively with the level of factor IX activity (r=0.74) and negatively with the relative amount of acarboxy-IX (r=−0.54). Crossed immunoelectrophoresis in the presence of calcium ions of patient plasma revealed a factor IX antigen with a faster electrophoretic mobility than that of normal factor IX. Factor IX antigen from the patient with the largest amount of acarboxy-IX had a lower affinity to BaSO 4 than factor IX antigen from normal plasma. Neutralization of factor IX in the patient plasma samples by antiserum gave increased percentage Thrombotest values but had no effect on the Normotest values. The effect of the antiserum on the Thrombotest values was about the same on the patient plasma samples as on undiluted and diluted plasma from normal persons.

A study of acquired inhibitors of factor IX by means of precipitating rabbit antisera against factor IX

Plasma from three patients with hemophilia B and acquired factor IX inhibitors interfered with the reaction between rabbit antiserum against factor IX and factor IX in immunoelectrophoretic techniques. The inhibitors did not precipitate factor IX in diffusion-in-gel experiments, but behaved like IgG on DEAE ion chromatography. Incorporation of inhibitor plasma in the agarose gel in the first dimension in crossed immunoelectrophoresis reduced the electrophoretic mobility of factor IX antigen. The reduction of the mobility was dependent on the concentration of inhibitor plasma in the agarose. Incorporation of inhibitor plasma in an intermediate gel in “rocket” immunoelectrophoresis gave a concentration dependent reduction of the size of the “rockets”. Both techniques could be used for determination of a titre of the inhibitors which was in agreement with the titre determined in a coagulation assay.

Ferritin labeling of concanavalin A by glutaraldehyde. Comparison of different molar con A/ferritin/ glutaraldehyde ratios and characterisation of the products

Optimal conditions were investigated for preparation and isolation of monomeric Con A ferritin, with one molecule ferritin bound to one tetravalent molecule of Con A. Con A and ferritin were covalently coupled with highly purified glutaraldehyde as a bifunctional reagent at different molar ratios. The effect of four different concentrations of glutaraldehyde and of two different ratios of 125I-Con A to ferritin on the yield of Con A-ferritin monomers were tested in one-step reactions. The different products were subjected to linear sucrose density gradient centrifugation and the amount of 125I-Con A or ferritin or conjugates in the fractions obtained were measured by optical density at 280 nm (protein) and 310 nm (ferritin), gamma-counting ( 125I-Con A) by quantitative immunoelectrophoretic techniques (free, ferritin, ferritin bound to Con A), and by counting relative numbers of ferritin monomers, dimers, trimers and larger oligomers under the electron microscope. In the 30–56% w/w sucrose gradient unlabeled Con A was optimally separated from ferritin or ferritin-labeled Con A monomers. Ferritin monomers and Con A ferritin monomers could be recovered at a sucrose concentration of 42 to 49% w/w. A high molar ratio of Con A to ferritin during the coupling reaction caused formation of high molecular weight Con A-polymers. Optimal yield of Con A-ferritin monomers was obtained with an equimolar ratio of Con A to ferritin, a glutaraldehyde to ferritin ratio of 26 : 1 and a final ferritin concentration of 310 nmoles/ml.

Immunochemical specificity of a benzidine technique proposed to catalases histochemical detection

As an attempt to test the specificity of an histochemical technique proposed to detect catalases, an investigation was carried out by immunochemical techniques. Purified catalases were used after analysed immunochemically by double immune diffusion test and immunoelectrophoretic technique. These pure catalases induced, after injecting into guinea-pigs, anti-serums that react specifically with catalase and does not give any cross reaction with peroxidases and haemic iron containing compounds. By the direct and indirect immuno-fluorescence techniques it was shown an intense catalase reactivity inside the cytoplasm of adrenal cortex and hepatic cells, that appears as a granular pattern. These results are very similar to those provided by the histochemical technique, either concerning to the reactive cells or to the granular pattern of the positive reaction. In such instances, the immunochemical results suggest the specificity of the histochemical reaction. This specificity is confirmed by the previous treatment of tissue sections by catalases anti-serum. After this treatment either the immunochemical or the histochemical technique to detect catalases provide negative results on cells that before the treatment were strongly reactive.

A Practical Method for Quantitation of Antibody Responses to Tetanus and Diphtheria Toxoids in Normal Young Children

Antibodies to diphtheria and tetanus toxoids were assayed in 46 children from 16 to 33 months of age following vaccination. A reversed rocket immunoelectrophoretic technique was used for antibody determination. This method is simple and consistent, and although the limit of detection for antibodies of both types (about 1 U/ml) is above the normal range for primary immune responses, antibody levels in immunized children five weeks after a second injection of vaccine were within measureable range in all but two of the children studied. Levels more than one standard deviation above the mean were found in one child for both antitetanus and anti-diphtheria and in four children for anti-tetanus toxoid antibody only. All four of the high responders to tetanus toxoid were Black, carrying both Black and Caucasian Gm haplotypes. The two apparent nonresponders were Caucasian. All children had normal immunoglobulin levels, except for one with low IgA, who showed normal antibody responses to both toxoids. The results suggest that measurements of precipitating antibodies to diphtheria and tetanus toxoids provide a practical index of humoral immune function in small children.

Crossed immunoelectrophoretic and crossed radioimmunoelectrophoretic studies employing a model allergen from codfish.

A standard reference pattern in crossed immunoelectrophoresis (CIE) of the parvalbumin fraction of a cod white muscle extract was established. Crossed line immunoelectrophoresis (CLIE), tandem CIE and crossed radioimmunoelectrophoresis (CRIE) were used to identify the major allergens, thereby establishing a basis for future work employing quantitative immunoelectrophoretic techniques in the study of codfish allergens. Rocket immunoelectrophoresis and rocket-line immunoelectrophoresis were used to demonstrate antigenic determinants on polypeptide fragments of the major allergen, Allergen M. The results show the usefulness of these techniques in studies of antigenic determinants on non-precipitating polypeptides. CRIE with 8 patients's sera showed radiostaining indicating IgE binding corresponding to at least 7 precipitates in the crude extract CIE preparations. The purified DS 22 fraction was shown to contain 2 CRIE-positive (IgE-binding) precipitates. When using Allergen M, these precipitates were also demonstrated, one of them in trace amounts only.

Proteins from disassembled microtubules characterized by oligospecific antisera

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.

Problems of conductor demonstration in haemophilia A: error possibilities in the determination of factor-VIII-associated antigen

Difficulties in the detection of haemophilia A carriers by the determination of the ratio factor VII coagulation activity to factor VII antigen concentration are shown in part one of our investigations. The second part evaluates methodical parameters of the quantitative immunoelectrophoretic technique. According to our results reliable data can only be obtained under the following conditions: 1. At least three determinations of each sample are required and the mean value should be used as the final result. 2. Each gel plate should be calibrated separately 3. The calibration line should include values less than 100%. Factor VIII concentrates are not suitable for determination of the calibration curve, as higer dilution results in lower calculated concentration of the undiluted concentrate. Storage of pooled normal plasma at--25 degrees C is possible without significant loss of antigen concentration over 5 months. However, plasma samples of a single person show an unpredictable variation in the antigen concentration during storage over the same time.

ORIGIN OF A DOMINANT BEER PROTEIN IMMUNOCHEMICAL IDENTITY WITH A β-AMYLASE-ASSOCIATED PROTEIN FROM BARLEY

Two-dimensional immunoelectrophoretic techniques were used to elucidate the origin of a dominant beer antigen 1. Immunochemical identity was found between a protein Z associated with β-amylase in extracts of barley and one form of the beer antigen 1a. Immunochemical identity was also found between a papain-modified form of protein Z and the antigen form 1b from beer. The results indicate that protein Z is very resistant to degradation and denaturation during malting and brewing.

Isolation and characterization of proteins associated with the lung surfactant system.

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretic techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.

Free and Protein‐bound β‐Amylases of Barley Grain. Characterization by Two‐dimensional Immunoelectrophoresis

AbstractThe β‐amylases of ungerminated barley (Hordeum distichum L. cv. Emir) were characterized by two‐dimensional immunoelectrophoretic techniques in order to elucidate the structure and physiological importance of the latent β‐amylase present in cereal grains. Two water‐soluble forms with partial immunochemical identity were detected. One of the enzyme forms was found to consist of aggregates between β‐amylase and an immunochemically distinct non‐active protein. Both β‐amylase and the protein could be released from the aggregates with β‐mercaptoethanol and, to some extent, with papain. β‐Mercaptoethanol increased the extractability of β‐amylase and of the non‐active protein. The aggregated form of β‐amylase was found to predominate in extracts made in the presence of papain. Possible functions of the non‐enzymatic protein in formation of latent β‐amylase is discussed.

Differentiation of Bwamba and Pongola viruses by agar-gel diffusion and immunoelectrophoretic techniques.

The agar-gel diffusion (AGD) and the immunoelectrophoretic (IEP) techniques were employed in the differentiation of virus strains of the Bwamba-Pongola group. These techniques were found to be more specific than either the complement fixation (CF) or the neutralization (N) test in the differentiation of Bwamba and Pongola viurses. Of 22 virus strains isolated in Nigeria and previously typed as strains of Bwamba virus by CF and N tests, 12 strains of mosquito origin were definitely classified as Pongola viruses and 10 of human origin as Bwamba viruses by the AGD and IEP techniques.

Alpha-l-antitrypsin Deficiency and Liver Disease in Adults

Thirteen adult patients (aged 16 to 73 years) from 12 families are described with liver disease and alpha-l-antitrypsin deficiency. Long-term observation of several of these patients suggests that the liver disease may be only slowly progressive, but review of possible factors aggravating this has failed to reveal any obvious clues. Progression to death from hepatic failure was the commonest outcome, but one patient developed a malignant hepatoma and two others died because of intraperitoneal haemorrhage due to ruptured cirrhotic nodules—a complication not hitherto described in association with this condition. Diagnosis of alpha-l-antitrypsin deficiency was based on serological, histological, immunopathological and genetic studies. The most useful screening test in liver disease was found to be the demonstration of PAS positive globules in liver biopsy material which is diagnostic when shown to be antigenically identical with alpha-l-antitrypsin by immunofluorescence or immunoperoxidase, the latter being a superior technique. Serum estimation of alpha-l-antitrypsin deficiency was performed by immunoelectrophoretic and immunodiffusion techniques, the former being preferred because it gave more consistent results. Both methods, however, were of limited value since wide variations in the serum values are commonly found in normal and abnormal states. Genotyping was carried out using starch gel electrophoresis and although of value in family studies, its value as a diagnostic aid is limited because of technical difficulties and also because alpha-l-antitrypsin accumulation in the liver may be found in both homozygous and heterozygous states. It is suggested that adult liver disease associated with abnormalities in alpha-l-antitrypsin may be more common than has hitherto been reported. This condition should be systematically sought in all cases of liver disease of uncertain aetiology.

Plasma protein profiling: The diagnostic evaluation of disorders in plasma protein composition by a new immunoelectrophoretic method

A new electroimmunoprecipitation technique is presented by means of which a great variety of antigens e.g. plasma proteins can be simultaneously and quantitatively determined with a single-step electrophoretic separation. The essential features of the new technique are: 1. (a) subdivision of the antibody gel into gel strips containing monospecific antibodies to individual plasma proteins. 2. (b) sample application as a “sample gel” filling a trough over the width of the immunopiate. Quantitation is based on the fact that the distance an antigen can migrate within a gel containing a defined amount of specific antibody directed against the antigen is determined by the concentration of the appropriate antigen within the sample. The area where the antigen is finally completely consumed by immunoprecipitation and antibody present in excess is sharply delineated. The applicability of the method in simultaneous quantitative determination of 15 plasma proteins is demonstrated with plasma from healthy blood donors and patients with various diseases. The advantage of the new technique as compared to commonly used clinical acetate folia electrophoresis is the high degree of specificity for the determination of a great number of individual, diagnostically meaningful plasma proteins. The advantage over common quantitative two-dimensional immunoelectrophoresis is its uncomplicated way of evaluation. The potential clinical application of the new quantitative immunoelectrophoretic technique in diagnostic screening and differential diagnosis is discussed.

Disc-electrophoretic protein pattern of cervical mucus in cases of humoral sensitization against spermatozoa.

Midcycle cerevical mucus samples from 20 fertile women with negative, and from 6 infertile women with positive serum-sperm agglutinating activity were subjected to a qualitative protein analysis by the Ouchterlony-technique, to polyacrylamide (PAA) discelectrophoresis and after purification on spermatozoa they were also run against rabbit antihuman serum in the Laurell immuno-electrophoretic technique. By the Ouchterlony method the main serum protein fractions were shown to occur also in cervical mucus. No significant differences could be found in the protein pattern of samples from fertile and infertile females as detected by polyacrylamide (PAA) electrophoresis. In all samples a split protein fraction corresponding to serum albumin and a band preceding the protein fraction corresponding to serum preablumin were observed. In 2 samples from infertile females it could be demonstrated that immunoglobulins occurring in the cervical mucus were directed towards antigenic sperm components.

Application of pepsin-digested horse antibodies for quantitative immunoelectrophoresis

The majority of the precipitating antibodies in hyperimmune horse serum belong to the beta globulins, as demonstrated by reversed immunoelectrophoresis. As these antibodies migrate in agarose gel during electrophoresis in conventional pH = 8.6–8.9 buffers, horse antiserum cannot be used satisfactorily for quantitative immunoelectrophoresis. On pepsin digestion of horse antiserum F(ab)′ 2 fragments are generated which migrate with the gamma globulins. This cleaved product works well in quantitative immunoelectrophoretic techniques.

Laboratory Diagnosis of Bence Jones Proteinuria in a Patient with Plasma Cell Leukemia

Kappa Bence Jones proteinuria was found by immunoelectrophoretic techniques in a patient with plasma cell leukemia, who presented with no M-proteins either in serum or urine. However, a significant decrease in normal immunoglobulins was observed. On microscopic examination of kidney sections obtained a autopsy, protein casts were seen in the distal tubules. Bence Jones protein was not detected by the conventional heat test.

THE SEROLOGY OF PSEUDOMONAS AERUGINOSA ANALYSED BY MEANS OF QUANTITATIVE IMMUNOELECTROPHORETIC METHODS

Serologic cross-reactions between 26 strains of Pseudomonas aeruginosa representing 13 O groups were studied by various quantitative immunoelectrophoretic techniques. As reference system was used a polyvalent Ps. aeruginosa antigen and a corresponding rabbit antiserum. Fifty-one (93 per cent) of the 55 Ps. aeruginosa antigens in the reference system were present in all the strains and corresponding antibodies in the reference system could be completely absorbed by all the strains. Complete cross-reactivity was also found between antigens of the reference system and 3 of the 4 antigens present only in some of the strains. The last of the 4 antigens not present in all the strains could only absorb part of the corresponding antibodies in the reference system. Absorption experiments with whole heat-killed bacteria indicate that this antigen is related to the O group antigens of Ps. aeruginosa. None of the antigens of the reference system were related to the mucoid substance produced by some strains of this bacterium.