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A method for the quantitative determination of plasma concentrations of ampicillin and cloxacillin in mixture is described. The antibiotics were separated by high voltage paper electrophoresis. The high affinity of cloxacillin for albumin seriously interfered with the electrophoretic separation of the antibiotics and the quantitation of cloxacillin. Therefore, prior to electrophoresis, the plasma proteins were digested with trypsin to release cloxacillin. Subsequent to the electrophoretic separation the antibiotics were developed micro-biologically by agar diffusion using the separated spots on the paper as diffusion centres. The inhibition zones were clearly visible after staining and could be quantitated. The method was evaluated statistically and showed good agreement with the paper disc diffusion method.

A subfragment (peak IV) of Fc from human IgG belonging to the CH3 region and found to be slightly different from both pFc and tFc' fragments was released after tryptic digestion of acid-treated Fc. Gel filtration at dissociating conditions of reduced and alkylated fragments indicated a molecular weight of 10,200. The Fc, pFc', tFc', and peak IV fragments all interacted with a rheumatoid factor having anti-Gm(1) specificity and showed a reaction of identity in agar diffusion against both anti-Fc and anti-pFc' sera. In immunoelectrophoresis, peak IV and tFc' fragments behaved similarly but with a more anodical movement than pFc' fragments. The probable location of the new fragment in terms of the sequenced gamma1-chain is proposed to be the residues 335 to 439.

Case report of a patient with chronic ulcerative dermatitis and monoclonal gammopathy. No signs of myeloma could be detected. The M-component was found to be of class G, L-chain type K and subtype 3.

Eighteen patients were studied, selected only on the basis of a persistently elevated cold haemagglutinin titre. The concentration of γM-globulin in the initial serum sample varied from 0.7–24.5 mg./ml. In all of 15 patients studied in detail, the cold haemagglutinin behaved as a monoclonal γM-globulin. In two patients, a homogeneous γ-globulin remained in the serum after absorption with red cell stromata in the cold. This was shown to be due to an additional monoclonal γG-globulin without cold haemagglutinin activity. The protein pattern remained virtually constant in each patient throughout the observation period.

Twenty-five patients with acute monosymptomatic optic neuritis were examined. Of primary interest were the changes in the cerebrospinal fluid as a possible indicator of future development of multiple sclerosis. The most common abnormality was mononuclear leuco-cytosis which occurred in 60 %. Other abnormalities were infrequent, which is in sharp contrast to findings in definite cases of multiple sclerosis. The total protein concentration was elevated in 6 of 25 cases (24 %) and the relative immunoglobulin G concentration was elevated in 4 cases (16 %). On agar gel electrophoresis bands in the gamma globulin region were found in 6 cases (24 %). Three patients have so far developed a definite multiple sclerosis.

Haemoglobinopathies are common in sub-Saharan Africa. As such haemoglobin electrophoresis are required to inform clinical decision making. However, haemoglobin electrophoresis is an assay that detects protein at either alkaline or acidic pH. Such assays do not interrogate gene sequences but rather the product of a gene. As many posttranscriptional and post-translational modifications impact the final output of the gene (i.e. protein), presentation of such protein-based assay must accurately reflect the technique employed. It is grossly misleading and scientifically inaccurate to report cellulose acetate and/or citrate agar haemoglobin electrophoresis results as ‘haemoglobin genotype’. We propose a new paradigm in which haemoglobin electrophoresis data would be presented as ‘haemoglobin phenotype’ at a specified pH. Funding : None declared Keywords : Electrophoresis, haemoglobinopathies, haemoglobin, cellulose acetate electrophoresis, citrate agar electrophoresis

Many Staphylococcus epidermidis strains possess capsule or slime layers and consequently the staphylococcal cell surface should be regarded as a soft, polyelectrolyte layer allowing electrophoretic fluid flow through a layer of fixed charges. The presence of such a soft layer decreases the energy barrier due to electrostatic repulsion in the interaction of the organisms with negatively charged substrata [Morisaki, H., Nagai, S., Ohshima, H., Ikemoto, E.Kogure, K. (1999), MICROBIOLOGY: 145, 2797-2802] and hence plays an important role in their adhesion. In this paper, the authors compare the electrophoretic softness and amount of fixed charge in the outer cell surface layers of 20 S. epidermidis strains, grown in a liquid medium or on a solid agar, as determined from the dependencies of their electrophoretic mobilities upon the ionic strength of a suspending fluid. Most of the staphylococcal cell surfaces were relatively soft, with a mean cell surface softness (1/lambda) for strains grown in liquid medium of 1.7+/-0.6 nm (standard deviation over all 20 strains) which is soft by comparison with a completely bald, peptidoglycan-rich streptococcal cell surface (1/lambda=0.7 nm). When the staphylococcal strains were grown on solid agar, the cell surface softness of 17 of the 20 strains increased, sometimes by a factor of two. On average for 20 strains, the cell surface softness increased significantly (P:0.05, Student's t-test) to 2.8+/-1.8 nm. The amount of fixed charge in the outer cell surface layer was -28+/-9 mM for bacteria grown in liquid medium and -24+/-12 mM for bacteria grown on agar. A soft, highly negatively charged polyelectrolyte layer was inferred by microelectrophoresis for all the staphylococcal cell surfaces, regardless of whether staining had indicated the presence of a capsule or slime layer.

We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions).Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF) followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin.In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by ∼50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%.Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.

Separation of proteins treated with sodium dodecyl sulfate (SDS) according to molecular size was achieved by discontinuous electrophoresis in vertical low-concentration agarose gels. A linear relationship was found between the migration distance and the square root of the molecular weight. This holds for proteins in the range of 7-200 kDa separated in 1.25% w/v agarose gel slabs (7 x 7 x 0.15 cm) with 0.1% w/v SDS and sulfate as leading ion. The linear regression coefficient was 0.998. The molecular weight and charge of coions influenced the separation. Small ions with low pKa values were found suitable as coions. The migration distance of proteins treated with SDS varied linearly with the agarose concentration of the gel. The agarose type and quality affected the resolution of the SDS-protein bands. We conclude that agarose gels can substitute polyacrylamide gels for the separation of proteins treated with SDS. A homogeneous agarose gel at a concentration of about 1% is a nonsieving support for electrophoresis. Therefore, the separation described here cannot be explained by the pore size of the gel. The results suggest that the separation is mainly due to the relative migration velocities of the coion and the proteins treated with SDS.

A gel electrophoretic method coupled with agar diffusion has been devised for detecting tumor antigens in human colon tissue. Separation of the antigens is achieved on duplicate electrophoretic gels. One gel is used for the location of the antigens by protein staining and the other gel is used for assaying of the antigenicity by agar diffusion against homologous antiserum. Analysis of perchloric acid extracts of colon tumors by this coupled method revealed the presence of carcinoembryonic antigen and two additional glycoprotein antigens. Analysis of KClHCl tumor extracts revealed two new tumor antigens.

Summary Neonatal screening for haemoglobinopathies utilizing cord blood samples is well established, although it has a high miss rate and has the inherent problem of possible misdiagnosis from maternal contamination of the sample. The use of dried Guthrie card samples which are taken at six days of age avoids these problems and has the advantage of using an established system of sample collection. Controversy exists as to the method of choice for analysis of dried samples, this study of 2406 samples found that Iso-electric focusing (IEF) analysis of dried specimens gives excellent correlation when compared with cellulose acetate/citrate agar electrophoresis of liquid cord blood samples. The IEF results were clear and relatively simple to interpret even when the samples had been stored at room temperature for 4 weeks. The commercial enzyme linked immunosorbent assay (ELISA) screening test JOSHUA reliably determines the presence or absence of haemoglobin S in dried specimens. It could therefore be used as a relatively cheap and simple method for the confirmation of sickle cell trait in neonatal screening programmes based on dried specimens.

Cryo- and pyroprecipitability of protein in the serum of a patient with multiple myeloma were both derived from the same polymerized IgA with lambda light chain, and were composed mainly of 16.1 S which migrated to the β2 region. A 6.7S component produced by reduction of the polymerized IgA did not reveal these properties. This suggests an important role of disulfide bonds in the conformation of IgA polymers with these thermoprecipitabilities.

The technique of counterimmunoelectrophoresis (CIE) has been adapted for the purpose of detecting and quantitating serum anti-DNA precipitating antibody. Ionagar No. 2 and Tris-HCl buffer were employed. Conditions for electrophoresis were 5.4 V per linear cm of agar, 20.0 mA per slide, and a 20 min migration interval. Pyronin staining was used to facilitate the presence of DNA in precipitin bands. Clear-cut, dense DNA anti-DNA precipitin bands were detectable by CIE using selected reference human sera from patients with systemic lupus erythematosus. Precipitin activity was absorbable with DNA and with a goat anti-IgG immunosorbent. Precipitins were also detected in ammonium sulfate-precipitated serum fractions and in IgG-rich fractions obtained by DEAE-cellulose chromatography. CIE would seem to offer advantages of simplicity and rapid reproducibility in screening large numbers of serum samples for anti-DNA precipitating antibody.

(1982). Hemoglobin Mobility in Citrate Agar Electrophoresis -Its Relationship to Anion Binding. Hemoglobin: Vol. 6, No. 2, pp. 199-208.

We have analyzed 140 hemoglobin variants in citrate agar electrophoresis and conclude that their separation from Hb A is greatly influenced by altered affinity of the anion binding site for citrate ions. We include the result of analysis in citrate agar electrophoresis of pyridoxylated Hbs A and S, both of which have the same mobility as Hb F.

We have described an application of a radioimmunoassay (RIA) method, known as radioelectrocomplexing (REC), which involves the anodal migration of antigen and the cathodal migration of antibody in agar electrophoresis. The agar is divided into zones of free antigen (DNP125I-HSA) and antigen bound with anti-DNP. Complete assays of anti-DNP can be performed in 2–4 hr since both immune complex formation and separation of free from bound antigen can be accomplished by electrophoresis in 60–90 min. Estimation of the weight of specifically-purified anti-DNP chicken antibodies in the nanogram range by REC is of the same order as the reported sensitivity of other RIA methods. The method was capable of demonstrating the higher avidity of the 17 S than 7 S antibody. Based on hapten inhibition the relative binding constants of DNP derivatives and anti-DNP were of the same order as reported from more definitive methods.

Blood glucose reacts with haemoglobin to form an unstable Schiffs base which is then converted by Amadori rearrangement to a stable keotamine [l]. This glycosylated haemoglobin (G Hb), formed over the life span of the erythrocyte, can be used as an indicator of long-term control in diabetic patients [2]. The glycosylation of haemoglobin and its clinical usefulness have been the subject of a number of reviews [3,4]. The methods available for the estimation of G Hb either measure the total HbAl (i.e. HbAla + b + c), or measure the main stable glycosylated fraction HbAlc after isolation from the other fractions. The former methods (including electrophoresis and mini-column ion-exchange chromatography) are more appropriate for use in routine clinical laboratories handling large work loads from diabetic clinics. However, agar electrophoresis [5] using commercially prepared agar plates and scanning densitometers is expensive although technically simple, and separation of G Hb by mini-column ion-exchange chromatography [6] is sensitive to pH and ionic strength of buffer and operating temperature. Affinity chromatography methods are claimed to be less sensitive to these factors [7]. We have investigated the affinity chromatographic medium Glycogel B (Pierce and Warriner, Chester, Cheshire, UK) and compared the results with those from commercially available kits based on agar electrophoresis and mini-column ion-exchange chromatography being used routinely in two other hospital laboratories.

In our hands the dianisidine technique for the staining of ceruloplasmin in polyacrylamide gels following disc electrophoresis has proven unsatisfactory for three reasons. First, staining of ceruloplasmin could not be achieved with physiological amounts of the glycoglobulin present in serum. Second, even when relatively massive quantities of pure ceruloplasmin permitted effective staining there was loss of stain intensity with increasing electrophoresis time. Third, the necessity of utilizing dianisidine in alcoholic solution produces undesirable shrinkage and distortion of the gels. However, a modification of the p-phenylenediamine technique for the staining of ceruloplasmin in agar gels has been found to stain ceruloplasmin very effectively in polyacrylamide gels following disc electrophoresis.

An immunoassay was developed which is capable of detecting IgA1 protease positive microorganisms from biologically derived samples grown on agar plates. Activity was detected by overlaying plates with agar containing insolubilized IgA and capturing Fab fragments on nitrocellulose disks coated with anti-λ light chain. Using cultures of human saliva, a comparison of this assay with a more traditional method involving electrophoresis and Western blotting of individual colony material revealed excellent agreement between the two methods. This assay should be useful in studying the distribution and ecology of organisms producing IgA1 protease, and in assessing its potential as a virulence factor.

The application of Triton X-100 in cellulose acetate electrophoresis and its effects on the separation of serum alkaline phosphatases is described. The cellulose acetate electrophoretic patterns of serum alkaline phosphatase found in liver and bone disease have been compared with the patterns found in agar, agarose, and polyacrylamide gel in the presence and absence of Triton X-100 and the differences described and discussed. Changes in the alkaline phosphatase electrophoretic pattern and the effects on certain electrophoretic variants have been noted following addition of Triton X-100 to all parts of the system.

The many factors that may be involved in the standardisation of isoenzyme assays are illustrated by reference to lactate dehydrogenase separation by electrophoresis and isoenzyme demonstration by tetrazolium staining. Some proposals are made for a standardised lactate dehydrogenase isoenzyme procedure by this technique.

A method for quantitation of the abnormal lipoprotein of cholestasis, LP-X, is described. After incubation of the serum with labelled free cholesterol, LP-X is separated from the normally occurring lipoproteins by electrophoresis in agar. By liquid scintillation counting the percentage of free cholesterol present in LP-X is determined. From the serum level of free cholesterol, the amount of free cholesterol in LP-X is calculated. The composition of lipids and proteins in LP-X has previously been found to be fairly constant, and from its content of free cholesterol the amount of LP-X may be calculated.

Urinary proteolytic activity and agar gel electrophoresis was done 3 months after complete gastrectomy in 7 patients, and the findings compared with those in 25 presumably normal subjects. It was found that 6 of the 7 gastrectomized patients showed proteolytic activity in the urine and that this enzymatic activity appeared as two or three bands on agar gel electrophoresis. The source of these pepsinogens is speculative.

Electroendosmosis is a complicating factor in gel electrophoresis. Determination of electroendosmotic mobility by the use of vitamin B12 as a marker in agar and agarose gels at different concentrations revealed that electroendosmosis was not reduced to zero by extrapolation of observed mobility values to zero gel concentration. It is shown that a Ferguson plot of the observed values of electrophoretic mobilities yields the correct values for KR; however the extrapolated values of electrophoretic mobility must still be corrected for electroendosmosis to obtain the true electrophoretic mobilities.

This paper describes experiments on the nature of humoral antibodies which suppress induction of delayed hypersensitivity in mice to purified proteins, i.e., contrasensitizing antibodies. These antibodies may be analogous to those responsible for afferent or early central immunosuppression in immunological enhancement phenomena and for similar forms of immunosuppression sometimes referred to as immune deviation, split tolerance, or preimmunization tolerance. CF-1 mice were sensitized with either human serum albumin or ovalbumin in incomplete Freund adjuvant, were injected with contrasensitizer-rich mouse antisera or fractions thereof, and subsequently were skin-tested to measure the relative capacities of antisera or their components to interfere with sensitization. Agar gel zone electrophoresis located Contrasensitizing activity among the γ 1 globulins, and this identification was confirmed by DEAE cellulose chromatography. Fractionation on Sephadex G-200 dextran columns showed the inhibitory activity to be among molecules of approximately 170,000 molecular weight, and a combination of chromatographic procedures further indicated that this activity is due to specific antibodies closely associated with mouse IgF or 7S γ 1 globulin. Contrasensitizing antibodies also were found to be heat-stable, and although precipitin and passive hemagglutination antiserum titers often correlated well with immunosuppressive activity, neither was equivalent to it.

The abnormal human low-density lipoprotein class characteristic of biliary obstruction (LP-X) was reacted with [ 14 C]succinic anhydride to an extent of 70–80 moles of succinyl groups incorporated per 10 5 g of LP-X protein. The modified lipoprotein retained the typical morphology and ultracentrifugal flotation and sedimentation properties of LP-X but failed to react with antiserum to the native lipoprotein. On agar and agarose gel electrophoresis the succinylated lipoprotein had an increased mobility toward the anode relative to LP-X, as a result of the increased negative charge on the protein component. Partial delipidation of succinylated LP-X and ultracentrifugal fractionation of the protein into a fraction containing phospholipids plus at least three relatively small proteins (Apo-X) and an essentially lipid-free protein, chemically similar and immunologically identical with albumin, permitted us to evaluate the extent of reaction of these two protein classes with succinic anhydride in intact LP-X. On the average, the Apo-X fraction had 72 moles of succinyl groups incorporated per 10 5 g of protein, whereas the albumin fraction incorporated 55 moles per 10 5 g of protein. Extensive reaction of susceptible amino acid residues (mostly lysines) with succinic anhydride, without disruption of the lipoprotein structure, indicates that these protein groups are accessible to the reagent and are not involved in critical protein-lipid interactions. Elimination of immunoreactivity upon succinylation of LP-X implies that, at least for the Apo-X component, lysine residues participate in the interaction with LP-X antibodies. Also, the present results strongly support the view that albumin is not merely adsorbed to LP-X, and suggest, furthermore, that protein-protein interactions are not directly responsible for the characteristic stacking of LP-X discs as seen in the electron microscope.

The binding characteristics of human TBG, TBPA and TBA of T4 and T3 were studied by agar gel electrophoresis in a variety of buffers. The highest maximal binding capacities of TBG and TBPA were found in Hank's balanced salt solution at pH 7.4. Phosphate and tris maleate ions at this pH resulted in the reduction of the thyroxine maximal binding capacity of TBG and TBPA. It was also shown that pH, ionic constitution and molarity of the system affect the distribution of tracer T4125I among the specific protein carriers. Thyroxine binding inhibitors were shown to affect TBPA, TBA and TBG at a physiological pH like the effect described at other pH levels. The relative binding potency of TBPA for thyroxine:DNP:salicylate:penicillin was in a ratio of 670:250:10:1, respectively. An attempt to deal with the binding capacity data by the Scatchard technique is described.

Paper electrophoresis of cerebrospinal fluid proteins has demonstrated that the content in gamma globulins may be elevated in certain inflammatory reactions of the central nervous system; it has also demonstrated that certain qualitative differences may exist in the gamma globulin fractions in different cases. We have pursued this line of investigation by biochemical determination cf glycoproteins as well as by electrophoresis. These preliminary results led us then to attempt to study more specifically the qualitative differences. Agar electrophoresis reveals a more detailed subdivision of cerebrospinal fluid proteins into a larger number of phases, and also demonstrates the presence of several phases within the gamma globulin fraction. With the same technique, we have also been able to study the water-soluble proteins of cerebral tissue. Comparison between the electrophoretic patterns obtained on agar from cerebrospinal fluid and from cerebral tissue reveals a number of differences and suggests that certain pathologic gamma globulins would seem to appear at the same time in the spinal fluid and in the brain. It is thus possible to speculate about the endomeningeal origin of those protein fractions. Additional studies comparing agar electrophoretic patterns obtained from human and from animal cerebral tissues reveals an essentially similar pattern.

The frequency of M-components was studied by agar gel electrophoresis in sera from 807 patients, 467 (57%) females and 340 (43%) males with histologically proven solid malignant neoplasms. M-components were found in the sera of 40 male and 20 female patients. Apart from two known cases of multiple myeloma and one case of Waldenström's macroglobulinaemia, none of the patients were found to be suffering from these diseases. The frequency of M-components increased with age, and this was more evident in males. Twenty-two of 60 patients with M-components did not exhibit abnormalities on immunoelectrophoresis. Of the 35 remaining patients, 27 had an abnormal component of the IgG class, 6 of the IgA and 2 of the IgM class. M-components were found in the sera of patients with a wide variety of neoplasms. There appeared to be no evidence of an increased frequency of M-components in the sera of patients with solid malignant neoplasms compared with normal adult population. ImagesFig. 1

A technique of agar-gel electrophoresis possessing a high resolving power is described whereby a minimum of nine fractions are revealed in normal human serum. This is achieved by using a steep voltage gradient so as to complete the electrophoretic separation in less than 30 min. A very efficient, inexpensive cooling liquid was found in petroleum ether. Water losses due to evaporation are completely avoided. The electro-endosmotic flow is regularized by interposing large agar blocks between the electrophoretic plate and the electrode vessels. The method seems to be especially promising for the study of tissue proteins and of animal serum proteins.

Creatine phosphokinase isozymes in sera of patients with progressive muscular dystrophy were studied by agar gel electrophoresis. A method of demonstrating these isozymes is described in detail. Sera of patients with progressive muscular dystrophy were found to have either the MM fraction or both the MM and MB fractions.

Practical prevention of coronary heart disease requires the screening of individuals at risk in a given population. The role played in the pathogenesis of this disease by the different lipoprotein fractions makes the mere determination of cholesterol and triglycerides an insufficient tool for the primary screening of individuals with different types of hyperlipoproteinemias. An automated procedure is described for the fast and accurate direct determination from serum of the cholesterol content of the β and pre- β -lipoproteins which not only will differentiate normals from abnormals but will detect abnormal individuals who otherwise will be missed by only cholesterol or triglyceride determination. In addition, evidence is shown demonstrating that this method is not affected by the post-prandial status of the sample, making this procedure ideally suitable for programs of primary screen for hyperlipoproteinemias.

Agar gel electrophoresis of human serum adenosine deaminase resulted in the separation into three major and two minor enzymatically active components. Changes in the electrophoretic pattern of the deaminase were investigated in normals and in three disease states : lung cancer, pulmonary tuberculosis and hepatitis. Each of the three diseases tested produced a particular pattern characterized by an increase in one of the major components. The importance of these patterns for the clinical diagnosis of the disease states was investigated and it appears that the patterns may have applications in lung cancer.

Problems concerning fixation of proteins after agar and agarose gel electrophoresis are briefly surveyed. The fixatives described so far have been found to be insufficient for fixation in agarose gel. An effective fixative, containing picric acid, is described. Proportionality between protein concentration and dye uptake is demonstrated using picric acid fixation upon agarose gel electropherograms.

The lipoproteins of pooled pig serum were separated into three distinct zones by electrophoresis in 0.45% agarose gel in Barbitone-acetate buffer, pH 8.6. High-density lipoproteins ( d = 1.09–1.20) moved with albumin, very low-density lipoproteins ( d = 1.007) with α 2 -globulins, and low-density lipoproteins ( d = 1.007–1.09) with β-globulins. The apparent absence of α 1 -globulin with the presence of high-density lipoproteins lends support to the use of lipoprotein nomenclature based on ultracentrifugal rather than on electrophoretic properties. The multiple components of low-density lipoproteins visible in the ultracentrifuge under certain conditions could not be resolved in Barbitone (diethylmalonylurea), borate, or Tris buffers. Mobilities of pig serum proteins and lipoproteins were compared to the mobilities of the corresponding components of human serum. Electrophoresis in agarose gel was also successfully applied on a preparative seale and individual lipoprotein classes were thus isolated from total lipoproteins. Improved methods for fixation and lipid staining were devised which led to better visualization of the components and better preservation for documentation purposes.

The behavior of serum albumin, IgG, IgM, IgA and salivary IgA was studied in the recently-introduced quantiative technique of electroimmunodifussion. This method by combining the modes of immunodiffusion through antibody-containing agar and electrophoresis of antigen achieved performance advantages over radial immunodiffusion in the quantitation of protein antigens, particularly when in dilute solutions. From the results obtained with these standard antigens of varying molecular size and electrophoretic mobility, principles have been derived which should allow the adaptation of this method to the measurement of any soluble protein.

Paper or agar gel electrophoresis is a simple and convenient method for the demonstration of the presence of Mb in a kidney extract or biological fluids. TLC on Sephadex may be used for the separation of Mb and Hb as well. Immunoelectrophoresis can serve for the detection of minute quantities of Mb in vivo.