Countercurrent Immunoelectrophoresis Research Articles

Human serum “parvovirus”: A specific cause of aplastic crisis in children with hereditary spherocytosis

1. Adair CV, Gould RL, Smadel JE: Aseptic meningitis, a disease of diverse etiology: Clinical and etiologic studies on 854 cases. Ann Intern Med 39:675, 1953. 2. Bland RD, Lister RC, Ries JP: Cerebrospinal fluid lactic acid level and pH in meningitis. Am J Dis Child 128:151, 1974. 3. Rutledge J, Benjamin D, Hood L, Smith A: Is the CSF lactate measurement useful in the management of children with suspected bacterial meningitis'? J PEO~ATR 98:20, 1981. 4. Pavlakis SG, McCormick KL, Bromberg K, Peter G: Cerebrospinal fluid anion gap in meningitis. J PEDIATR 96:874, i980. 5. Belsey M: CSF glutamic oxaloacetic transaminase in acute bacterial meningitis. Am J Dis Child 117:288, 1969. 6. Feigin RD, Wong M, Shackelford PG, et al: Countercurrent immunoelectrophoresis of urine as well as of CSF and blood for diagnosis of bacterial meningitis. J PEDIATR 88:773, 1976. 7. Fikrig SM, Berkovish S, Emmett SM: Nitroblue tetrazolium dye test and the differential diagnosis of meningitis. J PEDIATR 82:855, 1973. 8. Nachum R, Lipsey A, Siegel S: Rapid detection of gram negative bacterial meningitis by the limulus lysate test. N Engl J Med 289:931, 1973. 9. McCarthy PL, Frank AL, Ablow RC, et ah Vatue of the C-reactive protein test in the differentiation of bacterial and viral pneumonia. J PEDIATR 92:454, 1978. 10. Gill CW, Fischer CL: An evaluation of C-reactive protein assay using a rate immunonephelometric procedure. Am J Clin Pathol 75:50, 1981. 1 I. Sabel KG, Henson LA: The clinical usefulness of C-reactive protein (CRP) determinations in bacterial meningitis and septicemia in infancy. Acta Paediatr Scand 63:381, 1974. 12. Bayston R: Serum C-reactive protein test in diagnosis of septic complications of cerebrospinal fluid shunts for hydrocephalus. Arch Dis Child 54:545, 1979. 13. CorralI C J, Pepple JM, Moxon ER, Hughes WT: C-reactive protein in spinal fluid of children with meningitis. J PEDIATa 99:365, 198 I.

Commercial latex agglutination for detection of group B streptococcal antigen in body fluids

SEVERAL TECHNIQUES for the detection of bacterial antigens in body fluids have been shown to be helpful in providing a rapid, specific etiologic diagnosis prior to culture results in infants and children with meningitis and bacteremia. For the detection of group B streptococcal (GBS) antigen, these include countercurrent immunoelectrophoresis (CIE), ~4 latex agglutination 5-7 and slide coagglutination tests, 8 and enzyme immunoassay) Among these, latex agglutination is simple, rapid, and has the advantage of somewhat greater sensitivity than CIE and greater specificity than coagglutination. When a commercially available latex reagent (Streptex) was used for the detection of GBS antigen in cerebrospinal fluid (CSF), serum, and concentrated urine of infants with GBS infection, respectively 78.6%, 26.7%, and 93.3% of specimens were positive. 6 False positive results were rare. Recently a similar but more sensitive reagent (Wellcogen Strep B) has been prepared specifically for testing of body fluids. We describe the sensitivity and specificity of this latex agglutination assay in detecting GBS antigen in CSF and concentrated urine from infants with and without systemic GBS infection.

Occurrence of infection with a parvovirus-like agent in children with sickle cell anaemia during a two-year period.

The occurrence of infection with a parvovirus-like agent during the period April 1979-May 1981 in children attending a single sickle cell clinic in London was investigated. Virus was detected in serum by counter-current immunoelectrophoresis (CIE) and immunoelectron microscopy (IEM). Viral antibody was detected by CIE and specific IgM antibody by an IgM-antibody capture assay. Of the 68 children studied nine presented in aplastic crisis and evidence of infection with the parvovirus-like agent at the time of the crisis was found in all nine. Eighteen of the other children were antibody-positive at some time during the study. In 11 children there was no evidence of recent infection; however, two of these had a history of aplastic crisis in previous years. The other seven seroconverted during the course of the study but did not show any haematological effects. Five of these had a primary infection, one appeared to have reinfection and in the seventh there were insufficient data to distinguish between the two. Possible explanations for the difference between those presenting with aplastic crisis and those with asymptomatic seroconversion are discussed.

The sensitivities of different immunoassays for detecting leptospiral antigen

The detection of leptospiral antigen in biological fluids is important for the diagnosis of leptospirosis in animals and man. However the sensitivity of dark field microscopy, the usual detection method, is often inadequate. A comparison was made between the sensitivities of several immunological techniques for detecting Leptospira interrogans serovar hardjo. By staphylococcal coagglutination 10(8) leptospires per ml could be detected and by countercurrent immunoelectrophoresis 10(7) per ml. The best sensitivity obtained by enzyme-linked immunosorbent assay was 10(5) leptospires per ml, and by radioimmunoassay 10(4) to 10(5) per ml. Radioimmunoassay offers the prospect of improved diagnosis of leptospirosis through the detection of leptospiral antigen.

Phage influence on the synthesis of extracellular toxins in group A streptococci.

Phage conversion of group A streptococci to produce streptococcal exotoxins was shown to occur more widely than has been previously reported. Toxigenic conversion was found in 19 newly constructed lysogenic and pseudolysogenic strains resulting in synthesis of exotoxin types A and B. Conversion was accomplished by a positive conversion effector, which was a phage characteristic expressed by the prophage and vegetatively reproducing phage. Exotoxin production was determined by the rabbit skin test and by countercurrent immunoelectrophoresis with type-specific antisera. New lysogens and pseudolysogens were constructed with strains which failed to produce at least one exotoxin type. Phages were obtained from toxigenic strains isolated from cases of scarlet fever. Conversions were consistent and repeatable; loss of the recently introduced phage was accompanied by loss of the newly acquired toxin productivity. Conversion resulted in production of additional exotoxin type or types and never affected existing toxin synthesis. Converting phages were characterized by electron microscopy and negatively stained preparations and were all found to be of morphological class B1. All phage nucleic acid was double-stranded DNA. Though similar in structure, each converting phage had a different host range, and the nine new converting phages identified here did not react with antiserum prepared against the originally reported converting phage.

The development and use of an antibody capture radioimmunoassay for specific IgM to a human parvovirus-like agent.

An IgM-antibody capture radioimmunoassay (MACRIA) was developed for the detection of IgM antibody specific for the human parvovirus-like agent B19. Diagnosis of infection with this agent by either antigen detection or antibody seroconversion had been made by counter-current immunoelectrophoresis (CIE) in 18 cases of aplastic crisis occurring in children with homozygous sickle-cell disease. The MACRIA described here gave positive results in 17 of these 18 cases; in the remaining case only an acute specimen taken from the patient during viraemia and late convalescent specimens taken 184 and 247 days after onset of illness were available. The test was used to investigate 20 further cases of aplastic crisis in which neither viral antigen nor antibody seroconversion could be detected by CIE. Detection of virus-specific IgM permitted diagnosis of infection with this parvovirus-like agent in 17 of these cases. In the remaining three cases only single serum specimens taken late in convalescence, 82 days or more after the onset of symptoms, were available. In addition to these 34 cases of aplastic crisis in which primary infection with this agent was diagnosed by MACRIA, seven cases of apparent 'silent' infection detected by CIE were investigated. The test permitted the discrimination between primary infection and re-exposure to the virus in six of these patients. The use of this assay has added a considerable weight of evidence implicating primary infection with this parvovirus-like agent as an important cause of aplastic crisis in children with sickle-cell disease. Furthermore, MACRIA permits diagnosis of infection when only single serum specimens taken up to ten weeks after infection are available. Thus the use of this test will significantly facilitate the investigation of other clinical syndromes of presumptive infectious aetiology.

Advantage of Latex Agglutination Over Countercurrent Immunoelectrophoresis in the Detection of Haemophilus influenzae Type b Antigen in Serum

Detection of bacterial antigens has proved to be a valuable adjunct to early diagnosis of certain invasive infections. In the case of Haemophilus influenzae type b infections, capsular polysaccharide antigen (PRP) has been detected by a variety of techniques including countercurrent immunoelectrophoresis (CIE),1-3 latex particle agglutination (LPA),3,4 radioimmunoassay,5 and enzyme-linked immunosorbent assay (ELISA).6,7 There have been few prospective comparisons3,7,8 by which to establish the sensitivities and idiosyncrasies of these techniques. This report compares the two simplest techniques, CIE and LPA, in children with H influenzae b infections, drawing attention to a shortcoming of CIE in those with nonmeningeal infections. METHODS

Rapid Laboratory Diagnosis of Infectious Diseases

The countercurrent immunoelectrophoresis and latex particle agglutination tests are rapid, do not require expensive apparatus, and are easy to perform if specific antigens and antisera are available. Both tests have been shown to be of value in the diagnosis of bacterial infections (such as those caused by H. influenzae, Streptococcus pneumoniae, N. meningitidis, E. coli, Staphylococcus aureus and group B streptococcus) and of viral infections (HRVLA and hepatitis B); they may also be of value in fungal and parasitic infections. Etiologic diagnosis of commonly seen clinical conditions (such as pneumonia, meningitis, cellulitis, osteomyelitis, lymphadenitis, and arthritis) by practicing physicians can be made using these tests to detect antigens in various body fluids in a hospital laboratory.

Haemophilus influenzae Pneumonia

Haemophilus influenzae type b can cause pneumonia. Although the frequency has not been accurately assessed, this etiology is not rare and may represent as much as one third of hospitalized patients with bacterial pneumonia. Approximately half have pleural involvement. The majority of patients have associated manifestations including otitis, meningitis, purulent pericarditis and epiglottitis. Cultures from the blood and localized areas (pleural fluid, etc) are usually positive and establish the diagnosis. Countercurrent immunoelectrophoresis (CIE) may be helpful in the partially treated child. Comment: H influenzae b can cause a variety of diseases including, obviously more commonly than heretofore believed, pneumonia. Treatment of this illness depends upon the B lactamase production by the isolated Haemophilus.

Acidic protein in macrophages.

An antiserum to unstimulated rat peritoneal macrophages was produced in rabbits. The antibodies were directed against an acidic protein with a molecular weight of 35,000 and with an isoelectric point at 4.6. The macrophage acidic protein (MAP) was purified by gel filtration of rat lung soluble proteins, followed by preparative isoelectric focusing. The preparation of MAP was pure as assayed by agar gel electrophoresis and showed one precipitation peak in crossed immunoelectrophoresis against the crude antiserum directed against peritoneal macrophages. The purified MAP was used for immunization of rabbits, and the antiserum obtained was monospecific, assayed by crossed immunoelectrophoresis and Grabar-Williams immunoelectrophoresis. The titre was 4 times higher in the anti-MAP antiserum (1:80) than in the crude antimacrophage antiserum (1:20), tested against MAP by counter-current immunoelectrophoresis. The antigen (MAP) was demonstrated by direct and indirect immunofluorescence microscopy in rat blood monocytes, in spleen and lung monocytic cells, in clusters of cells in the thymus, and in adventitial macrophages around larger blood vessels in liver, kidney, lung and brain. Scattered meningeal macrophages showed fluorescence in the normal, brain. In stab-wounded areas of rat brain MAP was localized to perivascular and perineuronal macrophages with a morphology similar to that of microglial cells. The localization of the fluorescence was the same both for the antiserum against MAP and for the antiserum raised against crude peritoneal macrophages.

Experience in Northern Nigeria with countercurrent immunoelectrophoresis, double diffusion and indirect haemagglutination tests for diagnosis of hydatid cyst in camels

Sera from 36 slaughtered camels with proven hydatid disease, from 46 camels found to be free of hydatidosis at post-mortem meat inspection, and from 26 animals with confirmed caprine heartwater, bovine trypanosomiasis and cysticercosis were examined. The indirect haemagglutination was found to be the most sensitive but least specific of the three tests. Ouchterlony's double diffusion (DD) test was specific and fairly sensitive. Although the countercurrent immunoelectrophoresis was as specific as DD, it was the least sensitive of the three serological tests. All three serological tests were always positive in camels with hydatid cyst infection affecting both the liver and the lung. A combination of indirect haemagglutination and Ouchterlony's double diffusion test could be useful for diagnosing hydatidosis in camels and probably other animals.

Sialic acid polysaccharide antigens of neisseria meningitidis and escherichia coli: Esterification between adjacent residues

Colominic acid and meningococcal Goup B polysaccharide, both (2→8)-α-linked homopolymers of sialic acid, are made water-insoluble either by reaction with a carbodiimide in aqueous solution at pH 4.75 or by treatment with 48% aqueous hydrofluoric acid at 0° for 48 h. I.r. spectra of the products show a major band near 1750 cm −1, consistent with ester formation; this band virtually disappears after mild, alkali treatment. Esterification also occurs by incubating the native polysaccharides below pH 6.0, and their i.r. spectra show that the degree of esterification increases as the pH is lowered. The relatively low molecular weight of these partially esterified, water-soluble polymers is consistent with intra- rather than inter-molecular ester formation. Counter-current immunoelectrophoresis shows that esterification of ∼9% is sufficient to abolish immunoprecipitation. 13C-N.m.r. spectroscopy of fully esterified colominic acid provides strong evidence for cross-linking between the carboxyl group of one residue and HO-9 of an adjoining residue. Meningococcal serogroup C polysaccharide, a (2→9)-α-linked homopolymer of sialic acid containing O-acetyl groups at C-7 and/or C-8, does not undergo intramolecular esterification after carbodiimide treatment. However, upon O-deacetylation of the polysaccharide, esterification occurs.

Quantitation of Dengue Precipitating Antibody by Inhibition Countercurrent Immunoelectrophoresis

Quantitation of Dengue Precipitating Antibody by Inhibition Countercurrent Immunoelectrophoresis

983 USEFULNESS OF SERUM C-REACTIVE PROTEIN (CRP) DURING THE NEWBORN PERIOD

Among 100 consecutive infants admitted to a NICU, serum CRP val ues ⩾ 1.0 mg/dl occurred in 18 during any of the first three days of life. Of these 18 infants, 12 had one or more of these problems: intrauterine growth retardation, meconium aspiration pneumonitis, febrile mother, transient tachypnea, and low Apgar score and/or shock and/or fetal distress. Of 11 with serum CRP values ⩾ 2.0 mg/dl, 8 had maternal fever, meconium aspiration pneumonitis, shock and/or fetal distress. Serum CRP,values during the first 3 days of life among 35 infants of mothers with fever and/or prolonged rupture of membranes were not helpful in identifying the 2 with systemic Group B β hemolytic streptococcal disease (urine positive for antigen by countercurrent immunoelectrophoresis), there being 7 others with negative surface cultures and serum CRP values ⩾ 1.0 and 3 with elevated values of 6 whose surface cultures were positive. Serial serum CRP determinations of 10 infected neonates (7 septicemias due to a variety of organisms, 1 urinary tract infection 1 pneumonia and 1 scalp abscess) followed the expected rapid decline during successful therapy. Serum CRP values during the first days of life are often elevated because of non-infectious causes. Accordingly, this acute-phase protein cannot be used to identify infection during the first days of life. However, it affords an excellent means of evaluating the course of infections and effectiveness of therapy among term and preterm infants.

Quantitation of dengue precipitating antibody by inhibition countercurrent immunoelectrophoresis.

The inhibition countercurrent immunoelectrophoresis test was employed to detect dengue virus antibody in patients' sera. Anti-dengue type 2 titers determined by inhibition countercurrent immunoelectrophoresis correlated well with hemagglutination inhibition titers. In secondary cases, more than fourfold increases in precipitating antibodies were observed. The control sera were negative except for sera from a few patients with systemic lupus erythematosus, which showed low titers. Simultaneous detection of dengue virus antigen and antibody in sera collected during the acute phase could confirm at least 90% of cases. This method is recommended as a routine technique to quantitate antibody in sera from suspected cases of dengue hemorrhagic fever.

Serological reactivity of duck sera to three electrophoretically characterized extracts of Aspergillus

Three different antigens, whole culture, cell sap shaker culture and culture filtrate extracts of Aspergillus were characterized by crossed immunoelectrophoresis. Thirteen to fifteen precipitin lines were produced with their homologous antisera. Application of these extracts to the assay of wild duck sera showed that the whole culture extract was the most sensitive of the three antigens in detecting antibody. Countercurrent immunoelectrophoresis, used with a modified buffer, produced results comparable in sensitivity to immunodiffusion in detecting avian antibody.

Identification of bacterial antigens in patients pretreated with antibiotics using counter-current immunoelectrophoresis (author's transl)

The article describes the method of counter-current immunoelectrophoresis as a rapid and safe technique for the identification of bacterial antigens in body fluids. This technique has proved very useful especially in patients who had been pretreated with antibiotics, since the culture yields are usually negative in such patients, as experience has shown.

Limitations of Countercurrent Immunoelectrophoresis (CIE) in the Diagnosis of Empyema

Infectious agents are the most common cause of pleural effusions in children, developing more commonly during the course of bacterial pneumonias and less frequently associated with viral, mycobacterial, and fungal respiratory infections. Immunologic techniques, such as countercurrent immunoelectrophoresis (CIE), are often more valuable than cultures in establishing a specific etiologic diagnosis in children. In seven of the ten cases of empyema in childhood reported by Siegel et al,1 CIE was positive, while cultures of blood and pleural fluid were negative. Antibiotics had been given in each of these seven cases of sterile pleural effusion. The article by Holsclaw and Schaeffer in this issue of Chest (see page 867) reports the successful use of this technique to identify the specific etiologic agent in sterile pleural effusion in a puzzling case clinically suggestive of rheumatic fever or varicella prodrome. CIE is a rapid and sensitive method for identifying as little as .05 to 0.1 µg of several different types of pneumococcal antigens or Hemophilus influenzae, type B (Hi B) antigen. This is an especially useful diagnostic method when the patient previously has been given antimicrobial drugs or the infection has become subacute.

Antigen-Antibody Complexes Related to the Baboon Endogenous Virus in Humans With Acute Lymphoblastic Leukemia-Hand Mirror Variant (ALL-HMC)

AbstractHand mirror cells are a morphological configuration that are seen in immunologically stimulated lymphocytes and can be induced by antigen-antibody complexes. Therefore, the bone marrow and peripheral blood plasma of two patients with acute lymphoblastic leukemia-hand mirror variant were evaluated for the presence of antigen-antibody complexes. Both patients had antigen-antibody complexes in the bone marrow plasma and not in the peripheral blood plasma as determined by double counter-current immunoelectrophoresis. The antigen moiety of these complexes appears immunologically related to components of the baboon endogenous virus (BaEV), and the antibody moiety also appears related to structural components of the BaEV. Bone marrow plasmas from patients without leukemia were evaluated for the presence of antigen-antibody complexes and found to be negative. The antigen-antibody complexes may account for the presence of hand mirror cells in the bone marrow of patients with acute lymphoblastic leukemia-hand mirror variant.

Antibodies in rabbits immunized with staphylococcal lipoteichoic acid.

Fractionated rabbit antiserum to staphylococcal lipoteichoic acid (LTA) was tested for reaction with homologous antigen by precipitation in agar gel, countercurrent immunoelectrophoresis, immunoperoxidase technique and electron microscopy. The antibodies to LTA present in the rabbit antisera examined were found to be of the IgM class.