Feb 4, 1988 - LaRochelle T, North G, Stern P. A new extraction of arginine ... Radioimmunoassay of arginine vasopressin in human plasma.J ..... Hendrtlc J. Vreman, Ricardo B. Ronqulllo, Ronald L Magno, Herbert C. Schwartz, and David ...
2. Reigger GAJ, Liebau G, Kochsiek K. Antidiuretic hormone in congestive heart failure. Am J Med 1982;72:49-52. 3. Creager MA, Faxon DS, Cutler SS, Kohlmann 0, Ryan TJ, Gavras H. Contribution of vasopressin to vasoconstriction in patients with congestive heart failure: comparison with the reninangiotensin system and the sympathetic nervous system. JAm Coli Cardiol 1986;7:758-65. 4. Burnett Jr JC, Kao PC, Hu DC, et al. Ati-ial natriuretic peptide elevation in chronic congestive heart failure. Science 1986231:1145-7. 5. Atlas SA, Volpe M, Sosa RE, Laragh JH, Camargo MJF, Maack T. Effects of atrial natriuretic factor on blood pressure and the renin-angiotensin-aldoeterone system. Fed Proc Fed Am Soc Exp
Biol 1986;45:2115-21. 6. Borenstein HB, Cupples WA, Sonnenberg H, Veress AT. The effect of atrial extract on renal haemodynamics and urinary excretion in anesthetized rats. J Physiol (London) 1983;334:133-40. 7. Bichet DG, Kortas C, Manzini C, Baijon JN. A specific antibody to vasopresn in a man with concomitant resistance to treatment with pitressin. Clin Chem 1986;32:211-2. 8. Ysewijn-Van Brussel KARN, De Leenheer AP. Development and evaluation of radioimmunoassay of Arg8-vasopressin after extraction with Sep-Pak C18. Cliii Chem 1985;31:861-3. 9. Juppner H, Brabant G, Kapteina U, Kirschner M, Klein H, Heach RD. Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation. Biochem Biophys Res Conimun 1986;139:1215-23. 10. Gutkowska J, Bonan R, Roy D, et al. Atrial natriuretic factor in hwnan plasma. Biochem Biophys Res Commun 1986;139:287-95. 11. Miyata A, Kangawa K, Toshimori T, Hatoh T, Matauo H. Molecular forms of atrial natriuretic polypeptides in mammalian tissues and plasma. Biochem BiophysRes Commun 1985;129:24855.
12. Hartter E, Woloszczuk W, Stummvoll HK. Radioimmunoassay of atrial natriuretic peptides in human plasma. Clin Chem 1986;32:441-5. 13. Yandle TG, Espiner EA, Nicholls MG, Duff H. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J Cliii Endocrinol Metab 1986;63:72-9. 14. LaRochelle T, North G, Stern P. A new extraction of arginine vasopressin from blood: the use of octadecasilyl-silica. Pfiugers Archiv 1980;387:79-81. 15. Glanzer K, Appenheimer M, Kruck F, Vetter W, Vetter H. Measurement of 8-arginine-vasopressin by raclioimmunoassay: development and application to urine and plasma samples using one
extraction method. Acta Endocrinol1984;106:317-29. 16. Gutkowska J, BourassaM, Roy D, et al. Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma. Biochem Biophys ResCommun 1985;128:1350-7. 17. Husaun MK, Fernando N, Shapiro M, Kagan A, Glick SM. Radioimmunoassay of arginine vasopressin in human plasma. J Clin Endocrinol 1973;37:616-25. 18. Robertson GL, Mahr EA, Athar 5, Sinha T. Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma. J Clin Invest 1973;52:2340-52. 19. Camps J, Martinez-Vea A, Perez-Ayuso RM, Arroyo V, Gays JM, Rivera-Fillat F. Radioimmunoassay for arginine-vasopressin in coldethanol extracts of plasma. Clin Chem 1983;29:882-3. 20. Fyhrquist F, Wallenius M, Hollemans HJG. Radioimmunoassay ofvasopressin in unextracted plasma.Scand J Clin Lab Invest 1976;36:841-7. 21. Skowsky WR., Rosenbloom AA, Fisher DA. Radioimmunoassay measurement of arginine vasopressin in serum: development and application. J Clin Endocrinol Metab 1974;38:278-87.
CLIN. CHEM. 34/5, 973-975 (1988)
Identificationof HIV-SpecificOligoclonalImmunoglobulinsin Serum of Garnersof HIV Antibody N. N. Papadopoulos,’
R. Costello,’
N. Ceronl,2 and H. N. MoutsOpouIos3
Zone electrophoresis on agarose gel was performed on serum samples from H1V-antibody carriers and negative controls. Nitrocellulose strips precoated with an HIV preparation were then placed on top of the gels and developed by an immunoblotting procedure. A positive reaction was demonstrated between the HIV antigens and the HIV-antibodypositive serum samples with hypergammaglobulinemia and oligoclonal IgG bands. A negative reaction was found between the HIV antigens and HIV-antibody-negative serum samplesfroma normal person and a patient with monoclonal gammopathy. The presence of oligoclonal IgG bands in the serum of HIV-antlbody carriers, and their positive identIfIcation with HIV antigens, indicates a specific immune response of the host to the HIV infection and supports the use of oligoclonal lgG bands as markers to follow the course of HIV infection.
The phenomenon of oligoclonal banding is a new development in zone electrophoresis of serum proteins. Using a sensitive electrophoretic technique (1), we reported a high incidence of oligoclonal immunoglobulin bands (OIB) in serum samples of patients with acquired immunodeficiency syndrome (&ins) (2). Presence of paraproteins in sera of AIDS patients has been reported (3, 4). Also, we found OIB in serum samples from asvmptomatic carriers of human immunodeficiency virus (hlV) antibody. The OIB were identified as immunoglobulin G (IgG) heavy chain only and mixed ic and A light chains(S). In this report we present evidence of a positive reaction between OIB of serum samples from HIV-antibody carriers and HIV antigens. In contrast, no reaction was observed between the H1V antigens and antibody-negative serum samples from a normal patient with monoclonal gammopathy.
person
and a
Materials and Methods 1Clinical Chemistry Service, Clinical Center, and2 Laboratory of CNS Studies, NTNCDS, National Institutes of Health, Bethesda, MD 20892. 3Present address, University of loannina, School of Medicine, loannina, Greece. Received December 3, 1987; accepted February 4, 1988.
Elect ropho resis. In this study we used serum samples from asymptomatic persons who tested positive for HIV antibodies, first with an enzyme-linked immunosorbent assay (EU5A) and then with the Western blot test (Biotech Research Laboratories, Rockville, MD 20857). We also used serum samples from a healthy person and a patient with a CLINICALCHEMISTRY,Vol. 34, No. 5, 1988 973
strong monoclonal band that tested negative by the Western blot test. Zone electrophoresis of serum proteins was performed by an agarose gel technique that detects strong and weak protein bands in the gamma globulin region of the electrophoretogram (1) and is more sensitive than similar zone electrophoresis tests (6). The protein bands were identified by immunofixation as previously described (5). Immunoblotting. Immunoblotting of serum IgG antibodies specific to HIV was performed by a procedure used to identify virus-specific oligoclonal IgG in cerebrospinal fluid (7), modified as follows. The active H1V-infected cell culture preparation, banded in sucrose for concentration and purification, was obtained from Electro-Nucleonics, Inc., Silver Spring, MD 20904. The IflV was inactivated in a 1 g/L solution of Triton X100 surfactant before use. Nitrocellulose strips were incubated overnight at room temperature, with a 10-fold concentrated HilT preparation derived from an H-9 cell line infected with HIV. The entire procedure was performed under the hood. In the morning, the strips were briefly washed in phosphatebuffered saline and incubated at room temperature for 1 h in Tris-buffered saline, pH 7.4, containing, per liter, 60 g of bovine serum albumin and 5 g of Tween 20. Electrophoresis was performed in serum samples diluted 100- to 400-fold. Immediately after the end of the electrophoresis, the strips with the adsorbed H1V antigens were washed in phosphatebuffered saline and laid on top of the gels. Several layers of filter paper, a glass sheet, and a 0.9-kg weight were added. Passive transfer of proteins to nitrocellulose strips was allowed for 40 mm. The strips were briefly washed in phosphate-buffered saline and their nonspecific binding sites were blocked with a 200 g/L solution of normal goat serum in phosphate-buffered saline for 30 miii. After a brief wash, the strips were incubated at 37#{176}C for 2 h with rabbit anti-human IgG antiserum diluted 200-fold in blocking solution. The strips then were washed for 15 mm in a 0.5 g/L solution of Tween 20 detergent in phosphate-buffered saline, with three changes of the solution. The procedure was completed by using the peroxidase “ABC” kit (Vector Lab., Burlingame, CA 94010) and development with 4-chloro-1naphthol. To prove the specificity of the method, we included seronegative samples from a normal person and from a patient with monoclonal gammopathy. Seropositive samples were blotted to nitrocellulose precoated with the supernatant fluid from a non-infected H-9 cell line and with human T-cell lymphotropic virus type 1 (HTLV-1).
Globulins
A
B
C
0
FIg. 1. Serum protein electrophoretic patterns of HIV-antibody-negatlve samples and their corresponding immunoblots of nftrocellulosestrips precoatedwith HIV antigens A, tern a nOrmalperson;B, immunoblotof A; C, from a patent with monoclonal gammopathy;0, Immunoblotof
Globulins
A$4
B
cf.
Results Figure 1 shows serum protein electrophoretic patterns of HIV-antibody-negative samples and their corresponding immunoblots. The A pattern is a typical normal control obtained by the agarose gel electrophoretic technique showing a diffuse y.globulin zone. The C pattern, from a patient with monoclonal gammopathy, shows a strong monoclonal band in the y-globulin zone identified as IgG K. Their corresponding immunoblots, B and D, show no reactivity with the H1V antigens. In Figure 2, A is the serum protein electrophoretic pattern of an HIV-antibody-positive sample with diffuse hypergammaglobulinemia. B is the immunoblot of the same sample showing a positive reaction with the H1V antigens in the y. globulin zone. C is the serum protein electrophoretic pattern of an H1V-antibody-positive sample showing OIB in they. 974 CLINICALCHEMISTRY, Vol. 34, No. 5, 1988
C
ILl
Fig. 2. Serum protein electrophoretic patterns of HIV-antibodycamera and theircorresponding Immunoblotsof nitrocellulosestiips precoated with HIV antigens A showsa diffusegIobulin zone.B, the immunoblotof A, showsa weakpositive reactionInthe ‘yglobullnzone.C showsOIB,and0, theimmunoblot of C, shows a strongpositivereactionhi the yglobulln zone. E, an immunoblotcontrolof C, shows a negativereaction when precoatedwith the cell line preparationnot Infectedwith HIV
globulin zone identified as IgG K. D is the immunoblot of the same sample; it shows that both the OIB and the diffuse’ globulin reacted strongly with the 11W antigens. E is an immunoblot control of sample C, showing no reaction when the nitrocellulose paper was precoated with non-infected H9 cell culture supernate. Also, no reaction was evident with sample C when the nitrocellulose paper was precoated with an HTLV-1 preparation.
DIscus8Ion Electrophoretic analysis of serum samples from H1Vinfected persons demonstrated discrete OIB within an intense y-globulin zone.It indicates two simultaneous processes: (a) a polyclonal B-cell hyperactivation and (b) a selective B-cell oligoclonal proliferation to secrete uniform IgG bands. In these studies we presented evidence of both of these processes by the positive reactions between the HilT-antibody-seropositive samples and the viral antigens. While diffuse hypergammaglobulinemia is a general characteristic of viral infections, the OlE indicate a modified cellular and humoral immune response of the host to the HIlT infection. It is estimated at this time that between one and two million persons have been infected with HilT (8). The central question is: what is the clinical course of the HilT-antibody carriers? Biochemical markers are needed to study the course of HIlT infection, the effects of therapy, and to assess clinical prognosis. The direct association of OIB with HIV antigens suggests the use of OIB as markers to follow the course of HIlT infection. Because the interval between initial HilT infection and development of ios is lengthy, we are continuing periodic testing of asymptomatic HIV-antibody carriers, to determine if appearance, persistence, or disappearance of OlE
may predict the clinical course of HIV infection. In some cases, instead of strong OlE originally present in the serum protein electrophoretic patterns, weak bands have been observed on the H1V-precoated mtrocellulose blot. In one case, an extra band appeared on the blot that was not present in the original serum protein electrophoretic patterns. This is due to variable quantitative distribution of the HilT antibodies present in the y-globulin zone and their diverse specificity against the HilT antigens used. The use of individual antigens such as p24, against HIV-antibody-positive serum samples with OLE, may provide additional information of the specificity of OIB. References 1. Papadopoulos NM, Elm RJ, Wilson DM. Incidence of y.globulin banding in a healthy population by high-resolution electrophoresis. Cliii Chem 198228:707-8. 2. Papadopoulos NM, Lane HC, Costello R, et al. Oligoclonal iminunoglobulins in patients with the acquired immunodeficiency syndrome. Clin Immunol Iminunopathol 1985;35:43-6. 3. Herist K, Haliquist AE, Tomar RH. Paraproteinemia in patients with acquired immunodeficiency syndrome (AIDs)or lymphadenopathy syndrome. Clin Chem 1985;31:1224-6. 4. Crapper RM, Deam DR., Mackay IR. Paraproteinemias in homosexual men with HIV infection. Am J Clin Pathol 1987;88:348-51. 5. Papadopoulos NM, Costello R. Oligoclonal immunoglobulins in the sera of healthy subjects at risk for Ame. Clin Biochem 198720:257-8. 6. Costello R, Papadopoulos NM. Oligoclonal banding in hypergammaglobulinemia [Abstract]. Clin Chem 1987;33:905. 7. Domes R, Ter Meulen V. Detection and identification of virusspecific, oligoclonal IgG in unconcentrated cerebroapinal fluid by ixnmunoblot techniques. J Neuroimmunol 1984;7:77-89. 8. Mueller N. Epidemiology of ame. Law Med Health 1987;14:250-8.
CLIN. CHEM. 34/5, 975-977 (1988)
Interference of Fetal Hemoglobinwith the SpectrophotometncMeasurementof Carboxyhemoglobin Hendrtlc
J. Vreman, Ricardo B. Ronqulllo, Ronald L Magno, Herbert C. Schwartz, and David K. Stevenson
We measured the concentration of carboxyhemoglobin (HbCO) in blood samples from 32 neonates by spectrophotometry (IL282 CO-Oximeter) and gas chromatography,finding a strong positive correlation (r = 0.89) between the concentration of fetal hemoglobin (Hb F) and HbCO as measured by spectrophotometry, but not by gas chromatography.Thus, Hb F interferes with the determination of HbCO by spectrophotometric techniques by falsely increasing apparent HbCO in direct proportion to Hb F. We concludethat, when Hb F is known or suspected to be present, blood HbCO cannot be reliably determined by methods based on spectrophotometry. AddItional Keyphrases: neonates sessing hernolysisrate in newborns
pediatric chemistiy
‘
as-
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5119. Received January 4, 1988; accepted February 8, 1988.
When blood containing fetal hemoglobin (Hb F) is analyzed for carboxyhemoglobin (HbCO) by spectrophotometric methods, falsely high values are obtained for HbCO (1).’ Ryan et al. (2) suggested that the correlation between Hb F (as percent of total Hb) and spectrophotometrically determined HbCO (percent saturation) could be used as a simple indirect method to estimate the blood Hb F. However, this suggestion was based in part on a correlation between the transcutaneous oxygen saturation (TcSO2) readings of the pulse oximeter (3) and the sum of Hb02 and HbCO, determined spectrophotometrically; they assumed no true HbCO to be present. In fact, true HbCO was not measured. The present study correlates concentrations of Hb F with HbCO as determined in the same blood specimen by spectrophotometry and gas chromatography. ‘Nonstandard abbreviations: Hb, total hemoglobin; Hb F, fetal hemoglobin; HbCO, carboxyhemoglobin; TcSO2, transcutaneous oxygen saturation.
CLINICALCHEMISTRY, Vol. 34, No. 5, 1988 975
