Document 6BB4nG8O6YajqX56bm11oe60o

VOLUME ONE GRADWOHL'S CLINICAL LABORATORY METHODS AND DIAGNOSIS EDITED B Y ALEX C. SONNENWIRTH, Ph.D. Professor of M icrobiology and Immunology, and Pathology, Washington University School of Medicine; Director, Division of M icrobiology, Department of Pathology and Laboratory Medicine, The Jew ish Hospital of St. Louis, St. Louis, Missouri LEONARD JARETT, M.D. Professor of Pathology and Medicine and Head, Division of Laboratory Medicine, Washington University School of Medicine; Director of Laboratories, Barnes Hospital, St. Louis, M issouri \i t\ EIGHTH EDITION with 748 illustrations, including 50 in color The C. V. Mosby Company ST. LOUIS TORONTO LONDON 1980 t 13 MEASUREMENT OF PROTEINS IN BIOLOGIC FLUIDS John Savory John E. Hammond life process." They are involved as a basic part of the structure of the living cell and are also re sponsible for a major part o f its function. For example, proteins serve as structural components of cells, enzymes, some hormones and hormone receptors, transport molecules, antibodies and In the years b etw een the editions of this text, clotting factors, plus additional functions too rapid progress has been m ade in m ethods for the num erous to note here. quantitation of serum, CSF, and urinary proteins. The "building blocks" of proteins are the 20 No longer is it sufficient sim ply to determ ine the com m only occurring a-am ino acids.T hese amino total mass p er un it volum e o f biologic fluid, but acids differ from each other in the structure of rather the concentration of a distinct protein com their side chain. In proteins these amino acids ponent. At die p resent tim e it is possible to quan are linked through su b stitu te am ide or p ep tide titate 10 protein com ponents using im m uno bonds, which arise by the elim ination of the chemical nephelom etric technics. The clinical elem ents of water from the carboxyl group of significance of these m easurem ents is growing one amino acid and the a-am ino group of the rapidly, and in the near future their significance next. will be readily apparent. Hopefully, in the en Proteins differ from each other hv the kinds of suing years, new teclinologic advances will be amino acids present, their arrangement, their made that will increase the sensitivity of these quantity, molecular w eight, surface changes, etc. technics and allow the quantitation of proteins As a result, there are probably an almost infinite found in very low concentration. O f special in num ber of proteins. O ver the last If) years many terest will be attempts at combining specific pro proteins have been isolated and -physically tein determ inations with routine chemical characterized through use of such methods as analysis, such'as enzyme determ inations current ion-exchange chromatography, gel filtration, ly done in the chem istry laboratory. By choosing polyacrylam ide electrophoresis, agarose electron judiciously the specific proteins and enzyme phoresis, isoelectricfocusing, preparative ultra- tests it may be possible to establish a,battery of centrifugation, and analytical ultracentrifugal determ inations b etter able to assess accurately techniques. T he developm ent o f radioim m uno the biochemical state of the patient and detect assay techniques has lowered the limits of detec pathologic conditions. tion from the m illigram and micrograin range Tor In the following description of various proce som e of the previously nam ed technics to the dures for protein determ inations, a brief discus nanogram or picogram range. Further technolog sion of the physiology and interpretation of the ic advances that w ould allow a rapid automated results is included as a convenience to allow for approach to radioim m unoassay are eagerly antic a rapid appraisal of the usefulness of the proce ipated and currently under development. dure. Also note in some cases there are several Because of the size of the protein molecules, methods described for the same constituent, and they cannot be absorbed through the intestinal generally 1 m ethod may be included for his mucosa. To obtain the necessary amino acids for torical interest* along with the new er methods. protein synthesis, the ingested proteins must be Few of the procedures have been carried over < degraded, absorbed, transported into the cell, from the previous edition due to their obsoles ! and resynthesized into proteins, cence. T he Kjeldahl determ ination for total nitro j It is in tlie form of am ino acids that most of the gen has been.retained for historical interest, but necessary m aterials for protein 'synthesis are ab paper electrophoresis has been elim inated since sorbed through the intestinal mucosa of the small its value is extrem ely lim ited. intestine into die m esenteric venous blood. This Proteins, as a fundam ental constituent of all docs not exclude the possibility of larger m ole protoplasm, have been termed "the essence of cules such as polypeptides or native protein be- 256 CHAPTER 13 MEASUREMENT O F PROTEINS IN BIOLOGIC FLUIDS 257 ing absorbed in small quantities. This is thought to be a contributing factor in causing certain food allergies. The fact that nitrogenous products and meat fibers are found in the colon and feces demonstrates that digestion and absorption are not always com plete, especially in cases of diar rhea, pancreatic insufficiency, and common bile duct obstruction. Amino acids entering the bloodstream are quickly taken up by the tissues so that there is usually only an insignificant rise in the blood amino acid level even after a protein-rich meal.. Although the mechanics of this uptake of amino acids are not yet completely understood, it is gen erally thought that they are temporarily stored primarily in liver and muscle. Portions of these stored amino acids are later liberated for use by other tissue cells, an'd consequently little am ino acid is excreted in the urine. Excess amino acids are deaminated, and the ammonia formed, if not reused in die production of other amino acids or nucleic acid constituents, is converted in the liver to urea. T h e nonnitrog nons portion of the deaminated amino acid may be used as a source of energy in ways sim ilar to that of carbohydrates and fats. The dynamic state that xists betw een proteins and amino acids makes up part of w hat is term ed "nitrogen balance.'' This means, in short, that a normal healthy individual takes in an am ount of nitrogen that is equal to the am ount excreted. Proteins in cell are constantly being broken down into their constituent amino acids, a por tion of w hich is reutilized in the synthesis of new protein. T he fact that the body is not a perfect machine results in the need of an exogenous source of aniino acids, since some amino acids are deam inated in the liver and used for o ther purposes or are excreted directly in the urine. If the intake of nitrogen is below the level of that required for maintenance of destroyed protein, negative nitrogen balance results. Positive nitro gen balance, the opposite situation, exists in growing children, pregnant' women, and re cover)' from negative balance. Methods for the m easurem ent of proteins in biologic fluids are separated in this chapter into manual and automated procedures. In addition, some reference methods are included, although we recognize that their application to routine analyses is lim ited. The automated methods described use either the continuous-flow AutoAnalyzer system (Technicon Instrum ents Corp., Tarry town, N.Y. 10591) or the centrifugal fast analyzer system. T he latter was chosen as a representative of the discrete sample analyzers because of its flexibility in be ing able to make both end-point and kinetic m ea surem ents. In addition, it has been adapted to specific protein measurements, which consti tutes an important part of this chapter. The centrilugul last analyzer is based on the original con cept oTDr. Norman Anderson, working at the Oak Ridge National Laboratories.* At the present time 3 companies manufacture this type of automated analyzer, and details of their instruments are available from company literature: Centrifichem (Union C arbide Corp., Tarrytown, N.Y. 10591), GeMSAEC (Electronucleonics, Inc., Fairfield, N.J. 07006), and Rotochem (American Instrum ent Co., Silver Spring, Md. 20910). We will discuss the Rotochem system, which has a 15-place rotor; however, these determ inations may also be performed on the other centrifugal analyzers with minor modification of the gample and reagent volumes. Many other discrete sam ple analyzers also are capable of making both kinetic and end-point measurements. Thus the types o f m ethods described in this chapter for the centrifugal fast analyzer are in most cases adapt able to other discrete sam ple analyzers. TOTAL SERUM PROTEIN Most clinical chemistry laboratories employ the biuret reaction for the m easurem ent of total serum proteins and use the simple refractometric technic for confirmation of results. The biuret m ethod is technically straightforward and p re cise, and is accurate with an appropriate m eans of standardization. T he standard used in this determ ination may either be a human serum pool or a commercially available solution of crystalline bovine albumin. In both cases the concentration is usually d eter m ined by Kjeldahl analysis and expressed in terms of milligrams of nitrogen per m illiliter of solution. In order to relate this value to mass of protein per unit volume, usually grams per deci liter, one must employ a factor that relates m illi grams o f nitrogen to milligrams o f protein. T his factor depends on the protein source. It has been recom m ended that a factor of 6.54 (i.e., 15.3% by weight) be used for human serum pools, and a factor of 6.41 (i.e., 15.6% by weight) be used for crystalline bovine album in1(Armour Pharm aceu tical Co., Phoenix, Ariz. 85077). The automated procedures include the contin uous-flow technique, which is probably used more than any other total protein method. The procedure described, or modifications such as those for adaptation to the continuous-flow SMA system , is precise and relatively trouble free. A discrete sam ple autom ated m ethod is described using the centrifugal fast analyzer. Kinetic m ethods have been described for the centrifugal fast analyzer, b ut their use is to be discouraged, since precision is poor and accuracy is in ques tion. The centrifugal fast analyzer m ethod de scribed here allows the biuret reaction to come to completion before a spectrophotometric mea- sn rem ent is m ade. ' Manual methods for total serum proteins Biuret reaction3 Principle. Serum proteins react with copper sulfate in sodium hydroxide to form a violet " b iu ret" complex. T he intensity of the violet color is proportional to the concentration of protein. I 262 CLINICAL CHEMISTRY i I'AHT II turn (Amt's Co., D ivision o f M iles Laboratories, Inc., Elkhart, Ind. 46514). Procedure 1. Cheek all urine specim ens witli Albustix and dilute all samples greater than " 2 + ." 2. Into paired lest tubes, pipel 2.0 ml urine or stan dard. W hile mixing add slowly 2.0 ml T suchiya's solution. H eat to 56 C for 15 min and then cool. 3. Centrifuge precipitate and wash twice with 1 ml ethanol. 4. To washed precipitate add 3-0 ml of 0.75N NaOH (30 g/L) and mix until precipitate dissolves. 5. To I test tube of each pair add 0.15 ml biuret blank solution, and to oth er tube add 0.15 ini b iu ret rea gent. Mix by inversion and incubate at room tem perature 20 min. 6. Zero the spectrophotom eter at 540 nm w ith cuvette containing 3.0 ml of0.75N sodium hydrox ide plus 0.15 ml biuret blank reagent. Record ab sorbance of sample and standard blanks. 7. Zero the spectrophotom eter with cuvette contain ing 0.15 ml biuret reagent and 3.0 ml 0.75N so dium hydroxide. Record absorbance of standard ami sample. Calculation where C. = concentration of unknown; C, = concentra tion of standard; A,, = absorbance o f unknow n; = absorbance ofunknown blank; A, = absorbance of stan dard; A ,b = absorbance of standard blank. Remarks. Using a Bausch & Lomb Spectronic 20 spectrophotom eter w ith cuvettes 1.27 cm in diam eter, the linear range of this biuret assay is 0-2.0 g/L.. Automated immunochemical method13 An automated im m unochem ical m ethod using the continuous-flow approach is described. An appropriately d ilu ted urine sam ple is mixed autom atically w ith antiserum to hum an serum proteins, followed by incubation at room tem per ature and quantitation by light-scattering in a fluoronephelom eter. Apparatus 1. An uiLnmutic p ip et (e.g., M icroM edic Systems, Philadelphia, Pa. 19104) is used to prepare d ilu tions of urine samples. 2. A single-channel autom ated system is assem bled from components o f the Technicon AutoAnalyzer II. This system consists of a sampler, proportion ing pump, modified analytical cartridge, a fluoronephelometer, and a chart recorder. Reagents 1. Goat anti-w hole hum an serum can be obtained from commercial sources (e.g., T echnicon In stru m ents Corp., Tarrytown, N.Y. 10591). A ntiserum is diluted 25-fold with physiologic saline (9 g NaCI/L) containing 40 g^L polyethylene gly col 6000 (F ish er Scientific, Fair Lawn, N.J. 07410). 2. Standard. Commercial reference serum (Tech nicon Instrum ents Corp., T arrylow n, N.Y. 10591) is used fur standardization. 3. Albustix (Aines Co., D ivision of Miles Labora tories, Elkhart, Ind. 46514). Procedure 1. All urine sam ples are screened for protein content with the Albustix and diluted appropriately in order to avoid antigen excess. Albuxtix rending neg trace + ++ +++ ++++ Dilution 1:10 1:10 1:10 and 1:20 1:20, 1:40. ami 1:100 1:100 and 1:200 1:200 and 1:500 2. After dilution samples, controls and standards are aspirated into the system, mixed with antiserum, and the light-scattering intensity of the antigenantibody complexes is m easured in the fluoro nephelom eter and recorded. Calculations 1. Total protein concentration is determ in ed for each sam ple using a m inicom puter programmed to do linear interpolation betw een the points generat ing the standard curve. 2. If a m inicom puter is not available, a standard curve may be plotted with sam ple and control values read from this curve. Remarks 1. T he useful range of the m ethod is from 1.5-100 mg total protein/dl. 2. Precision studies show a coefficient of varia tion of about 3% for the m ethod. 3. Extrem ely high sam ples m ust be diluted and repeated to ensure that m easurem ent is m ade in the region of antibody excess. 4. This autom ated m ethod is capable of ana lyzing 70 sam ples/h. 5. T he standard curve is generated by making 7 solutions of the reference serum, covering the useful range of the method. 6. Blank readings are riot required, since urine possesses low intrinsic light-scattering. Measurement of serum albumin and globulins Serum album in can be m easured directly in serum by dye-binding methods, or indirectly by estimation of total globulins. Fractionation of serum proteins by salt precipitation or electro phoresis also offers a means of estimating albu min. Three manual methods are described, and the two that possess the greatest convenience are dye binding with bromocresol green and the m easurem ent of total globulins. Both are techni cally straightforw ard b ut are subject to errors. The bromocresol green method has been shown to overestim ate album in due to nonspecific b ind ing,14 and the total globulin m ethod occasionally gives aberrant results in some patients with dysproujinemia. Provided the limitations of die methods are recognized, these two approaches are recommended, since no odier technically sim ple and fast m ethod is w idely available. Salt fractionation of album in and globulins is d e scribed and gives excellent results when careful CM tec od / glo ant ing apj sul: ina tnu ser spe run cor spr led spe mo Ma Ser P to i rat] acit uni' sub enti Wsu resi is g dye pho fere that broi and Rt 1. 2. 3. 4. 5. M. 1. 2. , CHAPTER 13 MEASUREMENT O F PROTEINS IN BIOLOGIC FLUIDS 263 technic is exercised, hut unfortunately the m eth od is too tedious for the m odern busy laboratory. Automated methods for serum albumin and globulin are discussed using continuous-flow and centrifugal fast analyzer technics. Dye bind. ing with brninocresol green offers the simplest approach to autom ation of serum album in but is subject to the sam e lim itations of accuracy as the m anual m ethod. Autom ated light-scattering imm nnocheinical m ethods have been described for serum alb u m in 15 and appear to offer excellent specificity. However, the expense of the antise rum and the technical difficulties of obtaining consistent results preclude their use as a w ide spread routine method. Such light-scattering technics will be described later for other serunispecific proteins and can be applied with minor m odifications to serum album in. Manual methods Serum albumin by dye binding'* Principle. Serum album in generally is thought to function as a carrier protein for a num ber of rather insoluble organic substances such as fatty acids and bilirubin, and hence it is endow ed with unique binding properties. The binding of these substances to album in is thought to be due to .entropy factors of tile solvent (water) and van der Wnals interaction betw een aliphatic am ino acid residues and the hound molecules. This binding is generally term ed hydrophobic. A num ber of dyes have been show n to bind album in hydrophobically and absorb light at a wavelength dif ferent from the unbound dye. O f the various dyes that have been used in serum determ inations, brom ocresol green is the most sensitive, specific, and relatively free from interference. Reagents 1. Brij-35. T hirty-percent solution o f Brij-35 in water (F isher Scientific Co., Philadelphia, Pa. 19406). 2. Stock brom ocresol green solution. Dissolve 419 mg brom ocresol green (Sigma C hem ical Co., St. Louis, Mo. 63118) in 10 ml o f O.IN sodium hy droxide, and dilute to 1 L with glass-distilled water. 3. Buffer. D issolve 11.9 g citric acid m onohydrate in 8(X) ml glass-distilled w ater. Adjust pH of solution to 4,2 with 10% sodium hydroxide (wt/vol) and dilute to 1000 ml with glass-distilled water. 4. Working brom ocresol green solution. Mix 250 ml of stock bromocresol green solution with 750 ml citrate buffer and 4.0 ml of 30% Brij-35. NOTE: This solution is com m ercially available (F isher Scien tific Co., P hiladelphia, Pa. 19406). 5. Standard. W eigh out 5 g human serum album in fraction V (Sigma C hem ical Co., St. Louis, Mo. 63118), and dissolve in 100 ml o f 0.9% saline con taining 0.20% sodium azide. D eterm ine protein concentration o f this solution by biuret procedure using bovine serum album in for standard (Armour Pharm aceutical Co.). Method 1. Pipel 0.020 ml serum into lest tube, add 5.0 ml brom ocresol green working solution, and mix. 2. Reagent blank. Pipet 0.020 ml of 0.9% sodium chloride into test lube and add 5.0 ml of bromociesol green reagent. 3. M easure absorbance at 628 nm in suitable spectro photom eter using reagent blank to zero instru ment. Calculations w here C,, = concentration of unknow n; C,( = concen tration o f standard; A* - absorbance of unknow n; A,, = absorbance of standard. Remarks 1. T he reaction betw een album in ami bromo- cresoi green is extrem ely fast anti goes to com pletion in only a few seconds. 2. The 1:250 dilution of serum generally elim inates interferences from hyperlipem ia, hyperbilirubinemia, and hemolysis. Grossly lipemic sera can be blank corrected using 0.9% saline as a reagent blank m ethod. 3. T he m ethod is linear from 0-7 g/dl. \ 4. The human albumin standard should be divided into aliquots and stored at 4 C. This standard is stable up to 1 mo. Total globulin in serum17 Principle. In the presence of strong acids, glyoxylic acid reacts with tryptophan residues of proteins to form a purple color. C opper sulfate is added to enhance the color formation. Since human globulins are known to contain 2-3% tryp tophan, it is possible to derive an em pirical factor relating the concentration ofN-ncetyltryptophan to serum globulin levels. Reagent 1. G lobulin reagent. T his reagent consists of 800 mg glyoxylic acid, 600 mg copper sulfate, 15 mole ace tic acid, and 2.2 mole sulfuric acid per liter of solution and is available u n d er the trade name Diagnostest (Dow Chemical Co., Indianapolis, Ind. 46206). 2. Standardization. N-acetyl-DL-tryptophan 220 mg/ dl equivalent to 4.0 g/dl globulin (Sigma Chem ical Co., St. Louis, Mo. 63118). Procedure 1. Label three 13 x 100 mm test tubes as "stan d ard ," "control," and "test," Using volum etric pipet, transfer 4 ml globulin reagent into each tube. 2. T ransfer 20 I standard into tube m arked "stan dard," cover, and mix thoroughly. T reat control and test sam ples in a sim ilar m anner. 3. C over test tubes with alum inum foil and heat 5 min at 100 C. 4. Cool 3 min in tap w ater and remix test tubes. 5. Adjust w avelength of sp ectrophotom eter to 550 nm. Set zero with unused portion of globulin rea gent and measure absorbance of all tubes. Calculations w here C,, = concentration of unknow n; C, - concentra tion of standard; A,, TM absorbance o f unknow n; .4, = absorbance of standard. Remarks 1. T h e m ethod is linear to 7.5 g gluhulin/ril, 2. T h e (V-acetyltryptophnn is r e c o m m e n d e d as 264 CLINICAL CHEMISTRY L I'ART II L'HA the standard o f choice, since it can he pur chase cl in a highly pure form and is soluble in a dilute alkaline solution.* T he empirical factor relating globulin and N-acetyU tryptophan was based on 28 sera containing normal amounts of album in, thus the contri bution of album in to the colored product is included in the factor. 3. Bilirubin up to 20 mg/dl gives less than a 5% interference, and moderate hemolysis and lipcmia contribute no appreciable interfer ence. transfer contents to test lube marked "album in test." 5. Add 2.0 ml of 28.4% sodium sulfite to another test lube labeled "albumin blank." 6. To test tube labeled ``standard," add 0.1 ml bovine serum album in standard and 1.9 ml of 28.3% so dium sulfite. 7. Add 4.0 ml working biuret reagent to all tubes. 8. Mix and allow to stand 20 min. 9. M easure nhsorhances at 555 mn o f all solutions in appropriate spectrophotometer. Calculations inter is de U 1. 2. 3. 4. Seru Albumin by difference18 Tl Principle. The serum albumin concentration where C, = concentration of unknown; C. = concentra on tl can be calculated by subtracting the total globu tion of standard; A,, = absorbance oT unknown; A, = Don lin concentration from the total protein concen tration. Reagent and method. See biuret total protein method and total globulin m ethod. absorbance of standard. Remarks I. If serum blanks are req u ired for lipeiiiic samples, follow the above procedure and i abso crest for a dilu Remarks 1. This m ethod is relatively free o f interfer obtain the absorbance of all the unknowns. T hen add 1 mg potassium cyanide to the i Rei 1, 1 ence from m oderate l pernia, icterus, and album in standard as w ell as all the serum 2. : hemolysis. samples. This step will destroy the absorb i 2. Significant errors in serum album in deter ance due to the copper-protein coni])lex Api minations by this m ethod can arise in leaving only the absorbance due to inter cam, .{ patients with m ultiple myeloma where the tryptophan content of the globulins can vary markedly. fering substances. Re-re;td all the un knowns. Calculate as follows:' A002ferem is pro s Serum albumin by salt fractionation,a I Re Principle. T h e accepted reference m ethod for the determ ination ofalbum in is the salt fractiona tion method of Wolfson et al., which uses sulfite to precipitate globulins from serum . T h precipi tated globulins are rem oved .by shaking with diethyl ether followed by centrifugation. Al bum in rem ains in the aqueous phase and is quantitated by the biuret procedure. w here A s = absorbance'of' standard; ASKrx = absorbance of standard after addition of potas sium cyanide; A r = absorbance of unknown; Arxcx --absorbance of unknown after addition of potassium cyanide; Cx = concentration o f stan dard; C r = concentration oi unknow n. 2. Vigorous mixing of the sam ples alter the ad dition of the ether reagent may result in the 1. ] 2. 3. Serur Reugents dnaturation and precipitation of albumin, Th. 1. Sodium sulfite 28.3% (wt/vol). 2. E ther teagent. 1% triton X-100 in absolute diethyl resulting in low er album in values. been ether. Automated methods Reo 3. Working b iuret reagent. P repare as described under total protein method. 4. Standard. C rystalline bovine album in, approxi m ately 6.4 g/dl (Armour Pharm aceutical Co.). Procedure 1. Into a dry ground-glass stoppered tube, place in The direct spectrophotometric determination of globulin in serum by the m ethod of Goldenberg and D rew es1* adapted to the continuousflow system of automation"* is described. A dual channel system also can be em ployed for the order: sim ultaneous determ ination of total protein by a. 4.0 ml of 28.3% sodium sulfite using a class A the m odified biuret m ethod. T his com bination of volumetric pipet. b. 0.2 ml serum dispensed volumetrically. c. 4.0 ml e th e r reagent d isp en sed w ith serologic pipet. 2. S topper tuhe and invert gently 8 times. 3. C entrifuge at 2000 rpm for5 min. Ifaq u eo u s phase is not perfectly clear, add an additional m illiliter of ether reagent and recentrifuge for 5 min. After centrifugation, globulin precipitate forms a pellet determ inations allowed the calculation ol serum album in by subtracting the globulin value from tile total protein. Total globulin is m easured by reaction of the tryptophan residues of the globu lin m olecules with glyoxylic acid in a strongly acidic medium. The colored product of the reac tion absorbs strongly at 550 mn. ! I between the 2 phases. Reagents 4. Carefully lip centrifuge tube and insert 2 ml class 1. G lobulin reagent. See m anual m ethod. A volumetric pipet into aqueous phase, keeping 2. Standard. See manual method. 1. I 1 \ 2. < l Apr Rotoc time i Pro* LI ( 2. I fi 3. I f* 4. T r 5. ( top of pipet covered with index finger to prevent A pparatus. T he m ethod req u ires the Ini lowing ether reagent from entering pipet. Remove exactly 2.0 ml aqueous phase, w ipe off pipet lip, and continuous-flow equipm ent: samples, proportioning pump, manifold, heating hath, colorimeter with 550 mn a CHARTER 13 MEASUREMENT OF PROTEINS IN BIOLOGIC FLUIDS 265 interference fillers', and chart recorder. A flow diagram is described in the original publication.1* Remarks 1. T he autom ated globulin m ethod is linear to ' 6 g/dl. 2. Recovery of gamma globulin added to serum is essentially quantitative. , . 3. The day-to-day precision o f the m ethod has been found to be 4.5%. 4. T he method can be standardized w ith N- acctyltryptophun. Serum albumin by continuous-flow analysis20 The continuous-flow album in m ethod is based on the hromocresol green dye-binding m ethod of Douniiis. T his m ethod m easures the increase in absorbance at 630 nm due to th e album in bromocresol green complex formed at pH 4.2. The need for a blank channel is elim inated due to the high dilution of the sam ple, approxim ately 1:441. Reagents 1. Hromocresol green solution. See manual method. 2. D iluent that consists of 1.0 ini of 30% Brij-35 p er liter distilled water. Apparatus. This method requires a sampler; 60/h cam, 9:1; proportioning pum p, manifold no. 170A002-01; single-channel colorim eter with 630 nm inter ference filter; and chart recorder. F urther information is provided in Techm con m ethod no. SE4-0030FD4. Remarks 1. T he m ethod is linear to 6 g/dl. *2. Q uantitative recoveries are obtained. 3. Studies show a precision o f the m ethod (95% limits) is approxim ately 3% . Interfer ences clue to hyperlipem ia and hyperbiliru binemia are minimized because of the initial high dilution of the sample. Serum albumin by centrifugal fast analysis21 The hromocresol green method of Doumas has been adapted to the centrifugal fast analyzer. Reagents 1. Bromocresol green working solution. See manual hromocresol green albumin method modified with antifoum (see remarks). 2. Standard. See manual hromocresol green album in method. Apparatus. T he m ethod is described for the Amineo Rotochem II system. With m inor modification of vol um e i( may he used w ith other centrifugal analyzers. Procedure 1. Piput 600 il distilled w ater into reference position y of transfer disk. 2. Pipet 5 i! distilled water, standard or sam ple, 90 fil saline, and 400 ^,1 hrom ocresol green reagent into appropriate position of transfer disk. 3. Load transfer disk into analyzer and initiate pro gram. 4. Take absorbance reading 30 s after initiation of reaction. 5. Calculation o f sam ple concentrations is made automat cal ly by com puter using absorbance m ea surements of standard and samples taken 30 s after acceleration of rotor. Remarks 1. T he m ethod is linear to 7 g/dl of allmmin. 2. Day-to-day precision (95% limits) is about 3%. 3. Before using commercially available work ing bromocresol green solution, add 2 ml Dow antifoam B per liter of working solu tion. This process prevents interenvette transfer of sam ples during vacuum mix cycle. 4. T he m ethod is fre^ o f interference from icterus and hemolysis. Fractionation of proteins by electrophoresis Only 2 serum protein measurements have been described up to this point, total protein and albumin. These are the 2 most common protein m easurements made in clinical chemistry labora tories, and serum album in is of considerable value to the clinician. Total-protein m easure m ents in serum are o flim ited value, since there are so many individual specific protein constitu ents that change to different degees in d isease states. For example, in acute inflammation some serum proteins become elevated (haptoglobin, orosomucoid, oq-antitrypsin), while others are decreased (albumin, transferrin). Thus fractiona tion of serum proteins has becom e an extremely important laboratory test. Electrophoresis has been the most common means of fractionating serum proteins, and tradi tionally quantitation of the protein fractions has been accomplished by densitometric scanning. In recent years, high-resolution technics using agarose gels have been proposed with qualitative visual examination being preferred to quantita tive scanning. T he proteins visualized on agarose gel electrophoresis are shown in Fig. 13-1. E lectrophoresis is a technic for the separation of charged molecules, which is dependent on dif ferences in the m igration velocities of the mol ecules in a buffer solution through which an electrical current is passed. Differences betw een the rates of migration of various molecular spe cies in an electrical field are the result of inequal ities in their ionic charges at a given pH. Ab though electrophoretic fractionations may be perform ed in a liquid buffered m edium (i.e., free, T iselius, or moving boundary electrophoresis), it is usually advantageous to m inim ize the diffu sion of the separated molecules by conducting the electrophoretic migration in an inert stabiliz ing m edium that is saturated with buffer solution. Exam ples of the stabilizing m edia used for elec trophoresis include filter paper, cellulose acetate, sucrose gradient starch gel, acrylamide gel, agarose, dextran gel, and glass heads. A num ber o f synonyms for electrophoresis have been used such as ionophoresis, tonography, and electrochromatography. The common applica tions of electrophoresis in clinical laboratories include fractionations of proteins, lipoproteins, glycoproteins, isoenzymes, hemoglobins, amino acids, and catecholam ine m etabolites. In the 266 CLINICAL CHEMISTRY PART II IgA IgM IgG [IgE and igD) Agarose gel oelectrophoresis MW : *r !?.'}' *-**-vfc-- * j v' -* : Y-iVSi r Prealbirmin Albumin apA ntitrypiin aj-M acroglobultn H aploglobin (2-2) T ra n s fe rrin /?-Lipoprotein C3 Application Fig. 13-1. Electrophoretic separation of serum proteins on agarose gel. (Courtesy L. M. Kiltingsworth, Univer sity of North Carolina, Chapel Hill, N.C.) present discussion, attention will be focused on 2 electrophoretic methods currently employed in clinical laboratories for the separation and quantitative determ ination of serum protein frac tions. ; Cellulose acetate electrophoresis" P rinciple. C ellulose acetate m em brane is used as an inert supporting m edium for the separation of serum proteins. This technic separates the serum into 5 principal regions, which are then detected with ponceau S stain. Proteins general ly are quantitated by scanning the cleared, destnined m em brane. Results are reported as a per centage o f the total, or if the total protein is known, the protein concentration per region may be calculated. Apparatus 1. Many different cellulose acetate electrophoresis systems are commercially available. The Micro zone system will be discussed in detail (Beckman Instrum ents, Fullerton, Calif. 94302). 2. Tire M icrozone system consists of an electro phoresis cell, a regulated power supply, and a densitom eter with scanning attachment. 3. Cellulose acetate mem branes are supplied with the Microzone system. Reagents 1. Barbital buffer. Dissolve 15.40 g sodium diethyl barbiturate and 2.76 g barbituric acid in I L water. This produces a solution of pH 8.6 and ionic strength of 0.075. 2. Fixative-stain solution. Dissolve 2 g ponceau S in 30 g trichloroacetic acid and make up to 1 L vol ume with water. 3. Rinse solution. 5% (vol/vril) acetic acid in water. D ilute 100 ml glacial acetic acid to 2 L. 4. Alcohol rinse. Absolute methanol, A.C.S. reagent grade. 5. C leaning solution. Mix 20 ml glacial acetic acid with 80 ml methanol. Procedure 1. Follow m anufacturer's instruction for preparing electrophoresis cell. 2. Place small am ount o f buffer in shallow dish and carefully float m em brane on surface. M em brane will slowly absorb buffer and turn uniform gray in color. If air is en trap p ed in m em brane, uniform gray color will not be obtained and membrane should not be used. 3. Remove m em brane from buffer, blot gently with filter paper, and place m em brane in electro phoresis cell. 4. Apply approxim ately 0.25 /ri serum to m em brane with serum applicator supplied with Microzone system. 5. C onnect pow er leads and him on pow er supply. Samples are subjected to electrophoresis for 20 m in at 240 V. 6. Follow ing electrophoresis step, remove mem brane and float it serum side up in shallow dish containing 100 ml ponceau S stain. As soon as bands becom e stained, im m erse m em brane in dye solution for additional 7 min. 7. Remove m em brane from stain and rinse in 5% acetic acid until background is w hite. 8. D ehydrate m em brane by immersion in absolute methanol. 9. After dehydration, position membrane on glass plate and im merse for 45 s in m ethanol-acetic acid clearing solution. Air-dry m em brane 2-3 min and heat 20 min in oven at 70-80 C. 10. Transfer cleared, dry m em brane gently from glassplate to plastic storage.envelope and label. 11. M ount plastic envelope w ith m em brane in car riage d ensitom eter and scan at 520 nm. C alcu latio n s 1. Using areas o f each protein region derived from densitom eter scan, calculate total area, and per centage of total area of each given region then may be calculated. 2. Protein concentration o f each region may be determ in ed by m ultiplying percentage of total area for that region times serum total protein con centration. Remarks 1. C ellulose acetate electrophoresis resolves serum proteins into 5 multicomponent re gions. 2. Some errors arise in quantitation due to variation in dye-building affinities of the various protein components. 3. T he resolving capability of die system is less than agarose gel electrophoresis. A g a ro s e gel e le c tr o p h o r e s is 33 A p p aratu s 1. E lectrophoresis cell. W ater-cooled electropho resis'cell large enough to accom odate glass plate CHAPTER 13 MEASUREMENT O F PROTEINS IN BIOLOGIC FLUIDS 267 20.5 x 11.0 cm is used (W ater-cooled Agarose Electrophoresis C ell--Laurel I type, MRA Corp., Bos loti, Mass. 02121). T his size o f cell is large enough to perm it sim ultaneous electrophoresis o f 9 samples. 2. Water cooling. E lectrophoresis cells are cooled by a refrigerated liquid circulator utilizing diluted ethylene glycol at 4 C, which will provide sufficient cooling to dissipate heat generated dur ing electrophoresis (Forma, Scientific, Marietta, Ohio 45740). 3. Power supply. Any well-regulated power supply capable of producing 0-400 V and 0-150 mA may lie used (Model SP-17A, H ealth Co., Benton H ar bor, Mich. 49022). Reagents 1. Buffer. Dissolve 105.15 g sodium barbital, 16.5 g barbital, and 6.9 g calcium lactate in 2 L hot de ionized w ater and d ilu te to 8 L. All chem icals are reagent grade. *2. Agarose. Dissolve 1 g agarose in 100 ml barbital buffer by beating solution to boiling. Agarose will rem ain in liquid state at 50 C (Sea Kcm Brand Marine Collidssher, Springfield, N.J. 07081). 3. Fixative. Fixative is a solution com posed o f 1000 ml deionized water, ICXX) ml m ethanol, and 200 ml glacial acetic acid. 4. Stain. Dissolve 1 g naphthol blue blank dye in 100 ml fixative solution. F ilter once before use (Sigma Chemical Co., St. Louis, Mo. 63118). 5. Tracking dye. Dissolve 20 mg brom ophenol blue in 100 ml barbital buffer (Sigma C hem ical Co., St. Louis, Mo. 63118). Procedure 1. D ue to variabilily betw een electrophoresis cells commercially available, no attempt will be made to discuss assem bly of cell. Please see manufac turer's instructions. 2. Coat glass plates (20.2 x 10.8 cm) with 0.5% solu ti o n of agarose in barbital buffer (8 ml o f 0.5% agarose per-plate) and allow to dry com pletely. This procedure ensures binding of separation gel to plate. 3. To pour separation gel, make a sandw ich of a pre coated plate, a V-inch single-cham ber plastic frame, and a slot-forming plate. (Glass plate *92-2; plastic frame, 92-34; slot-fonning plates, *92-3D ; MRA Corp.. Boston, Mass. 02121.) 4. Place assem bly in oven at 60-70 C (for about 10 min). 5. Remove assem bly from oven and fill hollow space with hot 1% agarose as rapidly as possible. 6. Place filled assem bly in refrigerator to gel. D uring gelling process the agarose may shrink, and if this happens, add additional hot 1% agarose. 7. W hen plate has gelled com pletely, rem ove clam p, place plate on a bench, and carefully pry up and remove slot-forming plate. Sample wells will hold approxim ately 10 /d of liquid. 8. Sprinkle a few drops of distilled water on bottom of electrophoresis cell and piaee plate in cell. Press down on plate to rem ove air b u b b le s , (n ote: Du not substitute buffer for distilled water.) T he w ater ensures good heal* transfer from plate to cooling liquid pum ped through bottom of cell. 9. Mix 2 parts serum or other fluid with 1 part bromophenol blue. Blot sam ple wells in plate and fill each well with 10 /d sam ple containing bromophcnol blue. 10. C onnect plate w ith cell by means of W hatm an 3 mm filter paper wicks with buffer. Cov er cham ber with glass plate. 11. Subject plate to electrophoresis at 250 V (approximately 100 mA). After 15 min turn pow er off, moisten wicks, fill sam ple w ells with buffer, and continue electrophoresis for another 55 min. 12. W hen electrophoresis is finished, turn off power, rem ove plate from cell, and subm erge it in fixing solution 15 min. At end of this period transfer plate to another tray and place u n d er stream of gentle running water for 30 min to rem ove buffer salts from gel. Dry plate. 13. Stain plate for 5 min in 1% naphthol blue black. D estain in fix solution until background is clear and then dry. Plate is now ready for clinical inter pretation. Remarks 1. Agar may not be substituted for agarose. Agar contains highly charged polysaccha rides that may interact with protein com ponents of serum. 2. Thin and regular application w ells are ex tremely important for high-resolution elec trophoretic patterns. The slot-forming plate produces consistently well-defined applica tion wells, and it is superior to other methods. 3. After fixation of the proteins, plates should he washed with w ater to remove buffer salts. The plates are then dried and after staining may he stored indefinitely with out deterioration of the stained hands. 4. The high-resolution properties of the system are dem onstrated in Fig. 13-1. Fractionation of urine and CSF proteins by electrophoresis The electrophoretic technics for serum protein fractionation are applicable to urine and CSF analysis, provided the sample undergoes a con centration step. Concentration of CSF and urine samples can he accomplished simply and rapidly by employing the Amincon macrosolute concen trator (Amincon Corp., Lexington, Mass. 02173). This disposable device consists of an 8-chambered plastic case, with each cham ber hacked by a 15,000 mol wt cutoff m em brane b onded to a filter paper absorbent. The chambers hold a max imum of 5.0 ml, and the sample can be concen trated 100-fold to a final volum e of 50 p.1. T his system is technically sim ple to use and well suited for use in the routine clinical laboratory. D eterm ination of sp ecific p ro tein s In serum CSF and urine T here is probably little doubt that the clinical chemistry laboratory of die future will emphasize the m easurem ent of specific proteins and rely on electrophoresis as a m eans of detecting "M " com ponents in plasma cell dyscrusias and catliodal bonding as seen in lupus erylhematosus.11-2* The gel-diffusion m ethods for specific protein measurements are usually slow, .expensive, imprecise, and have limited applica tions in the modern laboratory- A more rapid and precise technic involves light-scattering or neph elometric measurements of antigen-anti body 268 CLINICAL CHEMISTRY cumpluxes. This approach allows specific protein measurem ents to he m ade in a matter of 1 or 2 m inutes with a precision of b etter then 5% versus tlie gel-diffusion m ethods, which take several hours with poor precision. In addition, the lightscattering technic can be automated and adapted to both continuous-flow and centrifugal fast nnaIvzcr systems. In the light-scattering technic the reagent used is a specific antiserum (antibody) to the human serum protein (antigen). On mixing diluted serum with antiserum the antigen reacts with the antibody, producing large antigen-antibody com plexes (hat will scatter light. Most commonly the light-scattering is m easured at 90 in a fluoroin' eter with the spectrophotometric system adjusted so that the w avelengths of the incident and em itted light are identical. Mostly 360 nm is used, although higher wavelengths can be em ployed if slightly reduced sensitivity is accept able. The angle of light-scattering measurements can be varied, and'increased sensitivity can be achieved at angles other than 90. H owever, for most routine m easurem ents 90 is most conve nient, since conventional fluorometers allow only this angle for measurements. Recently an adaptation of a commercial centrifugal fast ana lyzer has allowed near front surface light-scat tering m easurem ents to be made. In recent years polyethylene glycol 6000 (PEG) has been incorporated into the reaction mixture and has a profound effect on the rate of formation of antigen-antibody complexes. W hereas in the absence o f PEG the antigenantihody reaction takes 15 m inutes to 2 hours to come (o com pletion, the presence o f PEG achieves equilibrium in 2-4 m inutes. T he PEG does not appear to be part of the complexes and probably acts by steric exclusion of macromoleeules for the domain of the polymer. The m ethods described in this chapter include manual, automated continuous-flow, and centrif ugal fast analyzer technics. The manual m ethods described do not use PEG, since its incoiporation into the reaction requires very accurate tim ing, otherw ise flocculation of the precipitated antigen-antibody complexes occurs. Both the manual and continuous-flow technics allow the antigen-antibody reaction to achieve equilibrium before light-scattering measurements are made. However, in the centrifugal fast analyzer ap proach the rate of formation of .the complexes is measured by fixed-time kinetics. Here the change in light-scattering betw een 2 fixed times (e.g., 15 seconds and 90 seconds) is m easured and related to a standard curve. In all of these light-scattering methods the standard curve is nonlinear and follows the char acteristic precipitin curve (Fig. 13-2), w hich is divided into zones of antibody excess (region 1), equivalence (region 2), and antigen excess (region 3). All of the m ethods described operate in the antibody excess zbne. Exam ination of Fig. 13-2 reveals that one can obtain identical liglit- PART II l Fig. 13-2. Precipitin curve of antigen-antibody reac tions followed by light scattering. (Courtesy L. M. Killingsworth, University of North Carolina, Chapel Hill, N.C.) scattering intensity for w idely variable antigen concentrations depending on whether the con centrations fall in antibody or antigen excess. Thus some means of detecting antigen excess sam ples is a necessary part of any procedure. In the continuous-flow technic a bimodal recorder peak readily identifies antigen excess samples and alerts the analyst to make a dilution of the sample. In the manual and centrifugal fast ana lyzer m ethods a second m easurem ent with a 1:1 dilution of the sam ple is need ed to detect antigen excess. In practice, how ever, .serum protein elec trophoresis can he used in screening for antigen excess, and the conditions described in this text are adjusted so that only patients with mono clonal ganiniopathies can be expected to achieve antigen excess. Since CSF-specifie proteins can be measured using identical m ethods to serum with different dilutions, the method description includes pro cedures for both serum and CSF. Manual nephelometric methods for the determination of aVantitrypsin. !gG, IgA, and IgM Principle. Serum proteins are quantitated us ing immunochemical reactions with specific commercial antisera. A fluorom eter modified for nephelom etry is em ployed to make light scattering measurements of the antibody-antigen complexes formed during the immunochemical reaction, which is carried out in the region of antibody excess. A m ultipoint standard curve is required since the relationship betw een light scattering and protein concentration is nonlinear. Apparatus 1. Light-scattering m easurem ents can be m ade in any suitable fluorometer, and die instrum ent uti lized in the initial developm ent of this method j was the T u rn er M odel III Fhiom ineler (G.K. | T urner Assoc., Palo Alto, Calif, 94303). A recorder may he connected to the lluorom clcr; how ever, it is possible to take readings* from the readout dial on the fluorometer. T he fluorom eter is eq u ip p ed with a narrow bandpass primary filler, peak trans mission at 360 nm (T urner no. 110-811). No CHAPTER 13 MEASUREMENT O F PROTEINS IN BIOLOCIC FLUIDS 269 secondary filter is used. T he fluorom eter is also equipped with a quartz microscale flow-through cell (no. 176F-QS, Helhmi Cells, Inc., Jamaica, N.Y, 11431). T he light source is a general purpose lamp that provides peak emission at 350 nm (T urner no. 110-850). 2. Serum may be d iluted by an autom atic p ip e t (Model 2500; MicroMedic Systems, Philadelphia, Pa. 10104). Reagents 1. Saline. N ine grains of reagent grade sodium chlo ride and 15 drops o f triton X-100 are dissolved in 1 L deionized water. T h e solution is then filtered through a 0.45gm M illipore filter(M illipore, Bed ford, Mass. 01730) to rem ove particulate matter. T he filtered saline is used to make all dilutions of anti serum and standards. 2. Antiserum. Coat antisera specific for ^ -a n ti trypsin, IgG, IgM, and IgA are com m ercially avail able (Techicon Instrum ents Corp., Tarrytown, N.Y. 10591; A tlantic A ntibodies, W estbrook, Me. 04092; Meloy Laboratories, Falls C hurch, Va. 22046).. 3. Standard. Commercial reference serum (Technicon Instrum ents Corp., Tarrytown, N.Y. 10591) is used to provide standardization for these pro teins, Reference serum has been standardized versus WHO standard pool. Standard curve is gen erated by dilutions of this reference serum. Procedure , -A ni it ry p sin 17 1. Prepare working standards by making 4 :5 ,2 :3 ,1 :2 , 1:5, and 1:10 dilutions o f reference serum . 2. Antiserum. D ilute goat antiserum specific for a,-antitrypsin 1:25 with saline. 3. Using a 5 I syringe, add 1.5 d of standard or unknow n sam ple to 1-0 ml d iluted antiserum in 10 x 75 mm lest tube. To second tube add 0.75 /I o f sam ple to 1.0 ml d iluted antiserum us check for antigen excess. Cover all tubes, mix 10 tim es by gentle inversion, and incubate at room tem pera ture for 45 inin. 4. Prepare blanks by addition of 1.5 /d serum to 1.0 ml saline. Blank readings greater than zero are only encountered with grossly lipemic samples. 5. Samples, standards, or blanks are injected into flow cell with 3 m] syringe by injection into inlet port. Flow cell and syringe are washed with saline betw een injections. F luorom eter is adjusted to zero with saline for blank m easurem ents and with dilute antiserum for m easurem ents of sam ples or standards. . Blank values are subtracted from reaction values. N onlinear standard curve is constructed by plot ting lilank-corrected relative intensity of light scattering versus concentration of a,-antitrypsin calculated from dilutions of reference serum. All unknowns are calculated by interpolation from standard curve. 7. Standard curve m easurem ents assum e antibody excess, and if determ ination is carried out as d i rected, antibody excess is achieved up to 300 mg/dl. 8. Concentrations of a,-antitrypsin greater than 300 mg/dl can he d etected by the fact that the reaction containing iiue-half the amount of sam ple will not show a corresponding decrease in light-scattering. If this is observed, then appropriate dilution must be made and the determination repealed. fgC" 1. D ilute serum 700-fold by mixing 20 /d serum with 14 ml saline in 16 x 125 mm plastic screw-top tubes (Falcon Plastics, Oxnard, Calif. 93090). 2. Pipet 1.0 ml diluted serum sam ple into 10 x 100 mm disposable test tubes for reaction and blank m ixtures. At this time 0.5 ml diluted serum is pi p etted into test tube followed by 0.5 ml saline resulting in 2-fold dilution, which is used to screen for antigen excess. 3. M ake 1:25 dilution o f goat antiserum specific for hum an IgG, i.e., 1 part antiserum to 24 parts saline. 4. Add 1.0 ml antiserum to each (action mixture. L ikew ise, 1 ml saline is added to each blank. 5. C over tubes and mix by gentle inversion. 6. Standardization is accom plished by making ap propriate dilutions of reference serum (Teehnicon Instrum ents Corp., Tarrytow n, N.Y. 10591) such that IgC concentrations are approxim ately 14(X), 700, and 350 mg/dl. Standards are analyzed ns de scribed above. 7. Allow reach on tu b e s to incubate at room tem pera ture 40 min. During this period, blanks may he m easured. 8. Adjust fluorom eter to zero for blank m easurem ent w ith saline in How cell. Blanks are then intro du ced into cell with 3 ml syringe, reading is taken, and How cell is w ashed with saline. 9. At e n d o f 40 min incubation, fluorometer is zeroed with solution consistingof1 ml diluted antiserum and 1 ml diluted antisenim plus 1 ml saline. Reac tions' are then m easured. Blank values are sub tracted from reaction values for both standards as well as unknown samples. 10. A nonlinear standard curve is generated and un know ns, blank corrected, are read from this curve. /AM 1. Since concentration oT IgA is low er than IgG, initial dilution o f only 150-fold is made, i.e., 0.10 ml serum d ilu ted with 15.0 mi saline. 2. Procedure for m easuring IgA is identical to that of IgC. Standard curve is generated from dilution of reference serum such that IgA concentrations are 180, 90, and 45 mg/dl. /gM" 1. Initial serum dilution for determ ination of IgM is 100-fold, i.e., 0.10 ml serum diluted w ith 10.0 ml serum d ilu ted with 10.0 ini saline. 2. Procedure for m easuring IgM is identical to that o f IgG. Standard curve is generated from dilution of reference serum such that IgM concentrations are 220, 110, and 55 mg/dl. Remarks 1. Stock and diluted antiserum should he stored at 2-5 C. 2. D ilu te d a n tise ru m is sta b le for 2 .w k w hen s to re d as' d ir e c te d . 3. P recip itin cu rv es sh o u ld be co n stru cte d for each n e w hatch o f an tiseru m in o rd e r to e n su re that m e a su re m e n ts are in zo n e o f an ti body excess. Automated immunochemical determination of serum-proteins A utom ated im m unochem ical technics have b een d ev elo p e d that allow the (|unntitalion of se v e ra l s e r u m p ro te in s s u c h is o ro s o n m e o id , or,-antitrypsin, ftg-m ncroglnhulin, haptoglobin, tran sferrin , C3, C4, IgG, IgA, and IgM . 270 CLINICAL CHEMISTRY PART II Continuous-flow methods39 The automated specific protein determination most w idely used today is based on the conlintimis-fiow concept coupled with nepheItime try for quantitation of the im inunogohulin complex. T he basic system is sim ple and in volves (1) m ixing of the d iluted serum sam ple with diluted monospecific antiserum , (2) incuba tion at room tem perature in a delay coil, (3) m ea surem ent of light-scattering in a flow cell m ounted in a n ep h elo m eter w ith a recorder trac ing of the m easurem ents, (4) m easurem ent of blanks in order to correct for the intrinsic light scattering o f serum sam ples, and (5) construction of the nonlinear standard curves from appro priate dilutions of the reference serum. The im m unochem ical reaction is allow ed to come to completion lie fore light-scattering m easure ments are made. Apparatus 1. An autom atic pipet (M icroM edic Systems, Phila delphia, Pa. 19104) is used to make a 100-fold dilu tiou of sam ples, controls, and standards. 2. A 3-channel continuous-flow system is con structed from parts of the AutoAnalyzer II (Technicnn Instrum ents Corp., Tarrytow n, N.Y. 10591). T his system is com posed o f 1 sam pler, 1 proportioning pump, 3 modified analytical car tridges, 3 fluoronephelometers, 1 dual-pen re corder, and 1 single-pen recorder. T h e system is designed for 100 analyses p er hour w ith a 1:1 sainplc-to-wash ratio. 3. More detailed assem bly instructions are available froin Tech ni co n Instrum ents Corp., Tarrytow n, N.Y. 10591. Reagents 1. M onospecific antisera to orosoniucoid, a ,-an ti trypsin, tij-inacroglobulin, haptoglobin, trans ferrin, C3, C4, IgC, IgA, and IgM are available commercially (Technicnn Instrum ents Corp., Tarrytown, N.Y. 10591; Atlantic A ntibodies, W est brook, Me. 04092). 2. Antisera are diluted appropriately before use with physiologic saline (9 g/L) containing 40 g/L poly ethylene glvcol 6000 (Fisher Scientific Co., Fair Lawn, N.Y. 07410). 3. Standards. A ppropriate dilutions o f reference serum (Technicon Instrum ents Corp., Tarrytown, N.Y, 10591) are used to standardize system. Procedure 1. D ilute all standards, sam ples, and controls 1:100 with physiologic saline (9 g^L) using autom atic pipet. 2. D eterm ine blank values by aspirating sam ples into continuous-flow system using physiologic saline containing polyethylene glycol 6000 as rea gent. 3. Transfer 3 antiserum lines to appropriate antisera solutions and aspirate samples. Reaction values are recorded. In this m anner proteins may be quantitated in groups o f 3. Calculations. Data reduction can be carried out by a minicomputer or programmable calculator pro grammed to do linear interpolation betw een points making up the standard curve, subtract blank values, and print concentration of controls as well as unknown. These operations may be carried out manually. Remarks 1. The system utilizes only 5 8 0 /d of the 1:100 dilution of the serum sample for the blank ns well as for the 9 serum protein determ ina tions. 2. Precision (95% limit) for the 9 serum pro teins ranges from 2.5-12% with 9 of the 10 proteins less than 5%. 3. The polyethylene glycol 6000 is added to enhance the rate of reaction.31'''" 4. Antigen excess sam ples produce a doublet instead of a symmetrical [leak. This doublet is a consequence of the continuous-flow technic with its unique dilutions and mixing systems and makes the identification of an tigen excess samples simple. Centrifugal fast analyzer methodsTM33 T he centrifugal fast analyzer can he modified for near front surface light-scattering using a heli um-neon laser light source. T he laser is m ounted in place of the conventional light source and a quartz disk with a dark-field is placed b e hind the cuvette assembly. T he laser beam [lass es through the cuvette scattering light finm antigen-antibody complexes, and the incident beam then strikes the dark-field, which prevents its detection by the photom ultiplier tube. The scattered light is detected through the quartz disk nround the dark-field. T he methods described in this chapter are kinetic and obviate the use of a serum blank. Polyethylene glycol is em ployed to speed up the reaction rate and enable analyses to be com p leted in only a few m inutes.31 Apparatus 1. K inetic light-scattering m easurem ents are m ade with a laser-modified Amiiico Rotoi-hcm II cen trifugal fast analyzer (American Inst run lent Co., Silver Spring, Md. 20910). 2. Data processing is accom plished by die PD P 8M com puter of the Rotnchein II. Reagents 1. A ntiserum . Antihiiman IgG, IgA, and IgM are ob tained from a comm ercial source (Technicon In strum ents Corp.. Tarrytow n, N.Y. 10591). A 1:20 dilution of each antiserum in phosphate buffer saline containing p o lyethylene glycol is used. 2. Buffer solutions. Phosphate-buffered physiologic saline (PBS), (pH 7.4, 10 mM in phosphate, 0.15M in NaCI) is p repared bv dissolving 1.18 g of" N a,H PO ,, 233 mg 0 r NaHjl>0. and 9.0 g NaCI in 1 L distilled deionized water. 3. Polyethylene glycol solution (PEG-PBS). Solutions of polyethylene glycol (MW 6000-7500) are prepared by dissolving 40 g polym er in 1 L PBS. 4. -Standards. Four different dilutions ofcoinniercinlly available reference serum (Technicon Instrunients Corp., Tarrytow n, N.Y. 10591) spanning the | normal range are em ployed for standardization. I Procedure IkG 1. D ilute sam ples and standards 1:1 IK) with PBSPEG. 2. Transfer 250 pi diluted antihiim an IgC to reagent wells of transfer disk, and 50 pi diluted standard or 1 i ( . I j j I j ! | I i CHAPTER 13 MEASUREMENT OF PROTEINS IN BIOLOGIC FLUIDS 271 Sim ple plus 2(X) PUS-PEC (liliiuiit Ii> sam ple 6. Skeggs, L. T., and Hnchslrasser, II.: C lin. Oheiu. wells. 10:918, 1964. 3. On couimuikI from the com puter, the rotor con 7. Corna!!, A. G., Bardawill, C. J., and David, M. M.; taining transfer disk is accelerated and reactants J. lliol, Cliem. 177:751, 1949. are mixed. After initial mixing, solutions are again 8. Young, D. S., Thomas. D. W., F riedm an, R. H., mixed Tor 0.5 s, 5 s prior to each reading (first Pestouer. L. C.: C lin. Chem . 18:1041, 1972. reading at 15 s, second reading at 65 s). 9. C row ley, L. V.: T ech. Bull. Reg. M ed. T ech. 39:47, IgA. T he procedure for IgA diflers from that de- j 1969. scribed for IgC in the initial sam ple dilution (1:40) and 10. H enry, R. J., Sobel. C., and Sealove, M.: Proc. Sue. die fixed-time interval chosen (15 s and 115 s). Exp. Biol. Med. 92:748, 1956. /gAf. Initial sam ple dilution (1:15) is made in physio 11. Patrick, R. L., and Thiers, R. E,: C lin. C hem , 9:283, logic saline. Diluted antiserum, diluent, and sample 1963. volum es are the same as those used for the m easure 12. Rice, E. W.: Clin. C hem , 21:398, 1975. m ent o f IgC. T he fixed-time interval chosen for analysis 13. Killingsworth, L. M,( Britain, C. E., and W oodard, is 170 s (15 s a n d 185 s). L. L.: C lin. Chem . 21(10): 1465-1468, Sept. 1975. C alcu latio n s 14. W ebster, D., Bignell, A. H. C., and Attwood. E. C.: 1. T he difference b etw een the relative intensity of Clin. Chem . Acta 53:101-108, 1974. the first and second readings may he plotted as a 15. H ellsing, K.: Automated iinm uno-precipitin reac standard curve versus concentration of standards. tions new methods, new techniques and evalua U nknowns may lie determ ined based on the stan tions, Tarrytown, N.Y., 1972, T echnicon Instru dard curve. As in most im m unochem ical determ i m ents Corp., pp. 17-20. nations, this curve is nonlinear. 16. D m pt, F.: Le Pharm acien Biologiste, 8:777, 1974. 2. A lternatively, data reduction may he accom . 17. C oldenberg, H., and D rewes, P. A.: C lin. Chem . plished by a com puter programmed to do linear 16:537. 1970. interpolation based on standard curve. 18. Savory, J., H eintges, M. G., and Sohel, R. E.: Clin. Remarks Chem. 17:301, 1971. 19. Wolfson, W. Q., Cohn, C., Calvary, E., and Elchiha, ]. Tlu* major advantages o f the kinetic m ethod F.: Am. J. Clin. Pathol. 18:723, 1948. are the speed of measurement, eliniinadonof blank measurements, and ease of data reduc tion. 1 2. Precision (95% limits) for IgC, IgA, and IgM is 5%, 4%, and 9%, respectively, for satnples within the normal range. 3. Antigen excess samples must be detected us 20. D ounus, B. T., W atson, W., and Biggs, H. C.: Clin. Chem . Acta 31:87, 1971. 21. Hammond, J. ., H eintges, M. C ., and Savory, J., personal comm unication, May 1975. 22. Kaplan, A., and Savory, J.: Clin. C hem . 11:937. 1965. 23. Johansson, ` B. C.: Scand. J. Clin. Lab. Invest. 29{suppl. 124):7, 1972. ing a 1:1 dilution o f the sam ple (see manual 24. Laurel!, C. B.: Clin. Chem . 19:99, 1973. m ethods), although screening o f agarose 25. Laurell, C. B.: Scand. J. Clin. Lah. Invest. 30:233, electrophoresis gels serves as an adequate means of detecting antigen excess. 1972. 26. Buffone, C. J., Cross, R. E., Savory, J., and Soorlak, C.: Anal. Chem . 46:2047, 1974. REFERENCES 27. Savory, J., and Killingsworth, L. M.: Ann. Clin. Lah. Sci. 3:43, 1973. 1. A nderson, N. G.: C lin. Churi. Acta 25:321, 1969, 28. Killingsworth, L. M., and Savory, J.: C lin. Chem . 2. A nnonr Pharm aceutical Co., Phoeoix, Ariz. 85077. 18:335, 1972. Personal com m unication, May 1975. 29. Killingsworth, L. M., In P eelers, H., editor: Pro- 3. de la lluerga, J., Sm elters, C. W., and Sherrick, J. C. tides o f biological fluids (23rd colloquium , 1975), In Sunderm nn, F. W., and Sunderm an, F. VV,, Jr., N ew York, 1976, Pergam on Press, pp. 291-294. editors: Sem in proteins and dipproteinem ias, Phil 30. Lizana, j., and H ellsing, K.: C lin. C hem . 20:415, adelphia, 1964, J. 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